While their function, as immune checkpoint molecules, is well known, B7-family proteins also function as regulatory molecules in bone remodeling. B7–H3 is a receptor ligand of the B7 family that functions primarily as a negative immune checkpoint. While the regulatory function of B7–H3 in osteoblast differentiation has been established, its role in osteoclast differentiation remains unclear. Here we show that B7–H3 is highly expressed in mature osteoclasts and that B7–H3 deficiency leads to the inhibition of osteoclastogenesis in human osteoclast precursors (OCPs). High-throughput transcriptomic analyses reveal that B7–H3 inhibition upregulates IFN signaling as well as IFN-inducible genes, including IDO. Pharmacological inhibition of type-I IFN and IDO knockdown leads to reversal of B7–H3-deficiency-mediated osteoclastogenesis suppression. Although synovial-fluid macrophages from rheumatoid-arthritis patients express B7–H3, inhibition of B7–H3 does not affect their osteoclastogenesis. Thus, our findings highlight B7–H3 as a physiologic positive regulator of osteoclast differentiation and implicate type-I IFN–IDO signaling as its downstream mechanism. Osteoclasts are bone-resorptive cells derived from hematopoietic cells of the myeloid lineage that are central to physiological and pathological bone remodeling , 2. The The B7 superfamily of proteins comprises immunoregulatory receptor ligands that act as either costimulatory or co-inhibitory immune checkpoints by primarily regulating the activity of T cells . The funCertain members of the B7 family such as B7.1/B7.2 (CD80/86) and B7–H2 (ICOS ligand) act as regulatory molecules in osteoclast differentiation. Unlike in their function as immune checkpoints in which they utilize a forward-signaling mechanism to propagate their effects, B7.1/B7.2 (CD80/86) and B7–H2 (ICOS ligand) inhibit osteoclast differentiation using a distinct reverse-signaling mechanism , 13. FurRecombinant human M-CSF and soluble RANKL were purchased from PeproTech, NJ, USA. Recombinant human M-CSF receptor/Fc chimera and B7–H3–Fc were respectively obtained from Sino Biological, PA, USA and R&D System, MN, USA. Human IgG purchased from Millipore was used as the control for B7–H3–Fc. Human IFN-αR2-receptor antibody was obtained from PBL Assay Science. Human IFN-γ antibody, IL-27 antibody, and recombinant human IFN-β, IFN-γ, and IL-27 were obtained from R&D System. Mouse IgG2a purchased from R&D system was used as the isotype control. 1-D-MT, 1-L/D-MT, and CH223191 were purchased from Sigma, MA, USA.Human peripheral blood monocytes (PBMCs) were isolated from the blood of healthy donors using Ficoll gradient centrifugation . Monocytes were purified from PBMCs using CD14-positive magnetic beads  according to the manufacturer’s instructions. Inflammatory RA or ankylosing-spondylitis (AS) macrophages were purified by positive selection using CD14 magnetic beads from synovial-fluid mononuclear cells. The experiments using human cells were approved by the Ethics Committee of Hanyang University Hospital for Rheumatic Disease . All healthy volunteers and RA and AS patients provided their written informed consent to participate in this study.−/−) mice with the C57BL/6 background were purchased from Cyagen Biosciences. Eight-week-old mice were housed in a specific pathogen-free facility. All experiments were approved by the Institutional Animal Care and Use Committee at Hanyang University (HY-IACUC-2020-0131).Wild-type (WT) and B7–H3-knockout (KO) . The attached cells, which are capable of osteoclast differentiation, were seeded and cultured with 50 ng/ml M-CSF and 100 ng/ml RANKL for four days. Cytokines were replenished every three days. The cells were cultured in α-MEM  supplemented with 10% FBS . The cells were fixed with 4% paraformaldehyde and stained for TRAP using Acid Phosphatase Leukocytes diagnostic kit , as recommended by the manufacturer. Multinucleated  TRAP-positive cells were counted in duplicate wells of 48-well plates. Microphotographs of staining were observed at original magnification X100.B7–H3, the OCPs were transfected with human pCMV3–B7–H3 ORF-expression plasmid and pCMV3-negative control vector . For transfection, lipofectamine 3000  was used according to the manufacturer’s instructions. After siRNA or plasmid transfection, OCPs were cultured in α-MEM with 40 ng/ml M-CSF and 80 ng/ml RANKL for seven days to generate mature osteoclasts. Of note, M-CSF 20 ng/mL was used as the default concentration, while the 40 ng/mL concentration was used to increase the transfection efficiency.Before transfection, 40 ng/ml M-CSF was added to CD14-positive cells from PBMCs or RA synovial-fluid mononuclear cells for three days. For RNA interference, the OCPs were transfected with ON-TARGET plus SMARTpool siRNAs specific for B7–H3, IDO, and AhR (Dharmacon). ON-TARGET plus Non-targeting Pool was used as the control. To overexpress At the end of the culture period, the cells were fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 for 5 min, and then stained with FITC-conjugated phalloidin (Sigma-Aldrich) for 1 h at 37 °C.After siRNA transfection, OCPs were plated on dentin slices  in 96-well plates and cultured with M-CSF and RANKL. The cells on the dentin disc were removed by washing with 70% ethanol. The dentin discs were rinsed with water and immersed in 1% toluidine blue O (Sigma-Aldrich) to stain resorption pits formed by osteoclasts.Femurs from WT and B7–H3 KO male mice were fixed in 4% paraformaldehyde for micro-CT analysis. Trabecular and cortical morphometry within the femur were quantified using a benchtop cone-beam-type in vivo animal scanner . Samples were imaged with the following settings: 60 kVp, 166 μA, and an aluminum 0.25-mm-thick filter. The pixel size was 9.0 μm and the rotation step was 0.6°. The cross-sectional images were reconstructed using a filtered back-projection algorithm . Trabecular morphometry used the following measurement parameters: bone-volume fraction (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. No), and trabecular spacing (Tb. Sp).Total RNA was extracted from the cells using Trizol (Invitrogen) according to the manufacturer’s instructions. The RNA was reverse-transcribed using reverse transcriptase (Invitrogen) and random hexamers (Invitrogen). qPCR was performed in triplicate with Fast SYBR Green Master Mix  using CFX96 . The primers used for qPCR are provided in Supplementary Table Total cell extracts were obtained using 1X RIPA lysis buffer  containing 1X proteinase-inhibitor cocktail , 1 mM PMSF (Sigma-Aldrich), and 1X phosphatase-inhibitor . Cell lysates were fractionated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes , and incubated with specific primary antibodies. Immunoblotting was performed with the following antibodies: B7–H3 (R&D System); c-Fos ; NFATc1 ; c-Fms, IκBα, p-p38 MAPK, p-p44/42 MAPK, p44/42 MAPK, IDO, p-STAT1, STAT1, AhR, β-actin, and GAPDH ; RANK, p38α, and iNOS . Secondary antibodies (Jackson ImmunoResearch) were incubated before adding the ECL substrate (Invitrogen). For quantitative analysis of protein expression, the optical densities of the blot bands were measured using the Image J software.Cells were washed with 1X PBS containing 0.5% bovine serum albumin and 0.1% sodium azide and then incubated in Fc-receptor-blocking reagent (Miltenyi Biotec). Cells were stained with APC-conjugated anti-human B7–H3  and analyzed by using FACS Canto flow cytometer . The flow-cytometric data were analyzed using FlowJo software .Cells were seeded in 96-well plates and added Ez-cytox viability kit