Plx1 directly binds to the Apc1‐loop500 in a phosphorylation‐dependent manner and promotes the formation of APC/C‐Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A‐B56 is recruited to the Apc1‐loop500 and differentially promotes dissociation of Plx1 and PP2A‐B56 through dephosphorylation of Plx1‐binding sites. Stable Plx1 binding, which prevents PP2A‐B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1‐loop500 is controlled by distant Apc3‐loop phosphorylation. Our study suggests that phosphorylation‐dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle.The anaphase‐promoting complex/cyclosome (APC/C), a multi‐subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin‐dependent kinase 1 (Cdk1) promotes Cdc20 co‐activator loading in mitosis to form active APC/C‐Cdc20. However, detailed phospho‐regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo‐like kinase (Plx1) and PP2A‐B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1‐loop Crosstalk between remote flexible loop domains and their phosphorylation states facilitates fine‐tuning of APC/C‐Cdc20 activity with cyclin‐dependent kinase activity during mitosis. Reversible phosphorylation is crucial for regulation of the activities of proteins involved in the cell cycle. Cyclin‐dependent kinases (Cdks) are arguably the most prominent kinases in cell cycle progression. Cdks can trigger cascades involving kinases and phosphatases, which leads to activation of functional proteins or protein complexes involved in the execution of specific cell cycle processes  play pivotal roles in the Cdk1‐driven activation of APC/C. The Apc1‐loop300 is an auto‐inhibitory domain blocking Cdc20 binding. When Apc1‐loop300 is unphosphorylated, Apc1‐loop300 resides on a site on Apc8B and blocks access of the C‐box, an essential binding motif in Cdc20. Upon phosphorylation, the inhibitory domain Apc1‐loop300 is dislocated from the C‐box binding site, allowing Cdc20 access and APC/C activation. Thus, the Apc1‐loop300 serves as a phosphorylation‐dependent molecular switch for mitotic APC/C. Intriguingly, phosphorylation of Apc1‐loop300 is accelerated by phosphorylation of a distant subunit, Apc3 (Apc3‐loop). Cdk1‐dependent Apc3‐loop phosphorylation recruits Cdk1‐cyclinB‐p9/Cks2, and then, Cdk1 phosphorylates Apc1‐loop300 in a phosphorylation relay. Thus, Apc3‐loop functions as a scaffold to recruit Cdk1 on APC/C.The APC/C is a multi‐subunit complex composed of 14 subunits and requires one of the Cdc20 family of APC/C co‐activators, such as Cdc20 or Cdh1, for catalytic activation of the E3 ubiquitin ligase and substrate recognition of the APC/C  was also studied in the mitotic phospho‐regulation of the APC/C  is an essential serine/threonine kinase for cell cycle events and phosphorylates numerous proteins including APC/C in mitosis  binds to Apc1‐loop500 in mitosis and promotes APC/C‐Cdc20 complex formation. We also demonstrate how Plx1 and PP2A‐B56 loading to Apc1‐loop500 are coordinated through its site‐specific phosphorylation, which is important for rapid entry and exit from mitosis. This study reveals the mechanism of dynamic control of the APC/C by polo‐like kinase and PP2A‐B56 phosphatase and underscores the importance of flexible loop domains.Here, we show that 500: 515–584 in Xenopus] contains two tandem copies of the polo‐binding motifs “S‐[pS/pT]‐[P/X]” (PBMs), in which X indicates any amino acid. These are highly conserved among species and are located upstream of a PP2A‐B56‐binding motif  in Xenopus egg extracts. When wild‐type (WT) Apc1‐loop500 fragment fused to maltose‐binding protein (MBP) was incubated with anaphase extracts supplemented with non‐degradable cyclin B, to ensure continuation of the anaphase state even after activation of the APC/C, endogenous Plx1 binding to Apc1‐loop500 was observed, over and above binding to MBP itself , whereas the mutations within PBMs (T532A/T539A: 2A) abolished PBD binding  remained at nearly undetectable levels even if a higher concentration of PBD‐Pin was used  displayed a decreased band shift of Apc3 in anaphase  was shown to promote activation of APC/C by stimulating phosphorylation of another loop domain of Apc1  . In contrast, L557A/V560A mutation inhibited the binding of Plx1. Either L557 or V560 or both residues may be required for Plx1 binding as these two residues are close to the S558 site that is required for Plx1 binding  was observed  harbouring a mutation by which Plx1 is unable to bind PBMs failed to inhibit B56 binding to Apc1‐loop500  in PP2A‐B56‐binding site strongly inhibited not only PP2A‐B56 binding but also Plx1 binding. We speculated that Plx1 and PP2A‐B56 might share the same binding region presumably around S558. To verify this idea, we investigated whether Plx1 was able to compete with PP2A‐B56 for its binding to Apc1‐loop500, we hypothesised that PP2A‐B56 might promote dissociation of Plx1 from Apc1‐loop500 through dephosphorylation of Apc1‐loop500. We first sought to confirm that purified PP2A‐B56γ dephosphorylates phosphorylated T532, T539 and S558  in Apc1‐loop500  whereas Plx1‐bindng is dependent upon pT539, we wanted to investigate how these CDK sites on Apc1‐loopated Fig . Intriguated Fig , enablin 500 Fig . This waC/C Figs  and EV5.ment Fig . As showB56γ Fig . Apc1‐lop500 Fig . This isn WT Fig , presuma500 because the E562A mutation in Apc1 (1‐E562A) specifically recruits Plx1, but not PP2A‐B56 on Apc1‐loop500. When the mutant APC/C (1‐E562A) was incubated with anaphase extracts for 45 min, the binding of Cdc20 was significantly higher than that to WT APC/C  might cause premature activation compared with WT APC/C. The timing of the activation of the APC/C was monitored by the timing of cyclin B destruction in anaphase extracts in which the endogenous APC/C had been replaced by WT or mutant APC/Cs. With WT APC/C, the degradation of [35S]‐labelled substrate cyclin B initiates around 30 min after the addition of non‐degradable cyclin B into interphase extracts  during mitotic exit by addition of p27, a CDK inhibitor. In WT APC/C, phosphorylation of Apc3 peaked at 5 min after addition of p27 , that is unable to bind Plx1, showed less prominent Apc3 phosphorylation than WT APC/C at every time point , a key site for PP2A‐B56 recruitment, using a specific antibody to phospho‐T532, in anaphase extracts in which the endogenous APC/C had been replaced by WT or mutant APC/C. When nine Cdk1 sites on Apc3‐loop were mutated to alanine (Apc3‐9A), phosphorylation of T532 on this APC/C was significantly reduced by ˜ 50%, compared with that on WT APC/C  as well as MBP antibodies. After phosphorylation, MBP‐Apc1‐loop500 appeared as two bands  on Apc1‐loop500, which differentially take place. According to prerequisite phosphorylation sites for Plx1 and PP2A‐B56 recruitment, Plx1 is recruited to Apc1‐loop500 prior to PP2A‐B56 and promotes APC/C‐Cdc20 complex formation. Thus, we propose that in conjunction with Cdk1 activity, Apc1‐loop500 might function as a feedback control hub, fine‐tuning optimal APC/C activity in mitosis  on a short 69 residue‐Apc1‐loop500, which are highly conserved through vertebrates. Plx1 relies on PBM2 (STPAK), not PBM1 (STPLE), which matches to the genuine CDK consensus sequence [pS/pT]‐P‐X‐[K/R]. In mitosis, T539, a requisite phosphorylation site for Plx1 binding in PBM2, is phosphorylated earlier than T532, a requisite phosphorylation site for PP2A‐B56 binding, in PBM1  over S558  and B56‐binding motif , respectively , which is slightly similar to a genuine PBM (Ser‐pSer‐Pro). In contrast, as for PP2A‐B56, it is the sequence around the PBM1 including T532. These findings may perhaps be explained by either (i) an affinity model in which additional site contact increases its affinity for robust docking or (ii) more than one molecule binds to these sites for cooperative docking. The well‐known PBD consensus, S‐pS/pT‐P, has been determined using short 16‐residue peptides containing a single PBM  play a vital role in site‐specific binding of proteins. Plx1 and B56 are reported to bind to the ‐PBD Fig . Howeveret al, et al, et al, et al, et al, et al, Although we find that PP2A‐B56 recruitment relies on phosphorylation of T532, at the moment, we do not know the underlying mechanism for recognition or binding. In a crystal structure of the B56 bound to a peptide containing B56‐binding motif, the motif is recognised in a concave surface in B56 HEAT repeat domain , that the PP2A‐B56 associated with Apc1‐loop500 and initiate phosphorylation of Apc3‐loop, which in turn promotes phosphorylation of T532 responsible for PP2A‐B56 recruitment, leading to dissociation of Plx1. Phospho‐regulation is no doubt a key element in the APC/C system control, so this feedback control most likely helps to control APC/C activity not only during mitotic progression but also in response to environmental cues. Yet, we cannot exclude the possibility that Apc1‐loop500 could regulate phosphorylation of other APC/C subunits and binding partners, and vice versa. We are still far from understanding how the dynamic regulation of the APC/C is achieved with other mitotic kinases and phosphatases as well as cellular singling networks such as the spindle assembly