reagent  for the indicated number of days. The cells were incubated for 1 h at 37°C to allow the Ez-cytox viability kit reagent to metabolize to formazan. Absorbance was measured at 450 nm using a microplate reader and culture medium was used as a blank.TapeStation RNA screentape . Only high-quality RNA preparations, with RIN greater than 7.0, were used for RNA library construction. A library was independently prepared with total RNA (1ug) for each sample by TruSeq Stranded mRNA Sample Prep Kit . The libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol  and qualified using the TapeStation D1000 ScreenTape . Indexed libraries were then submitted to an Illumina NovaSeq , and the paired-end (2 × 100 bp) sequencing was performed by the Macrogen Incorporated. Differentially expressed genes between B7–H3-deficient and control transcriptomes were identified using DESeq2 using a p-value <0.05 as the significance threshold and enriched pathways were identified using IPA.Total RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen). To assess the integrity of the total RNA, samples are run on the P < 0.05, **P < 0.01, ***P < 0.001. Columns and error bars in figures indicate the mean values and standard errors for at least three independent experiments.The results are expressed as mean ± SD. The Mann–Whitney U test was used to calculate statistical significance. Statistical significance was defined as *To confirm the presence of B7–H3 expression during human osteoclast differentiation, we induced PBMCs from healthy controls (HC) to undergo osteoclastogenesis using M-CSF and RANKL Fig. . ExpressCTSK), tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), and β3 integrin (ITGB3), as well as the protein expression of NFATc1  synovial-fluid macrophages. B7–H3 mRNA levels were higher in RA synovial macrophages compared with HC PBMCs and no significant differences in B7–H3 mRNA levels were found between AS synovial-fluid macrophages and HC PBMCs Fig. . ConsistIn summary, we found that inhibition of B7–H3 using B7–H3 siRNA and soluble B7–H3–Fc treatment led to osteoclastogenesis inhibition and to increased type-I IFN signaling, STAT1 activation, and IDO induction. In addition, type-I IFN and IDO suppression reversed osteoclastogenesis inhibition, while type-I IFN blockade suppressed B7–H3 siRNA-mediated IDO induction. Taken together, we add to the emerging paradigm in which immune checkpoint molecules moonlight as regulatory molecules in bone remodeling whereby costimulatory molecules  inhibit, while co-inhibitory molecules (B7–H3) stimulate osteoclastogenesis , 13.The role of B7–H3 in innate immune cells, which share the same monocytic lineage as osteoclasts, remains controversial. In mouse-derived BMMs, soluble B7–H3 (muB7–H3) stimulates NF-kB activation and proinflammatory cytokine release, indicating that B7–H3 exerts costimulatory effects on murine innate immune cells . In a prType-I IFNs regulate osteoclastogenesis via several mechanisms, including the inhibition of c-Fos and iNOS/NO signaling and indirect inhibition of SOC1 and MITF through miR-155 induction –20. Our Furthermore, the reason for the discrepancy between the AhR siRNA experiment and the 1-MT and CH223191 experiments is that kynurenine is only one of many endogenous AhR agonizts. Consequently, 1-MT-mediated kynurenine inhibition may have been insufficient to inhibit other endogenous AhR agonizts. Also, CH223191, while known as an AhR inhibitor, is a ligand-selective antagonist that preferentially inhibits agonizts of the halogenated aromatic hydrocarbons (HAH). The action of endogenous AhR ligands may not be inhibited by CH223191 as they are more likely to bind AhR-like non-HAH agonizts . TherefoNo significant bone-phenotypic changes were seen in B7–H3 KO mice in our study. The most likely hypothesis for this finding is that B7–H3 KO has opposing effects on osteoclasts and osteoblasts, such that there is no significant alteration in their relative balance . To direWe show that unlike in PBMCs, B7–H3 knockdown does not result in osteoclastogenesis inhibition in RA synovial-fluid macrophages. This finding raises the possibility that the B7–H3–IFN–IDO axis in healthy peripheral-blood OCPs is dysfunctional in the RA synovium. We further showed that the concentration of IFN-β capable of inducing IDO in healthy peripheral blood OCPs does not induce IDO nor STAT1 activation in RA synovial macrophages. This suggests that RA synovial macrophages have an attenuated response to exogenous IFN-β, which is consistent with many previous studies that have established the attenuated responses of RA synovial macrophages to inflammatory cytokines such as IL-6, -10, and -27 . TherefoIDO transcription and IDO-dependent immune tolerance [Various mechanisms mediate the immune-regulatory effects of IDO . In dendolerance . The funolerance . Many clBoth B7.1/7.2- and CD200R1-mediated IDO induction depend on type-I IFNs –35. We sA previously reported study established that B7–H3 is required to induce bone mineralization and osteoblast differentiation . Adding Our study has several limitations. First, exogenous IFN-β and B7–H3 inhibition both led to IDO induction, whereas IDO knockdown led to reversal of osteoclastogenesis inhibition only in the setting of B7–H3 inhibition and not exogenous IFN-β administration. Second, the mechanism of IDO-mediated osteoclastogenesis inhibition in the setting of B7–H3 inhibition or deficiency remains unknown. Third, the reason that B7–H3 deficiency does not lead to osteoclastogenesis inhibition in RA synovial macrophages remains unclear. Fourth, the mechanism of osteoclastogenesis inhibition in B7–H3 KO mice and the impact of B7–H3 KO in specific disease-model mice remains unclear. Last, the direct role of B7–H3 as a direct positive regulator of osteoclastogenesis was not evaluated mainly because of the lack of available B7–H3 agonizts since sB7–H3–Fc acted as an antagonist in our experiments. Although our gain-of-function study revealed that B7–H3 overexpression led to STAT1 inhibition and STAT-1 dependent genes, the transient nature of B7–H3 overexpression precluded further assessment of the impact on osteoclastogenesis. Future research is warranted in examining the above questions to further elucidate the mechanism of B7–H3 inhibition in the negative regulation of osteoclastogenesis.In conclusion, B7–H3 deficiency leads to inhibition of osteoclast differentiation, and the type-I IFN–IDO pathway is an important downstream-regulatory mechanism in B7–H3-mediated regulation of osteoclastogenesis. Our study highlights B7–H3 as a strong potential therapeutic target in human diseases in which B7–H3 type-I IFN–IDO-mediated regulation of immune responses and osteoclast activation play important pathogenic roles.Supplementary figures and legendsTable S1Table S2


Chrysin, apigenin, and resveratrol were found to be the best inhibitors targeting the enzyme’s substrate access channel and were able to produce a competitive inhibition with IC50 values ranged from 1.7 to 15.8 µM. Moreover, they showed a more potent antiproliferative effect against ER+ (MCF-7) than ER- one (MDA-MB-231) cell lines. On the other hand, both pomiferin and berberine were the best hits for the enzyme’s haem-proximal cavity producing a non-competitive inhibition  and showed selective antiproliferative activity towards MCF-7 cell lines.Recent findings suggested several allosteric pockets on human aromatase that could be utilised for the development of new modulators able to inhibit this enzyme in a new mechanism. Herein, we applied an integrated  Several plant-derived natural products have shown potent activity against HA, notably flavonoids that were reported to exhibited competitive inhibition against HAIn this context, we proposed an integrated in-silico and in-vitro pipeline to find out potential HA inhibitors from a plant-based natural products library proposing their exact binding site and modes of inhibition.The compounds library prepared for this study included 52 different plant metabolites from several major classes of plant metabolites . Phenolics and flavonoids were the most represented class (63.5%) as they has been reported previously to be HA-specific inhibitorsAll of these metabolites are considered major metabolites in their corresponding plant source so that they can be readily purified for further processing. Hence, this library is considered a good representative for the common plant natural products. Additionally, it shows a good structural diversity to test the efficacy of our proposed in silico protocol in differentiating between active and inactive compounds.in vitro assays. Our findings confirmed that five compounds were found to inhibit HA in the low µM range through competitive and non-competitive mechanisms that putatively established by targeting allosteric sites (Sites A and B) on the enzyme. Consequently, our outcomes in the present study highlighted the potential of natural products-based therapeutics for ER + BC that probably able to delay or avoid the onset of resistance which frequently develops with the current therapies. The approach applied in this study is depicted in We utilised this library in a protocol that integrates a number of computational and experimental steps as the following: (i) structure-based virtual screening (VS) using our in house library of plant-derived natural products (52 compounds); (ii) selecting top-scoring hits that obey Lipinski’s rule of fiveFigure S1) were purchased  or isolated from their natural source following the previously reported proceduresAll compounds used in this study (https://www.rcsb.org/). A list of the residues of these binding sites together with their grid boxes is reported in Table 1S. To account for these binding sites’ flexibility, we used their MDS-derived conformers sampled every 10 ns for docking experiments (i.e. ensemble docking). Subsequently, the retrieved top hits were ranked according to their binding energies. The generated docking poses were visualised and analysed using Pymol softwareDocking experiments were performed using AutoDock Vina software,,+ and Cl- ions with 20 A˚ solvent buffer from the molecular surface of the centrally placed receptor. Afterwards, the prepared system was energy minimised and equilibrated for 10 ns. Desmond software automatically parameterises inputted ligands during the system building step according OPLS force field. For simulations performed by NAMD, the parameters and topologies of the compounds were calculated either using Charmm27 force field by the online software Ligand Reader & Modeller (http://www.charmm-gui.org/?doc=input/ligandrm)MD simulations were performed by Desmond v. 2.Simulations were run for 100 ns at 310 K in the NPT ensemble with the Nose-Hoover thermostat and Martyna-Tobias-Klein barostat using an anisotropic coupling. We used the best binding poses for each compound inside both Site A and Site B as starting systems to investigate their binding stability and mode of interactions.G) were performed using the free energy perturbation (FEP) method. We first prepared the input files and script NAMD by the online-based software CHARMM-GUI Free Energy Calculator (http://www.charmm-gui.org/input/fec). Afterwards, these inputs were loaded to NAMD for simulations, where the equilibration was performed in the NPT ensemble at 310 K and 1 atm (1.01325 bar) with Langevin piston pressure (for ″Complex″ and ″Ligand″) in the presence of TIP3P water model. 10 ns FEP simulations were performed for each system, and the frames of the last 5 ns of the free enegry were measured for the final free energy valuesBinding free energy calculations  towards the haem moiety to make each ligand moving towards each binding site with a velocity 0.75 Å/ns. Finally, generated trajectories were visualised and analysed by VMD softwareFor further confirmation of the initial docking and MDS experiments, we generated a binding event simulation by placing the 5 ligands close to each binding site (∼15 Å) and applying force  properties was calculated using the online website “v/v). Subsequently, each solution was diluted in the aromatase assay buffer to obtain a range of concentrations for constructing a dose-response curve. The reaction was conducted by mixing aromatase mix (containing Recombinant Human Aromatase (2X), Aromatase assay buffer and NADPH-generating system) with the test compound. The reaction mixture was pre-incubated for 10 min at 37 °C to allow the test compounds to bind to HA. Then, the reaction was initiated after adding 30 ml of aromatase substrate/NADP+ mixture . The assays were conducted in 96-well microtiter plates in a final reaction volume of 100 μL/well. The resulted fluorescence was measured using a microplate reader  for 60 min. Experiments were performed in triplicate, and the average values were used to generate the dose response curves. To calculate the concentration that was able to inhibit HA activity by 50% (IC50), each compound was tested at different concentrations . In order to analyse the inhibition mechanism of each inhibitor, we performed a number of kinetic experiments at constant test compound  and at different substrate concentrations ranging from 0.25 to 6 times the approximate value of Km. For competitive inhibition, the % inhibition decreases as the substrate concentration increases, while it remains constant for the non-competitive oneKi) values for each inhibitor were determined according to the manufacturer protocol, where the rate of substrate utilisation, using 2 mM of the tested enzyme and 0–250 µM of the substrate, was monitored in increasing amounts of inhibitor (0–50 µM).HA inhibition assay was performed using the Aromatase Inhibitor Screening Kit , which utilised a fluorogenic substrate that is converted by HA into a highly fluorescent metabolite. After reconstitution of the kit’s reagents, a standard curve was constructed a serial dilution of the fluorescent standard. Each test compound was dissolved in DMSO to reach a final concentration of 0.25% (50) was calculated.The cell lines MCF-7 and MDA-MB- 231 used in this investigation were obtained from the American Type Culture Collection . MCF-7 cell proliferation was induced by testosterone. Tumour cells were seeded in triplicate in 12-well plates. 