checkpoint. Future work is definitely required to address the precise molecular basis of this regulation.In summary, this study reveals how Cdk1 and Plx1 promote APC/C recruitment of Plx1 on Apc1‐loopX. laevis PP2A regulatory B subunits are gifts from Dr N. Sagata , Apc3/Cdc27 , Apc5 , Cdc20 , maltose‐binding protein , Plx1  and Flag . Anti‐Apc3 monoclonal antibody (MAb), AF3.1, was used for immunoprecipitation or immunodepletion of APC/C. Apc1 phosphopeptide antibodies  were raised in rabbits against LPH‐conjugated phosphopeptide pT532: CMPRPSpTPLESI; pT539: CLESISpTPAKPL; and pS558: CEAGMLpSPVPEL . Antibodies were affinity‐purified using a phosphopeptide column prepared with SulfoLink Kit (Pierce). After elution with ImmunoPure Gentle Ag/Ab Elution Buffer (Pierce), antibodies were dialysed against TBS and non‐phospho‐specific antibodies were affinity‐depleted by passing through a column cross‐linked with the corresponding non‐phosphopeptide. The eluted phospho‐specific antibodies were then enriched by dialysis against TBS containing 50% glycerol. Plasmids for the expression of X. laevis egg extracts were prepared as described  to interphase extracts and incubating for 30–60 min at 23°C   Murray, . To prepet al, et al, Xenopus APC/C genes and TEV‐cleavable tandem Strep II‐tag fused to Apc6 at C‐terminus (Apc6‐strep) were generated by MultiBac system . Mutant APC/Cs were generated by PCR‐based mutagenesis, and mutation sites were confirmed by sequencing. To express APC/C complex, High Five insect cells (Invitrogen) at a cell density of 1.5 × 106 were co‐infected with the two recombinant baculoviruses at an MOI (multiplicity of infection) of 1 for each virus and incubated at 27°C for 48 h with shaking (150 rev/min). The cells were harvested, frozen in liquid nitrogen and stored at −80°C. The recombinant APC/Cs were purified with Strep‐Tactin beads and further affinity‐purified by Dynabeads Protein A conjugated to anti‐Apc3 monoclonal antibody (MAb AF3.1). The APC/Cs bound to beads were flash‐frozen and stored at −80°C.Expression and purification of recombinant APC/Cs were performed as described previously  fused with PreScission protease‐cleavable maltose‐binding protein (MBP) at the N‐terminus and a TEV‐cleavable 6xHis at the C‐terminus were performed as described previously , spanning amino acids 362–598 of X. laevis Plx1, was fused with a PreScission protease‐cleavable 3xFlag‐tag at the N‐terminus and a TEV‐cleavable 6xHis at the C‐terminus and subsequently subcloned into pET vector. The mutants were generated by PCR‐based mutagenesis. The resultant plasmids were introduced into BL21‐CodonPlus (DE3) and the fusion proteins were expressed at 37°C for 1 h in the presence of 1 mM IPTG for Apc3‐loop or 18°C overnight in the presence of 0.1 mM IPTG for Plx1‐PBD. The cells were lysed by 0.3 mg/ml lysozyme and sonicated in lysis buffer . The proteins were purified from clarified lysate using Ni‐NTA agarose beads (Qiagen). Recombinant p27Kip1 protein tagged with 6xHis was expressed in BL21‐CodonPlus (DE3) and purified using Ni‐NTA agarose beads (Qiagen). To make Flag‐tagged ubiquitin‐fused lysine‐less cyclin B (Ub‐K0‐cyclin B), the N‐terminal 70‐residue fragment of fission yeast cyclin B (N70) with no lysine residue was fused with a PreScission protease‐cleavable Flag‐tagged ubiquitin at the N‐terminus and a TEV‐cleavable 6xHis at the C‐terminus, subcloned into pET vector and then expressed and purified using Ni‐NTA agarose beads (Qiagen). Flag‐tagged X. laevis B56γ was purified as described previously  by incubating at 4°C for 0.5–1 h. Beads were washed with Tris‐NaCl buffer [20 mM Tris–HCl pH 8.0 and 200 mM NaCl] containing 0.01% NP‐40, XBCSF buffer  containing 0.01% NP‐40 and XBCSF buffer. The resultant beads were incubated in interphase extracts in the presence or absence of non‐degradable cyclin B∆167 at 23°C for 60 min, separated from extracts on Micro Bio‐Spin columns (Bio‐Rad) and washed once with XBCSF buffer and then twice with XBCSF buffer containing 0.01% NP‐40. The bound proteins were eluted with SDS sample buffer and analysed by SDS–PAGE and immunoblotting. The binding assay of Apc1‐loop500 to Plx1‐PBD was performed above except that Plx1‐PBD was added after incubation with non‐degradable cyclin B for 60 min and further incubated for 15 min. To see the impact of Cdk‐dependent Apc1‐loop500 phosphorylation on Plx1‐PBD binding, the MBP‐fused Apc1‐loop500 fragment protein was incubated in the presence or absence of Cdk2/cyclin A in buffer  at 30°C for 2hr and was bound to amylose beads (New England Biolabs) by incubating at 4°C for 0.5–1 h in Tris‐NaCl buffer containing 0.01% NP‐40. The beads were washed by Tris‐NaCl buffer containing 0.01% NP‐40 and XBCSF buffer containing 0.01% NP‐40 without EGTA, resuspended into XBCSF containing 0.01% NP‐40 without EGTA, incubated with Plx1‐PBD at 4°C for 10 min, and then separated on Micro Bio‐Spin columns (Bio‐Rad), washed once with XBCSF buffer without EGTA and then twice with XBCSF buffer containing 0.01% NP‐40 without EGTA. The bound proteins were eluted with SDS sample buffer and analysed by SDS–PAGE and Coomassie blue staining. The binding assay using MBP‐fused Apc1‐loop500 fragments and [35S]‐labelled B56γ was performed as described previously  were first phosphorylated by Cdk2/cyclin A in 25 µl of buffer  at 30°C for 2 h and then mixed with purified Flag‐B56γ (5 µg) bound to anti‐Flag affinity M2 beads (Sigma) in buffer  at 4°C for 15 min. The beads were separated on Micro Bio‐Spin columns (Bio‐Rad) and washed once with XBCSF buffer and then twice with XBCSF buffer containing 0.01% NP‐40. The bound proteins were eluted with SDS sample buffer and analysed by SDS–PAGE and immunoblotting.For the binding assay of Apc1‐loop 500 Fig , MBP‐fusCSF_300 [XBCSF containing 300 mM KCl and 0.01% NP‐40], eluted with SDS sample buffer and analysed by SDS–PAGE and immunoblotting.The APC/C was immunoprecipitated using Apc3 MAb (AF3.1) immobilised Dynabeads Protein A beads. The bound proteins were washed twice with XBet al, 35S]methionine  in a coupled in vitro transcription–translation system , and destruction assays were carried out using Xenopus egg cell‐free extracts (anaphase or interphase extracts). The samples were taken at the indicated time points and analysed by SDS–PAGE and autoradiography. The images were analysed using ImageJ .Destruction assays were performed as described previously (Fujimitsu Xenopus egg cell‐free extracts in the presence of non‐degradable cyclin B at 23°C for 45 min or 60 min and immunoprecipitated using Apc3 MAb (AF3.1)‐immobilised Dynabeads Protein A. The beads were washed twice with XBCSF_300 and once with Ub buffer  containing 0.01% NP‐40. The resultant APC/C‐Cdc20 complexes were incubated at 23°C in 20 µl of Ub buffer containing 2 mM ATP, 50 ng/µl E1, 6 ng/µl Ube2S, 750 ng/µl methylated ubiquitin (MeUb) and 5 ng/µl Flag‐ubiquitin‐fused lysine‐less N‐terminal 70‐residues of cyclin B (referred to as Ub‐K0‐cyclin B) as a substrate. The addition of MeUb prevents polyubiquitin chain formation, and a monoubiquitylated substrate facilitates quantification of APC/C activity. The reactions were stopped at the indicated time points with SDS sample buffer and analysed by SDS–PAGE and immunoblotting.Recombinant APC/Cs were incubated with X. laevis Apc3‐loop fragment proteins (80 µg/ml) were incubated in the XBCSF buffer containing 0.01% NP‐40 and 4 µg/ml of purified Plx1 in the presence of 2 mM ATP or 100 µM [32P]ATP at 23°C. The samples (2 µl) were taken at indicated times and analysed using SDS–PAGE, Coomassie blue staining or autoradiography.The purified 500 fragment proteins were bound to amylose beads (New England Biolabs) by incubating at 4°C for 0.5–1 h. Beads were washed with Tris‐NaCl buffer containing 0.01% NP‐40, XBCSF buffer containing 0.01% NP‐40 and XBCSF buffer. The resultant beads were incubated in interphase extracts in the presence of non‐degradable cyclin B∆167 at 23°C for 60 min, washed twice with XBCSF buffer containing 0.01% NP‐40 without EGTA, resuspended into XBCSF buffer containing 0.01% NP‐40 without EGTA and incubated with purified PP2A‐B56γ at 23°C. After reaction, the beads were separated on Micro Bio‐Spin columns (Bio‐Rad) and washed once with XBCSF buffer and then twice with XBCSF buffer containing 0.01% NP‐40. The bound proteins were eluted with SDS sample buffer and analysed by SDS–PAGE and immunoblotting. Dissociation assay of Plx1‐PBD was performed as described above except that Plx1‐PBD was added after incubation with cyclin B∆167 for 60 min and further incubated for 15 min, and the bound proteins were analysed by SDS–PAGE and Coomassie brilliant blue (CBB) staining.For dephosphorylation assay, the purified MBP‐fused Apc1‐loopStatistical analyses were performed in GraphPad Prism v6.0. Quantification data are presented as the mean ± SEM from three independent experiments.KF and HY conceived and designed the project. FK conducted all the experiments under the supervision of HY. KF and HY wrote the manuscript.The authors declare that they have no conflict of interest.Review Process FileClick here for additional data file.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.