24 h later, they exposed for 72 h to an increasing concentration of each tested compound (from 1 to 200 µM) together with 10 µM testosterone undecanoate. Tumour cells were harvested using trypsin and counted. Three independent experiments were performed, and the concentration of the compound able to inhibit cell growth by 50%  and extends to reach a haem moiety at the end, where the synthesis of oestrogen occurs. It has a total volume of 2351 Åatalysed 10. Hence,Table S2). Non-glycosylated flavonoids (9 compounds) alongside resveratrol were found to be the best scoring compounds , where the increase in the molecule’s hydroxylation was associated with decreased binding scores. The remaining compounds in the library that got lower scores  dissociated from HA’s Site A during the course of 30 ns MDS. The top-scoring compounds were then relaxed by 100 ns-long MDS to get insight into their behaviours inside the binding site and to calculate their binding free energies. The smallest and least hydroxylated flavonoids chrysin, apigenin, and resveratrol  against the opening of HA’s substrate access channel’s opening (Site A) using an ensemble docking protocolveratrol  graduallveratrol .On the other hand, the more hydroxylated flavonoids (6 compounds) achieved lower binding free energies  that were also translated in their instability inside the binding site (average RMSD ∼ 10 Å) and significantly lower in-vitro activity . We obseS2). Such structural information may help in designing new HA competitive inhibitors based on the flavonoid scaffold.To further confirm these findings, we allowed five molecules from each compound to achieve binding events by applying direct forces towards site A during another 50 ns-long simulations. Chrysin, apigenin, and resveratrol were able to achieve bindings after 12.7, 18.8, and 21.8 ns, respectively that were similar to their docking poses. Concerning tangeretin, hesperetin, and taxifolin, they were not able to achieve stable bindings inside site A  and S2. 3) binding pocket than Site A . Only tanshinone IIA, pomiferin, silibinin, betulin, maslinic acid, and berberine got binding scores < −7.5 kcal/mol , where all compounds in our in-house library were docked against this allosteric binding cavity . Consequently, the side chains of Site-B’s aminoacids achieved the highest stability during the course of MDS in case the binding with pomiferin followed by berberine, while with tanshinone IIA, its average RMSD was almost identical to that of the free unbounded form  and the type of inhibition of each HA inhibitorTo validate our preliminary computational experiments, top-scoring compounds were tested for their HA inhibitory activity and their antiproliferative effect against both the ER + MCF-7 and the ER- MDA-MB-231. Additionally, we performed enzyme kinetic analyses to calculate the inhibition constant  were also the most potent HA inhibitors and were more active against MCF-7 than MDA-MB-231. Also, they were found to inhibit HA via a competitive mechanism, and hence such results were in good accordance with the site A-directed inhibition as the computational investigation suggested. Moreover, tangeretin that was unstable inside site-A was inactive against HA. However, it showed some activity against both MCF-7 and MDA-MB-231, indicating that it might deal with cancer cells through a different mode of action.As shown in 50 ∼ 2.1 µM), and such observation suggested a different mechanism of action for this compound. A previous study has reported that tanshinone IIA can inhibit both breast cancer cell lines via targeting ADP-ribosyltransferase like protein 1 (ADPRTL1)ΔG > −3 kcal/mol), and both of them were inactive either against HA or breast cancer cell lines.Regarding compounds that were suggested to target site-B, both pomiferin and berberine were the best HA inhibitors, while the less stable one, tanshinone IIA was far less active. MCF-7 cell line was also more sensitive to the former compounds than MDA-MB-231. Surprisingly, tanshinone IIA was the most active compound against both cell lines (ICP) of certain bioactive compounds could suggest its probable pharmacokinetics. Lipinski’s rule of five contemplates a small bioactive molecule as a drug candidate if it possesses these physicochemical parameters i.e. log P ≤ 5, molecular weight ≤500, hydrogen bond donor [HBD] ≤ 5, and hydrogen bond acceptor [HBA] ≤ 10. Hence, around 90% of orally active drugs that have passed phase 2 clinical trials obey such rulesCompounds that showed HA inhibitory activities  were furThe ADME profiles of the active metabolites resveratrol, chrysin, apigenin, pomiferin, and berberine were calculated by the online software SwissADME. As illustrated in ,BC remains a significant worldwide health issue, particularly for women. Besides the standard treatment protocols that rely on chemotherapeutic agents, ER + BC can be managed by other therapeutics that modulate oestrogen. Both SERMs and ArIs have introduced as good options in this regard; however, their prolonged use was associated with acquired resistance,Plant-based natural products are still a crucial and diverse source of potential therapeutics and nutraceuticals that have a good safety profile. Many plant-derived compounds have been reported as very good ArIs, particularly, flavonoids and other phenolic compoundsin silico-based protocol to identify the most promising HA inhibitors depending on their site of interaction. In our initial virtual screening, we focussed on compounds that can target the enzyme’s unusual binding sites . Subsequently, a series of MDS experiments were performed to refine our preliminary docking step and to explore the mode of interaction of the best candidates. Non-glycosylated flavonoids along with resveratrol were found to be the best hits for site-A (i.e. the opening of substrate access channel) that mediates the competitive-type of HA inhibition, and upon MDS, resveratrol, chrysin, and apigenin were found to settle inside the channel achieving the highest binding free energies.Herein, we applied an ,Despite these three compounds have been previously reported as HA competitive inhibitors, their exact mode of interaction with HA was illusivein silico investigation. All of the best-scoring hits showed micromolar inhibition of HA, where site-A-targeting hits were more potent than those targeting site-B. Moreover, site A-directed inhibition led to competitive inhibition, while site-B-directed inhibition led to a non-competitive one. The ER + BC cell line MCF-7 was also more sensitive to these inhibitors than the ER- one MDA-MB-231.On the other hand, tanshinone IIA, pomiferin and berberine were found to be the best ligands for site B  that mediates the non-competitive-type of HA inhibition. However, only pomiferin and berberine were able to achieve stable binding inside this binding cavity with high bending free energies during the course of 100 ns MDS. The subsequent experimental validation came to support the Despite being less potent against HA, site B-directed non-competitive inhibitors offered some advantages over the site A-directed competitive one: (i) they were able to bypass the developed resistance against active-site directed inhibition in which the enzyme mutates the binding domain to be inaccessible for the inhibitor, (ii) they will not abolish oestrogen production completely and thus the onset of resistance can be delayed, (iii) such allosteric inhibitors offer some selectivity over the active site ones, and hence, they might be with much lower side effectsBoth pomiferin and berberine have been shown anti-breast cancer activityin vivo testing to be the next generation of ArIs.Besides being good HA inhibitors, they also showed good bioavailability and drug-like properties according to both Lipinski’s and Veber’s rules of drug-likeness. Consequently, these plant-derived compounds along with the structural information presented in this study offer a good starting point for further development of more effective inhibitors via structural modification or the subsequent In conclusion, we introduced an effective workflow for the rapid identification of HA inhibitors describing their possible modes of action. Hence, using this protocol with more extensive databases can help in the discovery of different HA inhibitors, particularly the non-competitive allosteric ones that can be promising alternatives or supportive to the current ArIs.Click here for additional data file.