The decreased TOC and TN were also observed at 60- to 100-cm sediment depth in the oyster site. This indicated that the OBD significantly impacted the concentration of TOC and TN in the sediment. To confirm the alleviative role of OBD, we conducted stable isotope (δ13C and δ15N) analyses, which further demonstrated the presence of heavier and less lighter forms of organic carbon and nitrogen sediment. The surface sediment layer (0–20 cm) at the oyster site showed 8% more δ13C‰ compared with the control site (p < 0.05), reflecting the reduction in the TOC. In order to reveal the potential microbial mechanisms involved in OBD, we performed a functional analysis using the Geochip5 advanced microarray technology. Regarding carbon metabolism, we observed that genes  involved in the degradation of relatively labile C-based molecules  were highly represented in an experimental area (p < 0.05). In addition, microbes in the experimental area exhibited a greater capacity for degrading recalcitrant C , which involves glyoxal oxidase (glx), manganese peroxidase (mnp), and phenol oxidase. Among the genes controlling nitrogen metabolism, the genes involved in denitrification, assimilation, ammonification, and nitrification were differentially expressed compared with the control area. These results indicated that microbial metabolic roles might have enhanced the C/N-flux speed and reduced the overall nutrient status. We concluded that OBD alleviates sediment nutrient overload under oyster farming from a microbial ecological perspective in a rapidly urbanized coastal area.Oysters are ecological engineers, and previous studies have examined their role as competent facilitators of ecological restoration. However, the decisive role of oysters in the aquatic environment is still debatable because oyster biodeposition (OBD) may also increase the nutrients enriched in sediments. In order to better interpret this problem, we sampled sediment cores from representative oyster culture areas and uncultured areas in Shenzhen Bay. The results have shown that the TOC  and TN  decreased significantly (  Eutrophication and environmental pollution are severe threats to coastal ecosystems worldwide . AnthropFilter feeders have the ability to reduce eutrophication. When oysters and other bivalves feed, they remove particulate organic matter and subsequently decrease the turbidity of water . ShellfiThe effects of nutrient filtration by these filter feeders have been controversial for a long time. The main reason is that the excreta of filter feeders are concentrated in sediments. Sediments act as a sink of nutrients but also may serve as a source of nutrients in the water, leading to eutrophication . Hence, Carbon and nitrogen contained in oyster biodeposits may have different biogeochemical fates, depending upon their environmental situation. Documenting environmental drivers of carbon and nitrogen dynamics in sediments is required to foresee the effect of oyster biodeposition (OBD) on eutrophication. Organisms that feed on deposits can consume fresh organic carbon and nitrogen incorporated in biodeposits, and then disturb the sediments for a brief or an extended period, or decompose to dissolved organic carbon and nitrogen followed by mineralization .Microorganisms are the most direct driving force that affects the amount of matter in sediment. Sedimentary microorganisms can decompose and mineralize nutrients and the organic matter in the biodeposition process , and canin vivo.In the current study, we tested the hypothesis that the sediment carbon and nitrogen concentration are significantly altered under the influence of the OBD status. We suggest that the oyster farming at Shenzhen Bay might accelerate the metabolism of nutrients and other microbial activities. Our purpose was to confirm the positive role of OBD and to determine the related microbial mechanisms involved in the regulatory processes of the carbon and nitrogen cycle from the microbial perspective. We chose Shenzhen Bay as the study area because it is an ideal place to determine the status of anthropogenic influence and eutrophic events during the pre- and post-urbanization period using its sediments as an indicating biomarker. In addition, there is more than a 13-km-long oyster culture area, which supplied us with excellent experimental conditions As for the experimental tools, GeoChip 5.0 advanced microarray technology and continuous flow–isotopic ratio mass spectrometer (CF-IRMS) techniques were simultaneously employed to address two key questions: (1) What is the impact of OBD on nutrients  in the sediments of the oyster farming area, and (2) How do microbes participate in the alleviation of the nutrient overload state? The link between microbial functional genes and oyster biodeposits behind nutrient dynamics was also evaluated. This study may have potential implications on the ecological health of a bay in terms of eutrophication, C burial, and preservation.2 area of Shenzhen Bay. The main species of oyster is Crassostrea hongkongensis. According to research on the chronology of Shenzhen Bay, the average sedimentation rate ranges between 0.92 and 1.10 cm/a, and therefore, the 50-cm-long sediment core was dated back to the year 1958. The clay ranges from 54.4 to 70.0% with a mean of 66.3%, silt from 29.9 to 40.4% with a mean of 32.8%, and sand from 0.0 to 5.8% with a mean of 0.9%.The study site lies in Shenzhen Bay , which is located on the east shore of the Pearl River Estuary, Shenzhen, Guangdong Province, China . ShenzheSediment samples were collected from two distinct Shenzhen Bay sites: the oyster farming area (test site) and the non-farming area (control or reference site) during a cruise in November 2018. At each location, eight manual cores were taken by a 100-cm-long polyvinyl sediment collector, and each sampled core was rapidly divided into five distinct layers of equal measure (20 cm) from the top to the bottom end. The sediments were sealed in airtight plastic bags and maintained at 4°C until they could be immediately brought back to the laboratory for further analysis.a concentration were measured with an automated instrument .Each sediment sample was subdivided into independent portions for GeoChip analysis, stable isotope detection, and nutrient determination. Suspended particulate matter was collected by filtering 500–1,000 ml of surface water from the bay with a piece of mesh having a pore size of 200 μm. The temperature, pH, salinity, turbidity, dissolved oxygen, and chlorophyll-3–, NO2–, and NH4+) were determined by a discrete chemistry analyzer  following a standard colorimetric method (http://cmore.soest.hawaii.edu. Standards (purity 99%) were purchased from Shanghai Macklin Biochemical Co., Ltd. (China). The organic carbon and nitrogen were analyzed by drying the sediment in an oven at 60°C overnight, followed by an acid wash to remove an inorganic portion. The analysis was performed using an elemental analyzer  with analytic precision of < 0.5%.The levels of dissolved inorganic nutrients . The δ13C and δ15N values of OM in the surface sediments and biodeposited samples were analyzed using an isotope ratio mass spectrometer (DELTAplus XL). Stable isotope ratios were assigned in δ notation expressed by the deviation from a standard reference material in parts per mil (‰): δ13C or δ15N (‰) = (Rsample/Rstandard– 1) × 1,000, where R denotes 13C/12C or 15N/14N. PeeDee Belemnite was used as a reference standard for carbon with an analytical precision of < ± 0.1‰, whereas atmospheric N2 was used as a reference standard for nitrogen with an analytical precision of < ± 0.2‰  can be defined as:RIC =  × 100,RIN =  × 100 (N) × 100 ,where RI denotes the reference index of carbon/nitrogen, and OC and N denote organic carbon and nitrogen, respectively.2H25SO4 for cellular breakdown .Briefly, 0.5 g of each sediment sample was extracted to obtain the microbial community genomic DNA, and the samples were sequentially ground in liquid nitrogen. The fine samples were then further treated with NaC1reakdown . Total DGeoChip 5.0 was used to analyze the functional potential of sediment microbial communities. The purified DNA (500 ng) was labeled with Cy 3 as previously described . The lab1 and analyzed as previously described  the microbial metabolic potentials were further assessed by the relative signal intensity, which was the standardized signal intensity normalized again based on the number of probes per sample . The graphs were drawn using OriginPro 2021b software (OriginLab Corporation).Quantities are expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was carried out to determine the effects on each parameter, followed by Tukey’s tests. To analyze the differences between the oyster site and control site, 4+, NO3–, NO2–, and PO43–) are shown in The basic physical and chemical parameters  and nutrient parameters % values at the oyster site were 0.081 ± 0.007%, 0.075 ± 0.006%, 0.063 ± 0.004%, 0.056 ± 0.009%, and 0.073 ± 0.011% at 0–20, 20–40, 40–60, 60–80, and 80–100 cm of depth, respectively . These v13C‰ and δ15N‰ ranged from −21.55 ± 0.42‰ to −23.68 ± 0.53‰ and 6.92 ± 0.30‰ to 6.51 ± 0.13‰, with an increasing concentration of heavier forms of carbon and nitrogen as compared with their lighter forms .The results from the experimental CF-IRMS analysis showed the isotopic fractionation of light and heavy carbon as well as nitrogen elements. The values of δer forms . There wing site . The higter site . The sur12 and N14 mg/g, wherever the concentration of heavier forms, δ13C‰, and δ15N, was found to be higher  showed the similarity between TOC and TN in scatter plots regarding the distribution trend. Alleviated values of nutrients such as TOC and TN can be seen in ∗ ×) shows 0.04 ± 0.01 intercept for the oyster site and –0.03 ± 0.02 for the reference site  between TOC% and TN% at the site under OBD influence.After measurement of the statistical association between the two variables TOC% and TN%, we observed the alleviative pattern at the oyster culture site compared with the reference site . The resnce site . Therefo13C‰ and δ15N‰ was stronger at the reference site compared with the oyster site . A potentially moderate correlation  was also seen between δ13C‰ and δ15N‰ at the oyster farming site  showed an 8.78 ± 0.81 intercept for the oyster site and 20.59 ± 2.83 for the reference site  between δ13C‰ and δ15N‰ at the site under OBD influence.As far as the results of distribution and correlation of stable isotopes of carbon and nitrogen were concerned, it appeared that the Pearson’s coefficient correlation between δing site . The resnce site . The plonce site , which dt-test analysis showed that the number of genes detected at the oyster site was significantly higher than that at the reference site  at the oyster site.Microbial functional gene categories for major biogeochemical and metabolic processes were examined to understand the effects of oyster biodeposits on sediment microorganisms. One-way ANOVA and paired nce site . No signnce site , 6. Amonnce site . A totalt-test analysis showed that the gene pectate_lyase_Oomycetes that codes for labile pectin degradation emitted significantly higher signal intensity at the oyster site. The surface oyster site receiving direct biodeposition significantly degraded both labile and recalcitrant carbon (p < 0.05), but the genes for labile organic matter degradation were more abundant than those for recalcitrant; therefore, labile carbon degradation was occurring at a higher rate  signal intensity at the oyster site. However, an abundance of genes coding the enzymes for methane synthesis and oxidation was noted . For denitrification, only nirk, nosz, and narg exhibited a significant difference between the oyster and reference sites. For denitrification at the reference site, there were no significant differences in the abundances of the genes nirk, nosz, and narg.The paired her rate . Among tas noted . In N meas noted , key funPreviously, it was deemed that sedimentary OM could act as an internal source of nutrients and energy, which might have important implications for the ecological sustainability of coastal marine ecosystems and eutrophication. In this work, we observed a significantly lower quantity of total carbon and nitrogen at the oyster site compared with the reference site . A possi2 in the year 2013 also presented a report describing the role of OBD in alleviating nutrient status. They mentioned that the nutrients supplied through oyster biodeposits might be partially released into the atmosphere and buried in deep sediments. Thus, we found lower values of TOC% and TN% due to sediment–biodeposit mix-up that constituted a different textural characteristic of surface sediment by incorporating more labile or degradable organic matter. The linear regression analysis with Pearson’s coefficient correlation showed that the distribution pattern of nutrients was greatly affected by the OBD at the oyster site. A strong positive correlation  between total organic carbon and nitrogen with an intercept for oysters and for the reference site suggested their use of a common source at the places under OBD influence. The combination of OBD with sediment gave rise to the expedition and boosted the microbial communities to efficiently and actively participate in releasing nutrients from sediments back into the water column and the environment.The Chesapeake Bay Program Scientific and Technical Advisory CommitteePrevious studies suggested that the biochemical composition of oysters and other shellfish biodeposits could boost the rate of metabolic activities that convert excessively available sediment carbon and nitrogen into releasable and sequestered forms . Most fi13C than the reference site, suggesting that the sediment layer has been receiving a mixture of inputs from estuarine and OBD sources. However, the δ15N values from the surface sediment layer of the oyster site indicated that there was also an anthropogenic influence at the oyster farming area. There was an enhanced percentage of δ13C‰ (11–12%) at sub-surface and core sediments, which decreased to (9%) in the sub-bottom, and its lowest turnover was found in the bottom sediments of the oyster-farming site, indicating the additional presence of organic carbon.The surface sediment layer of the site under OBD influence showed less depleted values of δ13C values, suggesting that the mixture of inputs from estuarine and OBD sources not only existed at the surface layer, but it also settled with increasing sediment depths. The surface sediment layer that received direct biodeposition from hanging oysters also showed increased δ15N‰. The change in the isotopic signature of δ13C‰ and δ15N‰, and the lower values of TOC% and TN% at the oyster culture site compared with the reference site appearing in regression analysis indicated the utilization of lighter forms of carbon and nitrogen under OBD influence.The sub-surface sediment portion expressed less depletion and increased δ13C‰ and δ15N‰ in surface sediment, but lower N14 implied that the fractionation of carbon and nitrogen resulted in their fluctuating values. The lowest turnover of δ13C in the bottom sediments at the oyster-farming site indicated a greater presence of organic carbon. The possible reason behind the higher δ13C‰ concentration at the oyster site compared with the control site (p < 0.05) might be the OBD influence, which drove the surface microbial communities to expedite the metabolic process of organic carbon reduction and release.Higher δ13C and δ15N values are consistent with those reported in other research studies (13C = −22.4‰ to −26.5‰ and δ15N = 1.8‰ to 3.8‰) and marine organic matter (δ13C = −18‰ to −24‰ and δ15N = 4‰ to 9‰). In addition, a study on the Pearl River estuary (which is near Shenzhen Bay) also reported similar results that could support our current data , pectinase (pectin), xyla (xylose isomerase), lactase_fungi (lactase), cdh (cellobiose dehydrogenase), ccml , and csoS1_Ccmk . The gene cda coding for the protein enzyme alpha amylase catalyzes the degradation of starch carbohydrates; thus, the labile carbon from starch faces microbial degradation in sediments. Pectin and hemicellulose were also in the category of semi-labile and, therefore, were degraded by microbes. However, cdh, which codes for cellobiose dehydrogenase, acted on terpenes to bring about the microbial degradation of recalcitrant carbon. Therefore, the surface site of oysters (TO) that received direct biodeposition from oyster rafts was involved in the significant degradation of both labile and recalcitrant carbon (p < 0.05), but the genes for labile organic matter degradation were more abundant than those for the recalcitrant. This implies that labile carbon degradation was occurring at a higher rate , IcfA (carbonic anhydrase), MCM (mm-CoA mutase), and mch (mesaconyl-C1-CoA hydratase). These changes indicated that the C cycle was more active at the oyster site than the reference site. The gene MCM was found in the subcategory of the 3-hydroxypropionate bicycle, and the gene mch is related to the 3-hydroxypropionate bicycle. The cyanobacterial ccmM gene has recently been proven as a source of increasing photosynthetic CO2 fixation in crop species , fumarase_3HP4HB (fumarase), and adhc , showed downregulation at the site under OBD influence  compared with the OBD-less effective area (BO), suggesting more active carbon fixation in the upper sediment layers. From an ecological standpoint, changes in C utilization and fixation by the microbial community may have long-term effects on C storage and sequestration  were enriched, and these are involved in ammonia oxidation and catalyze ammonia oxidation to nitrite. Ammonium production from N2 and nitrate may have been enhanced in the microbial community through enrichment of nirk (nitrite reductase-copper-containing), nirb (nitrite reductases-b), nifh (nitrogenase reductase), napa (nitrate reductases-a), and nrfa (nitrite reductase-a), thereby increasing the ammonium conversion efficiency.As far as the abundance of genes related to the nitrogen cycle was concerned, the GeoChip array showed that multiple genes were involved in nitrogen cycling . The majgdh (glutamate dehydrogenase), urec (urease), hao (hydroxylamine oxidoreductase), nosz (nitrous-oxide reductase), narg (nitrate reductase), nasa (nitrate-assimilating genes), hzo (hydrazine oxidoreductase), and hzsa (hydrazine synthase)  . The nitSome previous studies reported sediment nutrient alleviation from an aquatic ecosystem due to sediment denitrification . These sin situ to assess the sediment nutrient status of an area under OBD influence. Oyster farming via its biodeposition ameliorated the distribution of total organic carbon, total nitrogen, and their stable isotopes (δ13C‰ and δ15N‰). The stable isotopes showed that different utilization forms (lighter or heavy) of carbon and nitrogen were observed in the test area and control area. The GeoChip data confirmed that the potential mechanisms of OBD on C and N cycling might have originated from increased carbon degradation ability and increased denitrification capacity of the sediments. Our results provided related evidence that the sediments were greatly influenced by OBD input, which resulted in a more obvious alleviative effect in return. The significant enrichment of sediment carbon and nitrogen cycle genes due to increased microbial metabolic activity leads to nutrient alleviation under OBD influence. Also, the OBD changed the carbon–nitrogen flux and reduced the nutrient levels, which might be beneficial for a healthy aquatic ecosystem. The molecular mechanisms of oysters as ecological engineers to mitigate nutrient load in an aquatic ecosystem deserve further study in the future.The current study addressed the issue of mitigating nutrient load by conducting fieldwork The original contributions presented in the study are included in the article/AM was responsible for the conceptualization, investigation, preparation, and writing of the original draft. QY organized the teamwork in the field area and analyzed the environmental factors. XS and JS were in charge of the data analysis and software application. XL collected the samples and participated in the field experiments. JZ and Z-HC read and revised the manuscript and were in charge of the funding acquisition and project administration. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The inhibition of glutamate release by ISL was prevented after removing extracellular Ca2+ or blocking P/Q-type Ca2+ channels. This inhibition was mediated through the γ-aminobutyric acid type B (GABAB) receptors because ISL was unable to inhibit glutamate release in the presence of baclofen (an GABAB agonist) or CGP3548 (an GABAB antagonist) and docking data revealed that ISL interacted with GABAB receptors. Furthermore, the ISL inhibition of glutamate release was abolished through the inhibition of Gi/o-mediated responses or Gβγ subunits, but not by 8-bromoadenosine 3′,5′-cyclic monophosphate or adenylate cyclase inhibition. The ISL inhibition of glutamate release was also abolished through the inhibition of protein kinase C (PKC), and ISL decreased the phosphorylation of PKC. Thus, we inferred that ISL, through GABAB receptor activation and Gβγ-coupled inhibition of P/Q-type Ca2+ channels, suppressed the PKC phosphorylation to cause a decrease in evoked glutamate release at rat cerebrocortical nerve terminals.Reduction in glutamate release is a key mechanism for neuroprotection and we investigated the effect of isoliquiritigenin (ISL), an active ingredient of Glycyrrhiza with neuroprotective activities, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes). ISL produced a concentration-dependent inhibition of glutamate release and reduced the intraterminal [Ca Glycyrrhiza glabra L. root. ISL has received attention because of its various pharmacological benefits, including antibacterial, antiviral, antidiabetic, antioxidant, anti-inflammatory, anticarcinogenic, analgesic, and neuroprotective effects pyrrolocarbazole-12-propanenitrile (Go6976), cis-N-(2-Phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride (MDL12330A), gallein, and nifedipine were purchased from Tocris Cookson . Fura-2-acetoxymethyl ester (Fura-2-AM) was purchased from Invitrogen . ω-conotoxin GVIA and ω-agatoxin IVA were purchased from Alomone lab . PTX, 8-bromo-cAMP and all other reagents were purchased from Sigma-Aldrich .Isoliquiritigenin  was purchased from ChemFaces . 