Streptomyces mobaraensis contains several unusual residues in positions that are otherwise highly conserved. Site-directed mutagenesis experiments for these residues are reported that showed different effects, resulting in some cases in an improved catalytic activity, but in other cases in a loss of enzyme function. For other enzyme variants a functional switch was observed, turning SmTS1 from a sesterterpene into a diterpene synthase. This article gives rational explanations for these findings that may generally allow for protein engineering of other terpene synthases to improve their catalytic efficiency or to change their functions.The sesterterpene synthase SmTS1 from  Type I terpene synthases (TPSs) activate these acyclic molecules by the abstraction of diphosphate to produce a reactive allyl cation that can initiate a cascade reaction through typical carbocation chemistry, including cyclisation reactions by intramolecular attack of an olefinic double bond to the cationic centre Wagner–Meerwein rearrangements, and proton or hydride migrations . White GPP, C as the pubstrate . Furtherubstrate . Additioubstrate –14, a paubstrate –14, and te FPP, C as sesquSE triad  that tarStreptomyces mobaraensis (SmTS1) represents the first identified type I sesterterpene synthase (StTPS) from bacteria ,,21,2+ caird Mg2+ , showingsor R178  is in Smsor R178 , that is−1), with the exception of the R228L variant that was obtained with very low yields in the soluble fraction and thus not further studied , suggesting that installation of the large Arg residue blocks the available active site space for GFPP and thus disturbs the sesterterpene production by SmTS1, while the relative proportions of the individual products were similar as for the wildtype enzyme. The Q227D exchange also showed an interesting effect, causing a ca. 1.5-fold increased production (155 ± 13%), but no changes in the product proportions. The reason for this increase is not clear, but the introduced Asp in the Q227D variant is homologous to D225 of SdS that hydrogen bridges to the pyrophosphate sensor R178 , showing that Asn can functionally fully substitute for the otherwise conserved Asp in this position. The relative proportions of the sesterterpenes were slightly shifted in favour of the main product sor R178 . As SmTSon 155 ± %, but noRegarding the active site contouring residues, the G184L resulted in a completely disrupted sesterterpene biosynthesis, which supports the hypothesis that SmTS1 exhibits an unusually large active site cavity capable of taking up GFPP, while the enzyme variants with larger active site residues cannot. The G184L variant also showed no activity with any other substrate , which underpins the previously described role of this residue for SdS as effector: Upon active site closure the main chain carbonyl group of this conserved Gly comes into close contact with C3 of the substrate G182 in  and assi8) and nephthenol (9) that were identified by GC–MS  was determined in comparison to the total production of sesterterpenes by the wildtype ) pointed to the structure of (R)-nepthenol  D20 = +12  [R)- and the (S)-cembranyl cation -A may be in close proximity to an active site water, which may be able to attack at (R)-A to form (R)-9, while the distance to the cation in (S)-A is too large, preventing its attack to form (S)-9. In contrast, the formation of 8 only requires deprotonation that seems to be possible for both intermediates (R)- and (S)-A, explaining why compound 8 is nearly racemic.To determine the absolute configurations of , CHCl3) ), while , CHCl3) ) revealeation A, . The cat8 or 9, their enantiomeric composition was determined using the enantioselectively deuterated substrates (R)- and (S)-GGPP [8 and 9 will lead to incorporation of deuterium into diastereotopic hydrogen positions that can be distinguished by NMR spectroscopy. Herein, the additional 13C-labellings allow for a sensitive analysis by HSQC spectroscopy (R)- and (S)-GGPP with the SmTS1 A222M variant showed the incorporation of deuterium into only one of the diastereotopic hydrogens, indicating that (R)-9 is formed in an enantiomerically pure form, while for 8 from (R)-GGPP an enantiomeric ratio of 1.00:0.68 (19% ee) and from (S)-GGPP an enantiomeric ratio of 0.77:1.00 (13% ee) was observed, pointing to the formation of (R)-8 with 16% ee (average of both experiments).As the gaschromatographic analysis using a chiral stationary phase did not show any resolution for the enantiomers of -2H)GGPP . Their ctroscopy . Convers8 and 9 from the other enzyme variants was analysed following the same strategy, revealing that (R)-nephthenol was produced with high enantioselectivity also by the SmTS1 derivatives with A222V, A222L, A222I and A222F exchange (R)-8 from the A222F variant with high selectivity (94% ee), but mainly (S)-8 from A222V (37% ee), A222L (7% ee) and A222I (32% ee).The enantiomeric composition of the products Streptomyces mobaraensis it is possible that some of the usually conserved residues are naturally exchanged, but the enzyme retains its activity. Site-directed mutagenesis with installation of the otherwise conserved residue can lead to an improved activity, as shown for the Q227D variant. In other cases, a loss of activity is observed, e.g., in SmTS1 a re-installation of the missing pyrophosphate sensor in the G180R variant reduced activity towards GFPP, likely because of the steric bulk introduced by this exchange. We have also demonstrated that exchanges within the non-polar residues lining the active site can lead to a functional switch. For SmTS1 these residues are comparably small, and their exchange by larger residues can lead to a loss of activity with GFPP, as demonstrated for the A222V variant. It is interesting to note that this exchange at the same time leads to DTPS activity, while for the wildtype no GGPP conversion is observed. These findings will assist in future protein engineering for improved activity and functional switches in other microbial terpene synthases.Terpene synthases contain several well-known highly conserved motifs and single residues that are believed to be generally important for structure and function. As we show here, in special cases such as the sestermobaraene synthase SmTS1 from File 1Amino acid sequence alignment, details about the mutagenesis, purification and analytical data.