4-aminopyridine (4-AP), baclofen, (3-Aminopropyl)(diethoxymethyl)phosphinic acid (CGP35348), bafilomycin A1, isindolylmaleimide I (GF109203X), 5,6,7,13-tetrahydro-13-methyl-5-oxo-12Male Sprague-Dawley rats (150–200 g) were maintained at 21–23 °C with free access to water and food, under a 12:12 h light/dark cycle (lights-on at 07:00 h). The procedures used in this study were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experiments were developed after the protocol approved by the Institutional Ethics Committee (A10814). In this study, all efforts were made to minimize the number of animals used and their suffering.g for 10 min at 4 °C. The supernatant was retained and centrifuged at 14,500× g for 12 min at 4 °C. The pellet was resuspended and layered on top of a discontinuous Percoll gradient and centrifuged at 32,500× g for 7 min at 4 °C. Protein concentration was determined using the Bradford assay. Synaptosomes were centrifuged in the final wash to obtain synaptosomal pellets with 0.5 mg protein   . Therefo2 for 30 min at 37 °C. After Fura-2 loading, synaptosomes were centrifuged for 1 min at 3000× g and pellets were resuspended in hepes-buffered medium containing 1.2 mM CaCl2. Fura-2/Ca fluorescence was monitored in a Perkin-Elmer LS55 spectrofluorimeter at excitation wavelengths of 340 nm and 380 nm (emission wavelength 505 nm), 340/380 fluorescence ratios (F) were calculated. [Ca2+]C (nM) was calculated following calibration procedures C (nM) was calculated by the equation ([Ca2+] = dK (F − Fmin/Fmax − F)) . The inhibition of 4-AP-evoked glutamate release by ISL was concentration-dependent, with a maximum inhibition of 78.6 ± 0.5% produced at 50 µM; the half maximal effective concentration for this inhibition was 17.2 µM . When bafilomycin A1 was present, 10 µM ISL failed to produce significant inhibition . Similarly, the glutamate release evoked by 1 mM 4-AP was reduced in the presence of extracellular-Ca2+-free solution that contained 300 µM EGTA . This Ca2+-independent release evoked by 4-AP was unaffected by 10 µM ISL  = 50.9, p < 0.001 vs. control group].To investigatewhether the inhibition of 4-AP-evoked glutamate release by ISL was mediated by an effect on exocytotic vesicular release, we examine the action of ISL in the presence of bafilomycin A1 (0.1 µM), which causes the depletion of glutamate in synaptic vesicles. As shown in of VGCCs . As illu2+ through both N- and P/Q-type Ca2+ channels . The results above described indicated that the ISL-induced inhibition of glutamate release involves a reduction in Ca2+ influx through VGCCs. To verify this hypothesis, we used selective VGCC inhibitors to characterize the role of individual Ca2+ channel subtypes in the observed ISL-mediated inhibition of glutamate release. As shown in p < 0.001). When ω-conotoxin GVIA was present, glutamate release was further inhibited by 10 μM ISL . Notably, in the presence of ω-agatoxin IVA, 10 µM ISL inhibition of glutamate release was completely abolished , indicating that L-type VGCCs are not directly support glutamate release from synaptosomes. When nifedipine was present, 10 µM ISL significantly reduced 4-AP-evoked glutamate release . In addition, ISL also inhibited the release of glutamate induced by 15 mM of external KCl, which is a VGCC-dependent process C and neurotransmitter release  in nerve terminals can activate PKC and subsequently phosphorylate MARCKS, which increases synaptic vesicle availability and glutamate release C associated with GABAB receptor activation and the Gβγ-coupled inhibition of VGCCs as well as the subsequent suppression of PKC and MARCKS phosphorylation. Our finding provided valuable new insights into the mechanism by which ISL operates in the brain and offered a potentially therapeutic treatment for neuronal diseases that involve excessive glutamate release.In conclusion, the inhibitory effect of ISL on glutamate release from cerebrocortical nerve terminals is linked to a decrease in [Ca


To investigate the feasibility of using cortical bone trajectory (CBT) screws for bridging fixation in revision surgery for lumbar adjacent segment degeneration and to provide a reference for clinical practice.Computed tomography scans of the lumbar spines of 36 patients in our hospital were used. Sixteen males and 20 females with an average age of 65.5 ± 10.5 years (range: 46 to 83 years) were included. Three-dimensional reconstruction was performed using computer software. Screws with appropriate sizes were selected for the L1 to L5 vertebral segments, and traditional pedicle screws were placed using the standard method. After completing screw placement, simulated placement of CBT screws was performed separately. No overlap occurred between the two screws in the process of CBT screw placement, and the placement point and direction were adjusted until screw placement completion. After all screw placement simulations were complete, according to the contact area of the cortical bone of the screw trajectory and the screw puncture position and distance through the trajectory, the screw placement results were categorized as excellent, good, general, and failure. Excellent and good ratings were considered successful, while a general rating was regarded as acceptable. Then, the success rate and acceptable rate of each segment of the lumbar spine were calculated.Three hundred and sixty screw placement simulations were performed in lumbar pedicles, and 72 CBT screws were implanted in each vertebral body of the lumbar spine. The success rates in the L1 to L5 segments were 73.6%, 80.6%, 83.3%, 88.9%, and 77.8%, respectively, and the acceptable rates were 91.7%, 97.2%, 97.2%, 100%, and 91.7%, respectively. The overall success rate and acceptable rate of CBT screw placement in the lumbar spine were 80.8% and 95.6%, respectively.CBT screws are feasible for bridging fixation in lumbar adjacent segment degeneration revision surgery, and the accuracy of screw placement in different lumbar vertebrae varies. Cortical bone channel screws are used for the upper vertebral body, pedicle screws are used for the lower vertebral body, and pedicle screws are used on one side, while cortical bone channel screws are used on the other side.–12 In the literature, cortical bone channel screws and pedicle screws have been reported to be inserted simultaneously in the same pedicle to improve fixation strength. In addition, CBT screws can also be used for bridging and fixation in revision surgery for diseases of the lumbar vertebrae adjacent to the vertebral body. In this way, bridging and fixation can be completed without removing the original internal fixation device, which never simplifies the operative steps, minimizes trauma, or promotes patients’ recovery. However, few reports on this method are available, and the operation is difficult. What is the accuracy of CBT screw placement for bridging fixation? Do various cones differ? At present, related anatomical research is lacking. In this study, we use digital technology to study the feasibility of CBT screws in the repair and bridging fixation of lumbar vertebra-adjacent vertebral body disease and provide a reference for clinical application.In recent years, cortical bone trajectory (CBT) screws have constituted a new method of posterior lumbar internal fixation. This technique not only can be used in patients with osteoporosis but also has the advantages of minimal trauma, strong holding power, and few complications.2This study was conducted in accordance with the Declaration of Helsinki and approved by the institutional review board of the 920 Hospital of the joint service support force of the Chinese people's liberation army. The need for informed consent was waived by the institutional review board.2.1The computed tomography (CT) data of 36 inpatients from December 2015 to June 2018 were obtained, including 16 males and 20 females with an average age of 57.90 ± 17.40 years (range: 46–83 years). Inclusion criteria:(1)patients aged greater than 18 years; and(2)complete lumbar CT scan data.Exclusion criteria:(1)lumbar deformity or vertebral dysplasia;(2)damaged vertebral structures due to fractures, infections, or tumors; and(3)a history of lumbar surgery.2.2The lumbar CT scan data from 36 patients were imported into Mimics 19.0 software for three-dimensional (3D) reconstruction in Digital Imaging and Communications in Medicine format. Since the height of the L1-L5 pedicles is greater than the width, the pedicle width is the main factor limiting the diameter of a screw. Therefore, we first measured the pedicle width (PW), pedicle spongy width, and pedicle trajectory reference width (TRW), as shown in Figure First, TP screws were positioned in the L1 to L5 pedicles Fig. . The cri2.3T test, including the parameters of the left and right pedicles. P < .05 was considered statistically significant.SPSS 22 statistical software (SPSS) was used for statistical analysis. The Kolmogorov-Smirnov test was used to assess whether the quantitative data conformed to a normal distribution. Quantitative data with a normal distribution are presented as 3In this study, 360 screw placement simulations were performed in lumbar pedicles, and 72 CBT screws were implanted in each vertebral body of the lumbar spine. L1 pedicle screw results: 33 screws were excellent, 20 were good, 12 were general, and two failed; the success rate of screw placement was 80.6%, the acceptable rate was 97.2%, and the failure rate was 2.8%. L2 pedicle screw results: 44 screws were excellent, 14 were good, 12 were general, and two failed; the success rate of screw placement was 80.6%, the acceptable rate was 97.2%, and the failure rate was 2.8%. L3 pedicle screw results: 44 screws were excellent, 16 were good, 10 were general, and two failed; the success rate was 83.3%, the acceptable rate was 97.2%, and the failure rate was 2.8%. L4 pedicle screw results: 50 screws were excellent, 14 were good, eight were acceptable, and 0 failed; the success rate of screw placement was 88.9%, and the acceptable rate was 100%. L5 pedicle screw results: 38 screws were excellent, 18 were good, 10 were general, and six failed; the success rate was 77.8%, the acceptable rate was 91.7%, and the failure rate was 8.3%. The success rate of lumbar spine screw placement was 80.8%, the acceptable rate was 95.6%, and the failure rate was 4.4% , and patients with severe symptoms must be treated with revision surgery.,16,17 Different studies have reported that the incidence of LASD varies, and the incidence of LASD varies between studies. According to imaging evidence, the prevalence of LASD exceeds 40%, and the rate of surgical intervention ranges from 5.2% to 18.5%.,8 LASD repair often requires removal of the original internal fixation device, resulting in a prolonged operative time, intraoperative blood loss, many postoperative complications, and increased difficulty and risks of surgery and thus leading to substantial challenges for many surgeons.,19With the aging of the world population and the development of the pedicle screw technique, the number of patients undergoing lumbar fusion with internal fixation has increased rapidly each year. Between 1994 and 2006, the National Survey of Ambulatory Surgery revealed 5.4- and 2.6-fold increases in the number of surgeries for patients with intervertebral disc disease and spinal stenosis, respectively, and the number of patients requiring intervertebral fusion surgery increased by 340%. used an O-arm navigation system to complete LASD repair in five patients by placing CBT screws in the pedicle of the fused upper vertebra without removing the original internal fixation device. During the follow-up of 10 to 15 months, no surgical complications occurred, symptoms were significantly improved, and interbody fusion was achieved. Wang et al also reported satisfactory results for 12 patients with lumbar spondylitis who underwent revision surgery with CBT screws without removing the internal fixation device. Chen et al performed revision surgery on six patients with LASD using a C-arm machine alone, and satisfactory results were also achieved. Based on these studies, CBT screws can be used for revision surgery in patients with LASD; however, related reports are rare in the literature. Due to differences in the pedicle anatomy of different segments of the lumbar spine, the same lumbar segment displays some differences between individuals. Can CBT screws be used for revision bridge fixation in patients with LASD? What is the accuracy rate of lumbar revision surgery? The use of CBT screws in bridge fixation to complete revision surgery has been challenging in an increasing number of patients with LASD.The advent of the CBT screw provides a new option for fixation in LASD repair. CBT screws can be used for bridge fixation of adjacent vertebrae to achieve revision fixation, which reduces the technical difficulty and risk of traditional revision surgery. In 2014, Rodriguez et al selected lumbar CT scan data from 47 patients and divided them into two groups according to their history of lumbar surgery. In one group, CBT screws were placed based on the locations of the original pedicle screws. In the other group, two types of screws were placed concurrently, and the authors reported the feasibility of inserting two screws at the same time. However, the results obtained from the study have very limited value because of the nonuniform distribution of the surgically fixated segments in the group with a surgical history, namely, only three patients underwent original fixation in L1. In the present study, we used digital technology to simulate CBT screw placement based on TP screws and verified the feasibility of CBT screw placement. Using this approach, we successfully solved the problem of the “nonuniform distribution of surgically fixated segments” in the study by Jeffrey et al.Mullin et al used CBT screws with a diameter of 4.5 mm (L1-L5: 4.5 mm) and TP screws with a diameter of 5.5 to 6.5 mm (L1-L2: 5.5 mm and L3-L5: 6.5 mm). Rodriguez et al selected CBT screws with a diameter of 5.5 mm and TP screws with a diameter of 7.0 to 7.5 mm  for revision surgery. Different screw sizes might have different effects on the success rate and biomechanical strength. Screws that are too large might also increase the risk of fracture of the pedicle isthmus. Thus, further studies are needed. Based on conventional criteria, we simulated TP screw placement and then CBT screw placement. This study has good repeatability and had a sufficient sample size. At the same time, the screw trajectory, the cortical bone puncture, and the distance to the screw's exit point can be observed intuitively, and the result of screw placement is simple to judge. When evaluating screw placement, we classified screw placement according to the contact area of the CBT screw locus and the distance between the screw and the cortex. Then, the results of screw placement were classified as excellent, good, general, and failure and combined with the location of the puncture path . Excellent and good ratings were considered successful, and a general rating was regarded as acceptable, and then the success rate and acceptable rate of each segment of the lumbar spine were calculated. The evaluation method fully considers the characteristics of the CBT screw track and the safety of screw placement such that the evaluation of screw placement results is more reasonable and scientific. The results showed that the success rate of lumbar spine screw placement was 80.8% (73.6% ∼ 88.9%), and the acceptable rate was 95.6% (91.7% ∼ 100%). Some differences in the success rate and acceptable rate of screw placement were found in different segments of the lumbar spine. The accuracy rate of screw placement from high to low was L4 > L3 > L2 > L5 > L1, which is related to the screw size and anatomical structure of the pedicle.In the present study, lumbar CT scan data from 36 patients were used to simulate screw placement after 3D reconstruction. According to the parameters of the pedicle, the diameter of the selected CBT screw was 4.0 to 5.0 mm, and the diameter of the pedicle screw was 5.5 to 6.5 mm. These screw sizes were not the same as those selected in previous studies. Ueno et alThis design has some shortcomings because pedicle screws are placed in accordance with conventional standards, but in clinical practice, pedicle screws can be located in the upper, middle, or lower walls of the pedicle in the sagittal position and in the outer, middle, or inner walls of the pedicle in the coronal position. In the latter study, we classified pedicle screws according to the different positions of the pedicle screw in the pedicle and further improved the accuracy of screw placement. Because the operative technique, pedicle screw position, and individual differences in patients affect the results of screw placement, some differences in screw placement simulations in the software will occur. In addition, we explored only the feasibility of using CBT screws to bridge and fix vertebral bodies with upper and lower fixation with respect to posterior fixation for LASD repair. In addition to posterior fixation, the curative effect of LASD revision is also related to interbody fusion and nerve root decompression. A computer navigation system can be used to facilitate screw placement in clinical practice. Based on the CBT screw channel, we can also design a guide plate to assist with screw placement. When conditions are not sufficient, we can reconstruct 3D images according to patients’ initial CT data, simulate screw placement according to the position and size of the fixed screws, and then simulate CBT screw placement based on the pedicle screws again to provide a reference for the operation.5In this study, CBT screws were feasible for bridging fixation in LASD revision surgery, and the accuracy of screw placement accuracy in different lumbar vertebrae varied. However, the feasibility of using different pedicle screw positions requires further exploration. In addition, further experimental research and clinical application verification are needed.Conceptualization: Long wang, Yonghui Zhao, Sheng Lu.Data curation: Long wang, Yonghui Zhao, Xingbo Cai.Formal analysis: Yonghui Zhao, Xingbo Cai, Jinlong Liang.Funding acquisition: Yongqing Xu, Sheng Lu.Investigation: Yonghui Zhao, Xingbo Cai, Yulong Ma.Methodology: Long wang, Yonghui Zhao, Haotian Luo, Sheng Lu.Writing – original draft: Yonghui Zhao.Writing – review & editing: Long wang, Sheng Lu.


Introduction: Pin site infection is a common complication to external ringfixation. While the aetiology is well described, monitoring of onset,location, and the distribution of infection among the pin sites still needsfurther attention. The present pilot study evaluates the feasibility of aprospective registration procedure for reporting, evaluating, and monitoringof pin site infections in patients treated with external ring fixation. Thismay promote communication between team members and assist decision-makingregarding treatment.Methods: A total of 39 trauma, limb deformity, and bone infection patients ) treated with external ringfixation were followed in the outpatient clinic using the pin siteregistration tool. Pin site infection (Checketts and Otterburn (CO) grade,onset, location), use of oral or intravenous antibiotics, and any unplannedprocedures due to pin sites complications  were registered until frameremoval.Results: The mean (SD) frame time was 164 (83) d (range: 44–499). Weperformed 3296 observations of 568 pin sites. Pin infection was registeredin 171 of the 568 pin sites (30 %), of which 112 (65 %) were categorizedas CO 1, 42 (25 %) as CO 2, 9 (5 %) as CO 3, and 8 (5 %) as CO 5. Neither CO 4 norCO 6 was observed. A total of 35 patients (90 %) encountered CO 1–3 at least onceduring the observation time, while 1 patient (2.5 %) developed a majorinfection at eight pin sites (CO 5). Antibiotics were administered to Conclusion: In an effort to monitor pin site infections in this complexpatient group and to ensure the best clinical outcomes, our registrationprocedure in the outpatient clinic helped to recognize pin site infectionsearly and eased communication between team members providing a conciseoverview of the treatment course. However,there is a risk of acquiring pin site infections, which can occur during theentire treatment period. Pin site infection is the most frequentcomplication for external ring fixators with a reported incidence rangingfrom 10 % to 100 % depending on the indication, patient-relatedfactors, pin site care, as well as the duration of treatment . Superficial pin site infections can progress tomajor deep complications, e.g. severe soft tissue infection and acute andchronic osteomyelitis. Ultimately, this can result in frame abandonment withsignificant morbidity and mortality risk for the patient. In 1993, Checkettsand Otterburn (CO) published a classification system for pin site infectionsto support treatment decision-making  (Table 1).The CO-classification grades pin site infections into two essential groups:minor infection (CO 1–3) and major infection (CO 4–6), with involvement ofbone tissue in the latter, leading to pin loosening.Whereas the causes of pin site infections are adequately described in theliterature, monitoring of onset, location, and the distribution of infectionamong the pin sites still needs further attention. In order to monitor pinsite infections, facilitate communication between team members, and assisttreatment decision-making, we aimed to develop a registration tool. Thepresent pilot study evaluates the feasibility of a prospective registrationprocedure for reporting, evaluating, and monitoring of pin site infectionsin 39 trauma, limb deformity, and bone infection patients with external ringfixation.2In this prospective, single-centre case series of pin site infections were monitored with a novel registration tool, which was introduced at theDepartment of Orthopaedic Surgery, Aarhus University Hospital, as a part ofstandard medical documentation in November 2017 (Supplement).Between November 2017 and January 2019, 49 consecutive patients were treatedwith an external ring fixation and included in this study. A total of 10 patients wereexcluded due to missing data: 2 patients were postoperatively followed atanother hospital, 2 paediatric patients  were followed in the paediatric clinic, 5 patients  had no pin site registrationsheet due to administrative missteps, and 1 patient died 3 weeks afterthe operation for reasons unrelated to primary surgery.Follow-up data were available for 39 patients  untilframe removal, with a mean age of 49 years (range: 12–88) at the time of theprimary operation. In total, we report 3296 pin site observations of 568 pin sites. All pin sites were registered and named unambiguously in thefollowing sequence: anatomic region , proximalmetaphysis, proximal and distal diaphysis, distal metaphysis, and side. Each trans-osseous wire had two pin site registrations:entry and exit point. When several wires were inserted at the same level, numbering of the pin sites startedfrom the most posterior-medial site and continued anti-clockwise on theright extremity and clockwise on left extremity.The primary underlying diagnosis was tibial fracture: 34 cases, 15 openfractures  consisting of 14 proximal tibial fractures(AO type 41), 12 diaphyseal fractures (AO type 42), 10 distal metaphysealfractures (AO type 43), and 2 malleolar fractures. Four of these fracturesextended in two levels: three combined fractures of type All patients were treated with ring fixators, and no monoliteral frames wereapplied: Taylor spatial frame , Ilizarov ringfixation , and TrueLok Hexapod . System choice of external ring fixation was at the discretion ofthe surgeon.The majority of the frames consisted of three tibial rings . Eight patients had two tibialrings and two had four rings. The adjacent joint was temporarily spanned in 20cases . At metaphyseal level, theexternal rings were predominantly fixated with four trans-osseous 1.8 mmIlizarov olive wires (Orthofix) of stainless steel, whereas at diaphyseallevel, the rings were mainly fixated using 6 mm hydroxyapatite-coatedhalf-pins (Orthofix) per ring. The combination of wires and half-pins, aswell as their orientation and number, was at the discretion of the surgeonand surgical preference. At least three to four fixation points per bone segmentwere applied to secure the proper stability of the frame.2.1Staphylococcus aureus . No culture swabs from pin siteswere performed in CO 1 or in CO 2 cases. If no effect was achieved withincreased pin site care and oral antibiotics, pin site specimens werecollected for microbiological analysis and intravenous antibiotics wereadministered guided by the microbiological growth (CO 3 cases). The decisionof wire/pin removal or replacement was guided by infection grade, theduration in frame, and anatomical position and at last was left to thediscretion of the treating surgeon.Perioperatively, non-touch pin technique with intermittent drilling underirrigation was performed when inserting the pins. Every pin site wascarefully inspected for skin tension and released if necessary. Pin siteswere then covered with foam sponges soaked in 70 % alcohol and 0.5 %chlorhexidine solution and gently pressed to the skin with a rubber stopper. Thefoam sponges were removed on the first postoperative day, and all pin siteswere cleaned from remaining bloodstains and covered with new, figure of 8,split gauze dressings moistened with chlorhexidine solution. Occlusive gauzedressing was rolled above the bungs, which covered the pin sites completely.The pin dressings were changed at the day of discharge and repeated once aweek by a municipal home nurse. Frequent dressing changes and increased pinsite care (2–3 times per week) were our first line of management uponinflammation/superficial infection (CO 1–3). If no improvement of CO 1 wasachieved or further development of infection was observed, oral (CO 2) orintravenous (CO 3) antibiotics were administered. Following our localguidelines, first choice of antibiotics was peroral dicloxacillin, 1 g, fourtimes daily, as the majority of pin sites infections are caused by2.2An individual pin site registration sheet followed the patient throughoutfollow-up. The standard postoperative follow-up programme included pin siteinspection in the outpatient clinic at 2 and 6 weeks and was subsequentlyfollowed with 6-week intervals until removal of the external ring fixation.All unexpected visits were registered. Outpatient pin sites care was carriedout by an expert nurse who systematically evaluated all pin sites accordingto the CO classification. A database was created from both the individualregistration sheet and from the electronic medical report (MidtEPJ version30.1.6), registering pin sites status, outpatient visits, re-operations, useof antibiotics, and radiographics.Missing data in the registration sheet were handled as follows: if no sign ofpin site infection was noted in the electronic medical report, the pin sitestatus was registered as CO 0. If electronic notes regarding pin siteinfection status were missing, the CO grade was registered as a missingvalue.2.3Outcome measures were pin site infection , useof antibiotics, treatment duration, and any unplanned procedure due to pinsites complications . Descriptive statistics were applied.3In all patients, the external ring fixation had been removed at the time ofthe data analysis. The mean (SD) frame time was 164 (83) d (range:44–499). Six femoral pin sites had missing CO registration. Pin infection wasregistered at 171 of the 568 pin sites (30 %), of which 112 (65 %) werecategorized as CO 1, 42 (25 %) as CO 2, 9 (5 %) as CO 3, and 8 (5 %) asCO 5. Please refer to Table 2 for a detailed overview of anatomical pin sitelocations, quantity, and CO grading.A total of 35 patients (90 %) encountered a minor infection (CO 1–3) at least onceduring the observation time. A total of 19 of these 35 patients (54 %) were treatedsufficiently with increased pin site care (The mean (SD) time to onset of pin site infection was 34 (33) d (range:7–149). In 10 patients, the infection started at a single pin site, andthe remaining 26 infections were registered at several pin sites. The proximaltibia diaphysis was the most common anatomical location for infection start,while the foot accounted for the fewest cases.Antibiotics were administered to 4In this single-centre prospective evaluation of pin site infections in 39trauma, limb deformity, and bone infection patients treated with externalring fixation, we employed a simple registration procedure combining pinsite locations and CO grade, offering a feasible and easy-to-use instrumentfor health-care professionals to monitor pin site infections and providing aconcise overview of treatment course.Treatment with external ring fixation involves several health-careprofessionals forming a multidisciplinary team; therefore viable inter-teamcommunication is of utmost importance. While several pin site infectionclassification systems exist , theoutpatient registration tool presented in the present study is combined withthe validated CO-classification system . Thisprovided us with an overview of all frame pin sites and treatment course,which proved to be efficient for the workflow in the outpatient clinic.Early recognition of infection and initiation of relevant treatment iscrucial to prevent major late complications, e.g. frame abandonment andsevere soft tissue and bone infections. Therefore, besides providingadequate surgery, all centres treating trauma, limb deformity, and boneinfection patients with external ring fixation should strive for sufficient postoperative and outpatient monitoring to ensure the best clinicaloutcomes.The current inconsistent evidence of pin site infection rates is primarilydefined by two key factors: (1) treatment length and (2) the infection ratevary if expressed as the number of pin sites  or the number of patients.Prudently, treatment length correlates with higher risk and incidence ofinfection. Of the included 568 pin sites, 171 were infected (30 %), but 36patients (92.5 %) in this cohort developed a pin site infection accordingto the CO classification. Of these, 16 patients (41 %) were only graded asCO 1, in which infection was resolved in 7 of the patients with increased pinsite care. In 12 of the patients (31 %), the highest CO grade registeredwas CO 2, in 6 of the patients (15.5 %) CO 3, in 1 patient (2.5 %) CO 5,and in 1 patient (2.5 %) no CO grade was reported. Our findings parallelthe existing literature demonstrating that pin site infections are commonbut also that most pin site infections are categorized as minor and only fewlead to major infections .Staphylococcus aureus frequently responding readily to oral antibiotics if locatedsuperficially . The mean (SD) timeto onset of pin site infection was 34 (33) d, most often starting atseveral pin sites, necessitating antibiotic administration to 56 % of thepatients. In 15 of the infected patients (42 %), a wire was removed, whichis suggestive of an insufficient effect of antibiotics and possible infectioninvolvement of the deeper tissue layers. However, we frequently remove wiresin the later stages of healing when the activity level of the patientincreases and the pin site troubles the patient due to mechanicalirritation. In accordance with this notion, no radiolucency was observedaround the wires/pins on radiographs; therefore the pin site infectionregistration remained Evidently, the cause of pin site infection is multifactorial. Amongimportant factors are surgical and pin care performance, anatomicallocation, bacterial aetiology, and patient-related factors, e.g. comorbidity,intake of medication, nutrition status, and smoking. As illustrated by ouranatomical distribution of results, pin sites near joints have a larger riskof becoming infected possibly due to movement of skin around the pin sitescausing irritation of soft tissue and accumulation of fluid . Notably, we report nointraarticular infections. The most common aetiology of pin site infectionsis This study has a number of limitations. The included study population represents a heterogenous but genuine unselected (i.e. consecutive) cohort,as no strict exclusion criteria were employed. The registration procedure istime-consuming and requires trained and experienced health-careprofessionals, both in terms of evaluating the pin sites for CO grade andfilling out the registration sufficiently. In this context, we failed to includefive patients due to administrative missteps, illustrating the necessity ofcomplete inter-team communication. Moreover, evaluation of pin sites andsurrounding tissue is often subjective and can differ between clinicians,which may lead to either over- or underestimation of the results. However, CO grading can beused to ensure a reliable and reproducible assessment and to collect thedata for further development of treatment strategies.In conclusion, pin site infection is a common complication with externalring fixation. During a mean frame time of 164 d, we registered infectionin 30 % of the pin sites but in 92.5 % of the patients according to theCO classification. In an effort to monitor all pin site infections in thiscomplex patient group and to ensure the best clinical outcomes, ourregistration procedure in combination with the CO classification helpedearly diagnosis of pin site infections and eased communication and workflowin the outpatient clinic by providing a concise overview of treatmentcourse. Future studies should include further understanding and optimization ofof post-operative pin site care protocol and prospectively evaluate theinfluence of external factors on the development of pin site infections.10.5194/jbji-6-135-2021-supplementThe supplement related to this article is available online at: https://doi.org/10.5194/jbji-6-135-2021-supplement.