The implantation of screws is a standard procedure in musculoskeletal surgery. Heat can induce thermal osteonecrosis, damage the bone and lead to secondary problems like implant loosening and secondary fractures. The aim of this study was to investigate whether screw insertion generates temperatures that can cause osteonecrosis.We measured the temperature of twenty human femur diaphysis in a total of 120 measurements, while screws of different material  and different design (locking and cortex screw) were inserted in three different screwing modes  with 6 thermocouples (3 cis and 3 trans cortex). Each was placed at a depth of 2 mm with a distance of 1.5 mm from the outer surface of the screw.The screw design , the site of measurement (trans-cortex > cis-cortex) and the type of screw insertion (hand insertion > machine insertion) have an influence on the increase in bone temperature. The screw material (steel > titanium), the site of measurement (trans-cortex > cis-cortex) and the type of screw insertion (machine insertion > hand insertion) have an influence on the time needed to cool below critical temperature values. The combination of the two parameters (maximum temperature and cooling time), which is particularly critical for osteonecrosis, is found only at the trans-cortex.Inserting a screw hast the potential to increase the temperature of the surrounding bone tissue above critical values and therefore can induce osteonecrosis. The trans-cortex is the critical area for the development of temperatures above the osteonecrosis threshold, making effective cooling by irrigation difficult. It would be conceivable to cool the borehole with cold saline solution before inserting the screw or to cool the screw in cold saline solution. If possible, insertion by hand should be considered. The success of an osteosynthesis in trauma and orthopedic surgery depends on numerous factors. Some of them can be influenced by the surgeon and the technique used. Drilling holes and placing screws is an essential step in most osteosynthesis. This happens so often and routinely that most surgeons are not aware of effects on bone. In particular, the possibility of thermally induced bony necrosis during screw insertion might be underestimated.The heat generation during several surgical procedures (e.g. drilling) has the potential to irreversibly change and therefore weaken the mechanical competency of the bone and could be one factor leading to damage such as screw and implant loosening resulting in construct failure, re-fracture, and delayed union or malunion , 2.Bone tissue necrosis occurs immediately, if the temperature of 60 °C is exceeded . At loweDrilling is a process that has already been extensively investigated with regard to heat development and optimization. It was shown, that under special conditions a temperature of 70 °C was exceeded . The folWhile Augustin et al. were able to demonstrate that the optimal method of drilling to generate as little heat as possible was the use of lower drill speeds and higher feed rates , there hTherefore, the aim of this study is to investigate if osteonecrotic temperatures can be induced by the screw insertion. Additionally, different conditions and their influence on temperature generation have been examined. Hence, the screw material, the screw design and the screwing mode have been investigated in a standardized method.We measured peak temperature and duration while screws of different material  and design  were inserted into the human femoral diaphysis in three different screwing modes , for a total of 120 measurements. Each single combination of parameters was tested 5 times.Ten pairs of fresh-frozen human femoral diaphysis (length 15 to 20 cm) were used. The mean age of the donors was 77.4 years (range 60–93 years). Six of the donors were female, four male.Bone mineral density (BMD) of the cortical bone was determined for all specimens within the diaphyseal region by means of a peripheral quantitative computer tomography (pQCT). Therefore, all specimens were packed under vacuum and thawed to room temperature. CT scan with a slice thickness of 0.63 mm was performed . Image data given in Hounsfield unit (HU) was calibrated to volumetric BMD (vBMD) values given in mg hydroxyapatite per ccm (mgHA/ccm) using a BMD phantom . The BMD was measured within a region of interest (ROI) over 12 slices.30 × 5 mm head locking screws  and 30 × 4.5 mm cortical screws  were used for this investigation  or 3.2 mm  drill bits. For all drilling operations we used a commercially available non-surgical cordless screwdriver .In order to consider the working length of each screw, we measured the cortical thickness for the cis- and the trans-cortex at each drill hole using a depth gauge prior to inserting the screw. Additionally, all parameters  were examined on one pair of specimens in order to minimize the influence of bone density/thickness as far as possible.To measure temperature near the screw hole, a custom-made jig was inserted into each of the pre- drilled screw holes. It was thus possible to drill six 1 mm holes around each screw hole (three at the cis- and three at the trans-cortex). These holes had a depth of 2 mm and were placed at a distance of 1.5 mm from the screw outer surface  into the 1 mm pre-drilled holes and adapted them with “Arctic MX-2 Compound” insulation gel to ensure temperature conductivity. A seventh sensor was placed next to the bone as a reference value with the room temperature.All measurements were performed at room temperature. Three screwing modes were tested on each bone: manual insertion, full speed and reduced speed machine drilling. For manual insertion a standard surgical screwdriver was used. For insertion with the drilling machine, standard compressed air-operated surgical drilling machine was adapted, so that a standardized free-running speed could be guaranteed with the help of a rotary wheel. Two different power settings (688 rounds per minute (rpm) and 787 rpm; the compressed air line was adjusted to 8 bar) were used to simulate a full speed insertion as well as a reduced speed insertion.Before each measurement, the correct position of the thermal sensors was checked. Each individual test lasted for 120 s. By placing the sensors, it was possible to derive the temperature at the cis- and the trans-cortex separately.To calculate the cooling time until the temperature increase falls below 10 Kelvin, the measured temperature was mathematically interpolated (linear range in the cooling phase), and the resulting time determined. For this purpose, only the values of the sensors with a temperature rise of more than 10 K were used and averaged for further calculation. Under physiological conditions (body temperature 37 °C), the evaluated cooling time means the time required for the bone tissue to fall below the temperature of 47 °C –5. To esIn addition, a modified temperature coefficient for bones (mTCb) was calculated, which allowed us to investigate the temperature development in relation to the density of the tested bone. The coefficient is based on the assumption that there is a correlation between the temperature development and the amount of bone(mass) around the predrilled canal Fig. . The coeUsing the BMD and volume of the cylinder  and cortical thickness (t)), the mass of the region of interest can be calculated.The influence of the variation in bone density and cortical thickness is thus eliminated and statements can be made independently, e.g., on the material of the screw.The mTCb does not describe the temperature gradient over a wider area in the bone.With the implemented 7 thermocouples the temperature while screw insertion was recorded using a 8 channel thermocouple data logger by Pico Technology . The software used for data recording was the PicoLog-Recorder .The temperature was recorded in degrees Celsius with a frequency of 10 Hz. For each side (cis- and trans-cortex), an average of the three sensors/cortex was used in order to