It was observed that salicylate MBIs could maintain or improve enzyme inhibition and selectivity. To probe salicylate MBIs as fragments for fragment-based drug discovery (FBDD), an MBI that showed good inhibitory activity against GLO1 was derivatized and a rudimentary structure–activity relationship was developed. The resulting elaborated fragments showed GLO1 inhibition with low micromolar activity.Metalloenzyme inhibitors typically share a common need to possess a metal-binding pharmacophore (MBP) for binding the active site metal ions. However, MBPs can suffer from physicochemical liabilities, impeding the pharmacological properties and drug-likeliness of inhibitors. To circumvent this, problematic features of the MBP can be identified and exchanged with isosteric replacements. Herein, the carboxylic and hydroxyl group of the salicylic acid MBP were replaced and a total of 27 salicylate metal-binding isosteres (MBIs) synthesized. Of these 27 MBIs, at least 12 represent previously unreported compounds, and the metal-binding abilities of >20 of the MBIs have not been previously reported. These salicylate MBIs were examined for their metal-binding features in model complexes, physicochemical properties, and biological activity. It was observed that salicylate MBIs can demonstrate a range of attractive physicochemical properties and bind to the metal in a variety of expected and unexpected binding modes. The biological activity of these novel MBIs was evaluated by measuring inhibition against two Zn Metal-binding isosteres (MBIs) of salicylic acid have been developed for metalloenzyme drug development. Starting from a metal-binding pharmacophore (MBP), fragment growth strategies have been applied to identify hits from which lead-like compounds with good potency and selectivity can be obtained.6–8Metalloenzymes have been found to play important roles in the propagation of various diseases, ranging from microbial infections to cancer, which makes them an important class of targets for drug development.9–11Unfortunately, many MBPs are highly polar molecules that contain functional groups that can suffer from pharmacological liabilities, such as poor membrane permeability, challenging solubility, or toxicity. An attractive approach to circumvent these problems is to install isosteric replacements. Problematic functional groups can be exchanged with alternative, isosteric groups with improved properties while maintaining similar biological activity. This strategy is frequently applied in modern medicinal chemistry and drug development and it has been demonstrated that isosteric replacement can improve solubility, lipophilicity, cell permeability, and reduce toxicity. Furthermore, isosteres can be used to produce novel compositions of matter for intellectual property purposes.12,13Recently, isosteric replacement has been applied to MBPs for metalloenzyme inhibition. In a proof-of-concept study, the carboxylic acid moiety of picolinic acid was exchanged for known carboxylic acid isosteres to obtain a library of metal-binding isosteres (MBIs). These picolinic acid MBIs showed a broad range of physicochemical properties while maintaining similar metal-binding properties and biological activity as the parent MBP. This demonstrated the potential value of the MBI concept to allow for modulation of properties in the development of more drug-like metalloenzyme inhibitors by FBDD.14 A library of 27 salicylate MBIs was designed, synthesized, and studied for metal-binding and physicochemical properties. Furthermore, their inhibitory activity against two Zn2+-dependent metalloenzymes – human glyoxalase I (GLO1) and matrix metalloproteinase-3 (MMP-3) – and a dinuclear Mn2+-dependent metalloenzyme – influenza H1N1 N-terminal endonuclease (PAN) – was investigated. A selected salicylate MBI, which showed good inhibitory activity against GLO1, was computationally docked into the enzyme active site and a rudimentary SAR study was performed. It was demonstrated that salicylate MBIs not only cover a wide range of physicochemical properties and chemical space but also showcase various metal-binding modes, opening new avenues for metalloenzyme inhibitor development. It was observed that salicylate MBIs not only maintain, but also can surpass the parent salicylic acid MBP in terms of metalloenzyme inhibition and selectivity in select cases. Finally, it was shown that salicylate MBI fragments are easy to derivatize, which makes them strong candidates for lead development and rational drug design.Herein, MBIs of salicylic acid, a known metal binding ligand and metalloenzyme inhibitor, were prepared.14 Two sites of the molecule – the hydroxyl group and the carboxylic acid group – are generally involved in metal binding. Consequently, these functional groups were the target of isosteric replacement Zn(MBI)] borate) model complexes were prepared and characterized by X-ray crystallography Zn(MBI)] complex models the tris(histidine) active site of Zn2+-dependent metalloenzymes  and has been widely used to evaluate the structural characteristics of MBPs and MBIs as potential metalloenzyme inhibitors.15–17To evaluate the structural features of metal binding by the salicylate MBIs, [Zn(MBP)] complexes has been observed for salicylate ligands18 and it was hypothesized that the differences in binding behaviour observed here can be explained, in part, by charge neutrality. To obtain an overall neutral [Zn(MBI)] complex, monoanionic MBIs are required, as dianionic MBIs will produce charged species. However, some functional groups, such as carboxylic acids, alcohols, or sulfonamides must be deprotonated  to effectively bind to the metal centre. MBIs that combine two ionizable groups, such as 2, 3, 16, 19, 23, and 24, consequently can only bind as 6-membered chelates when double deprotonated , which disfavours charge neutrality. It should be noted that this is a limitation of the [Zn(MBI)] model complex and does not necessarily correlate within the context of a metalloenzyme active site. Indeed, several protein crystal structures of dianionic MBPs  bound to active site metal ions in metalloenzymes have been reported.19–21Both monodentate and bidentate coordination in [Zn(MBI)] model complexes, the physicochemical properties of the MBIs were also studied. The acidity (pKa) and lipophilicity (log P) were measured using potentiometric methods and the lipophilicity at physiological pH (log D7.4) was calculated from these two values  and are deprotonated at physiological pH, which explains the high polarity of these MBIs at this pH (log D7.4 approx. −2). When the carboxylic moiety was exchanged for a less acidic isostere, the lipophilicity at physiological pH increased significantly. Interestingly, this was also observed for the tetrazole MBIs – despite the rather low pKa of this functional group , lipophilicities at physiological pH were in the range log D7.4 ∼0. This highlights that by careful selection and combination of isosteric groups, MBIs with tailor-made physicochemical properties can be designed.Overall, a wide range of acidities and lipophilicities was observed among the MBIs. Except for MBI 4 , entry 4e.g., 4-(benzo[d]thiazol-2-yl)phenol, log P = 1.86, determined by a HPLC based method) suggests higher lipophilicity compared to other salicylate MBIs. This was confirmed by computationally calculated physicochemical data  could not be measured with the applied method due to the poor aqueous solubility of these compounds. Literature data of structurally related compounds  and is in proximity to two hydrophobic pockets.1,32 Previous studies (unpublished) found salicylic acid 1 to be a weak GLO1 inhibitor (IC50 >50 μM). This result was confirmed in our screening and around half of the salicylate MBIs showed similar or better inhibitory activity compared to salicylic acid. MBIs 11, 22, and 27 inhibited GLO1 significantly better, with 22 showing 50% inhibition and 11 almost complete inhibition at 100 μM. The good inhibitory activity of 11 compared to the other salicylate MBIs could be rationalized by hydrophobic  interactions of the benzothiazole moiety with the hydrophobic pockets in proximity to the catalytic Zn2+.8,33 This indicates that isosteric replacements in MBPs could not only maintain, but also improve enzyme binding for metalloenzyme inhibition.To evaluate if the salicylate MBIs could serve as starting points for future FBDD campaigns, the MBI library was screened for inhibitory activity against human glyoxalase I (GLO1) at fragment concentrations of 100 μM . GLO1 is2+-dependent metalloenzyme that is inhibited by a variety of MBPs – however, salicylic acid 1 is not a known MMP-3 inhibitor.1,14,34,35 Except for MBI 22, which showed 74% inhibitory activity against MMP-3, the salicylate MBIs showed only very weak or no inhibition at all. These results indicate that salicylate MBIs could serve as selective fragments for the development of more elaborate metalloenzyme inhibitors, even against enzymes that contain common active site metal ions .To probe the selectivity of the salicylate MBIs, the library was screened against matrix metalloproteinase-3 (MMP-3) at fragment concentrations of 100 μM . MMP-3 iN), a dinuclear Mn2+-dependent metalloenzyme.38 PAN plays a major role in the viral lifecycle and is highly conserved among various influenza strains, which makes it an interesting target for the development of influenza antivirals.1,7 While the inhibitory activity of salicylic acid 1 was rather low at a concentration of 100 μM, more than half of the MBIs showed some significant inhibition Zn(MBI)] model system. Indeed the majority (>20) of the MBIs generated had no previously described coordination chemistry. Unexpected binding modes were observed for salicylate MBIs capable of forming intramolecular hydrogen bonds, which will open new avenues in the development of metalloenzyme inhibitors. The physicochemical properties of salicylate MBIs are tuneable with a wide range of acidities and lipophilicities achieved. Importantly, the lipophilicities of most salicylate MBIs tested are in a desirable range for FBDD. The screening of their inhibitory activity against three metalloenzyme targets revealed that isosteric replacements in MBPs cannot only maintain, but potentially improve inhibitory activity and that salicylate MBIs can inhibit various metalloenzyme targets containing different metals. Supported by computational docking studies and rudimentary SAR, the enzyme binding of a selected salicylate MBI was investigated and the inhibitory activity improved. This demonstrates that salicylate MBIs are an attractive starting point for FBDD and lead development. In conclusion, salicylate MBIs are an interesting new class of metalloenzyme binding fragments with beneficial physicochemical properties and will be useful for the development of new metalloenzyme targeting inhibitors with more drug-like properties.Salicylate MBIs are easy to synthesize and allowed for rapid preparation of 27 compounds, of which nearly half were wholly new compositions of matter. They show a variety of metal binding modes in a  was prepared according to literature known procedures.37 [ZnOH]  was dissolved in 15 mL of CH2Cl2 in a 50 mL round-bottom flask. The MBI  in 10 mL of MeOH was added, and the reaction mixture was stirred overnight under a nitrogen atmosphere. The resulting mixture was evaporated to dryness and subsequently dissolved in a minimal amount (∼1 mL) of benzene. The solution was filtered using a syringe filter to remove any undissolved solids. The resulting complex in benzene was recrystallized using vapor diffusion with pentane. Crystals typically formed within a few days. was used to monitor enzymatic activity. After addition of substrate, fluorescence  was monitored over 45 min at 39 s intervals at 37 °C. Negative control wells contained no inhibitor and were set to 100% activity. A reported pyridinone-based inhibitor was included as a positive control for inhibition.38 Percent inhibition was determined by comparing the activity of wells containing MBIs to the activity of those without.Endonuclease was expressed and purified as previously reported.M. K. J. and S. M. C. outlined the project, interpreted results, and wrote the manuscript. M. K. J. and S. M. C. designed the MBI library, M. K. J. planned and executed the synthesis of all compounds. M. K. J. and H. S. performed the enzyme activity assays and interpreted the results. H. S., M. K., and M. K. J. performed X-ray crystallographic measurements and edited the data. J. K. performed computational docking studies.S.M.C. is a cofounder of and has an equity interest in Cleave Therapeutics, Forge Therapeutics, and Blacksmith Medicines, companies that may potentially benefit from the research results. S.M.C. also serves on the Scientific Advisory Board for Blacksmith Medicines and serves on the Scientific Advisory Board and receives compensation from Forge Therapeutics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies.SC-013-D1SC06011B-s001SC-013-D1SC06011B-s002SC-013-D1SC06011B-s003SC-013-D1SC06011B-s004