approximate the average temperature increase. In the event that the screw directly touched a thermocouple, this sensor was excluded.Additionally, for all measurements with a temperature increase of more than 10 K the time necessary to fall below the 10 K values was evaluated.Statistical evaluation was performed using Microsoft Excel 2016  and SPSS software package .p = 0.05 for all statistical tests.The parameters were each determined using a Man-Whitney-U test or a Kruskal-Wallis). Significance level was set at A summary of all results is given in Table Mean bone mineral Density was 1125.13 mgHA/ccm .The average heating over all measurements was 12.04 K (SD 6.67 K), the mean value for the mTCb is 0.159 K/g bone (SD 0.86). The mean time necessary to fall below the critical 10 K values was 61.27 s (SD 32.81).p = 0.447 for delta K and p = 0.295 for the mTCb). The cooling time was significant longer for the steel screws (p = 0.013). This is especially the case with the combination of steel and cortex screw .No significant difference in temperature development was seen when comparing steel and titanium screws . .When comparing cortical and head locking screws, a significant higher temperature increase for the cortical screw design could be found (b). Fig. . There wp = 0.000 for both delta K and mTCb, Fig. p = 0.000).On average, the temperature sensors on the trans-cortex heated up more than those on the cis-cortex (p = 0.541) and full speed .Manual screw insertion was associated with the highest heat generation followed by machine screw insertion at reduced speed (p = 0.645), whereas both machine insertions showed significant higher increase at the trans-cortex . The same is true for the consideration of the coefficient instead of the temperature in K.However, manual screw insertion showed similar increase at cis- and trans-cortex .Regarding the cooling time manual insertion showed similar times at the cis- and trans-cortex (below 60 s.), whereas both types of machine insertion showed significant longer cooling times at the trans-cortex . Assuming normal body temperature conditions of 37 °C for the bone in vivo and considering the osteonecrotic temperature limit of 47 °C set by Eriksson et al. 7 °C. Ass.In a preliminary test, we were unable to determine any significant influence of a second use of the screw (cortex and locking screw) with regard to an increased temperature development. This might be important also for intraoperative application. The surgeon can replace a screw and reuse it at another location without having to fear an increased risk of thermal induced osteonecrosis.Our investigation showed that the screw material  has no significant influence on the temperature development, but steel screws showed a significant longer cooling time. A possible reason for this result could be the different properties of the materials in terms of thermal conductivity. Due to their better thermal conductivity, steel screws absorb and release more heat. Titanium screws, on the other hand, absorb less heat due to their lower thermal conductivity and can therefore also release less heat.However, the screw design has significant impact on the heat generation. Higher temperatures were achieved by the cortical screw design compared to the head locking screw design. The cortical screw design has a larger surface area, which would actually favor heat dissipation, but it has only about 60% of the mass of the head locking screw design. Thus, the heat can be dissipated less into the screw and remains in the bone.Additionally, the trans-cortex showed significantly higher temperatures over all measurements compared to the cis-cortex. Furthermore, the cooling time was significantly longer for the trans-cortex, too. It can be assumed that the greatest frictional forces occur at the screw tip (thread cutting) and accumulate in the trans-cortex. The heat is then transmitted to the bone tissue of the trans-cortex and leads to a significantly higher heating. Additional attention must be given to the medullary canal. In the present study, this was no longer completely filled with medullary material due to sample preparation. This influences the temperature development and dissipation.We can identify some parameters that lead to a critical combination at the trans-cortex (> 15 K temperature rise and > 70 s. cooling time). The screw material steel, the screw design cortex screw and the insertion of the screw with machine at reduced and full speed are to be regarded as unfavorable.An investigation by Manoogian et al. confirmed the significant influence of the screw design. They investigated the heat generation the during insertion of standard and self-drilling Schanz-Pins into mid-diaphysis bovine femora. This group showed the highest temperature rise for the self-drilling pins followed by the insertion of self-drilling pins into a pre-drilled hole. The lowest rise was found for the insertion of standard pins into a predrilled hole .The mechanism of screw insertion also seems to have a substantial impact on the heat generation. The highest temperature rise was found when the screws were inserted by hand, as opposed to the lowest in the group using full speed machine insertion. But, the cooling time shows uncritical values for the manual screw insertion. The relatively high temperature increase can be explained by an increase in both the cis- and the trans-cortex . Thus, manual screw insertion can be defined as safe method of screw insertion.Both steps of placing a screw, the drilling and the screw insertion, have the potential to increase temperature of the surrounding bone tissue above critical values and therefore can induce osteonecrosis. This can finally result in screw loosening or ultimate failure of the osteosynthesis , 16, 17.One effective strategy to prevent osteonecrosis during drilling is sufficient irrigation. Sener at al. investigated the effects of irrigation on heat generation in different drilling depths. Interestingly, they found the highest rise in temperature without irrigation in a depth of 3 mm. With irrigation the highest temperature was found in 12 mm depth, but the temperature maximum was only 36.4 °C. Using saline solution with a temperature of 10 °C all measurements were below body temperature . This teThis study also has limitations. The experiments have been performed at room temperature and not at body temperature. This was done in accordance with the study of Matthews and Hirsch, who found no difference in the temperature rise during bone drilling in in vivo and in in vitro . AdditioBoth steps of placing a screw, drilling and screw insertion, have the potential to increase the temperature of the surrounding bone tissue above critical values and therefore can induce osteonecrosis.Relevant influence on temperature development is the screw design , the location of measurement (trans-cortex > cis-cortex) and the mode of screw insertion (hand insertion > machine insertion).Relevant influence on cooling time have the screw material (steel > titanium), the location of measurement (trans-cortex > cis-cortex) and the mode of screw insertion (machine insertion > hand insertion).Critical combination of both parameters (> 15 K heating AND > 70 s. cooling) can only be found at the trans-cortex. Parameters that lead to these critical values being exceeded are the screw material steel, the cortical screw design and the screw insertion with the machine both at reduced and full speed.The screw design and the location of measurement cannot directly be influenced. The higher temperature development at the trans-cortex makes effective irrigation problematic. It would be conceivable to cool the borehole with cold saline solution before inserting the screw or to cool the screw in cold saline solution.The surgeon, however, should be aware that the technique of insertion of the fixation elements may influence the later outcome of the entire construct. If possible, insertion by hand and irrigation should be considered.


The subcutaneous screw rod system, commonly known as the internal pelvic fixator (INFIX), is useful in managing unstable pelvic ring fractures. Conventional INFIX and transiliac–transsacral (TITS) screw techniques are performed using C-arm fluoroscopy. There have been problems with medical exposure and screw insertion accuracy with these techniques. This work describes new INFIX and TITS techniques using intraoperative computed tomography (CT) navigation and C-arm fluoroscopy for pelvic ring fracture.A typical case is presented in this study. An 86-year-old woman suffered from an unstable pelvic ring fracture due to a fall from a height. INFIX and TITS screw fixation with intraoperative CT navigation were selected to optimize surgical invasiveness and proper implant placement.The patient was placed in a supine position on a Jackson table. An intraoperative CT navigation was imaged, and screws were inserted under the navigation. Postoperative X-rays and CT confirmed that the screw was inserted correctly. This technique was less invasive to the patient and had little radiation exposure to the surgeon. Rehabilitation of walking practice was started early after the surgery, and she was able to walk with the assistance of a walker by the time of transfer.The technique employed in our case study has the cumulative advantages of safety, accuracy, and reduced radiation exposure, together with the inherent advantages of functional outcomes of previously reported INFIX and TITS screw techniques. Further experience with this approach will refine this technique to overcome its limitations and facilitate its wider use.The online version contains supplementary material available at 10.1186/s13018-022-02920-0. Pelvic ring fractures are commonly caused by high-energy trauma due to a car accident or a fall from a height. Fracture patterns determine conservative and surgical treatment . Severe To our knowledge, there are three types of internal fixation methods to be considered in the stabilization of pelvic ring fractures: anterior plate placement using an ilioinguinal approach; iliosacral (IS) screw fixation; and posterior lumbopelvic fixation using pedicle screw systems. In recent years, advances in spinal implants have allowed a minimally invasive spinal treatment (MIST) procedure for posterior pelvic ring fractures to be developed –6. A recBased on this background, the subcutaneous pedicle screw–bar fixation (INFIX) procedure, an anterior procedure using a spinal instrument, has been reported , 11. INFUnfortunately, intraoperative C-arm fluoroscopy is repeatedly taken during the procedure for positioning the screw and the level of radiation exposure to both the patient and surgical staff is extensive. In addition, the obtained images are blurred and cannot clearly show the anatomical structure around the sacroiliac joint. This is especially the case of anatomical variation where the rate of screw malpositioning is increased, adding considerably to the stress of the surgeon and the surgical team.In recent years, there have been some reports of spinal surgery using surgical support devices, such as intraoperative computed tomography (CT) navigation and robots, to solve the problem of C-arm fluoroscopy –16. SimiUnder the intraoperative CT navigation system, we will explain the techniques for the pelvic ring fractures using the INFIX technique and TITS screw fixation.Stabilization of the anterior pelvic ring was performed using intraoperative CT navigation in a manner based on the INFIX technique described by Vaidya et al. .The patient was operated on under general anesthesia based on the anesthetists’ decision. She was placed in a supine position on the operating carbon table  for the CT scan by O-arm . Standard spine instruments and navigated spinal instruments were used for the surgery. After proper painting and draping, the navigation reference frame was fixed to the iliac crest Fig. . Then, tNavigation was confirmed, and skin markings were made to identify the anterior superior iliac spine and pubic symphysis. After every navigated spinal instrument was verified, the best entry point on the anterior inferior iliac spine (AIIS) was marked by the navigated pinpoint probe.Through a 2–3 cm vertical incision centered on the AIIS, the entry point on the AIIS was approached by careful dissection through the interval between the sartorius and the tensor fascia lata, taking utmost care to protect the lateral femoral cutaneous nerve.The navigated high-speed burr Stealth–Midas System™ (Medtronic Sofamor Danek), pedicle probe and tap were used to make the screw hole, and then a CD Horizon® Ballast™ screw (Medtronic Sofamor Danek) was inserted Fig. .Fig. 2INIn the present study we used the Ballast™ screw, which is a polyaxial head screw. The ring notch at the base of the Ballast™ screw head provides a swing angle of up to 40°, making it easy to connect the rods. While looking at the virtual line on the navigation monitor, the Ballast™ screw was advanced from the AIIS. Even though it decreases the overall strength, it provides greater adjustments for the placement of the rod to decrease abdominal impingement.Navigation was also used to size the screw according to the patient’s physique, but most surgeons use multiaxial pedicle screws with a diameter of 8.5 mm and a length of 80 mm, with at least about 60 mm of the screw being intraosseous and about 20 mm outside the bone. We ensured that the pedicle screw head remained above the deep fascia to avoid compression on the femoral nerve and inguinal ligament.To confirm the screw placement, the C-arm fluoroscopy was adjusted to show the iliac view, and the awl was advanced directed towards the ischial spine Fig. .Fig. 3FlThe required rod length was measured and the rods were bent in semblance with the contour of the anterior pelvis. A 5.5-mm titanium rod was tunneled subcutaneously, connected to Ballast™ screw heads, and locked at one end Fig. . The rodReduction of the anterior pelvic ring injury was achieved by compression or distraction over rods as appropriate prior to locking the remaining screw head. The reduction was also done by distraction/lateral compression of the pelvic ring/traction and internal rotation.The quality of reduction and implant position was confirmed on C-arm fluoroscopy anteroposterior, inlet, and outlet views. The final tightening was done after confirming a satisfactory reduction. Finally, the position of the rod was checked by inserting two fingers between the rod and bone. At least 1 cm of the rod was kept proud on each side to help in removal.We present a video about the INFIX technique with intraoperative CT navigation   was performed with intraoperative CT navigation together with C-arm fluoroscopy depending on the morphology and displacement of the sacrum Fig. : Video 2Although the patients' walking start time varies, we allowed our patients to ride in a wheelchair and walk according to pain from the day after the operation during the early postoperative period.An 86-year-old woman with dementia was hospitalized following a fall from a height. Her diagnosis was a pelvic ring fracture (C1–3 type) with a sacral fracture and a bilateral upper and lower fracture of the pubis. Her preoperative X-ray and three-dimensional (3D) CT are shown in Fig. The day after the injury, INFIX and TITS fixation using intraoperative CT navigation was performed for the pelvic ring fracture. The operation time was 161 min and the estimated blood loss was 137 mL. In terms of radiation exposure, the patient organ doses were 0.140 mGy in the neck, 0.287 mGy in the chest, and 2.675 mGy in the abdomen measured using a dosimeter. In addition, a chest dosimeter inside the surgeon's protector recorded 0.018 mGy.A postoperative X-ray and 3D CT scan showed that the screws were in the correct position after the operation Fig. . The patUnstable pelvic fractures often require concurrent stabilization of the anterior and posterior pelvic rings. In addition, symphyseal plating leads to more blood loss when compared with INFIX . This isINFIX is one method that uses the already established principle of external fixation and is useful for pelvic ring fractures. Biomechanical studies have shown that INFIX has superior stability with respect to axial stiffness and stiffness at the pubic symphysis in the management of vertically and rotationally unstable pelvic ring injuries . AccordiThe technique of conventional INFIX and TITS screws using C-arm fluoroscopy entails radiation exposure. In addition, multiplanar repeated images are necessary for their accurate insertion, which increases radiation and operating time. On the other hand, in previous studies, intraoperative CT showed the highest screw accuracy at the screw position, but intraoperative CT navigation led to significantly longer total procedure and fluoroscopic times. Moreover, the patient’s radiation dose was significantly higher compared to that under C-arm fluoroscopy. It has also been reported that the irradiation dose is significantly higher than that of C-arm fluoroscopy . Our stuThere are still some limitations of this study. First, the surgeon's learning curve can affect the techniques. In addition, we did not compare the radiation exposure or accuracy of screw insertion with INFIX and TITS techniques to traditional fluoroscopy-only procedures in this study. Finally, this paper is a technical note, not an original article, and it may be necessary to evaluate the usefulness of this procedure in the future. All these limitations should be addressed in the future. To confirm the advantages of this method, a prospective multicenter randomized controlled trial compared with the current standard of care will be necessary.The INFIX and TITS screw technique can be considered a viable alternative to ORIF for unstable pelvic ring injuries as discussed in previous systematic reviews. In addition, we showed that our reported technique might be able to improve screw insertion accuracy and reduce medical radiation exposure.Additional file 1: INFIX technique with intraoperative CT navigation.Additional file 2: TITS technique with intraoperative CT navigation and fluoroscopy.


MOAP1), also known as MAP1 and PNMA4, belongs to the PNMA gene family consisting of at least 15 genes located on different chromosomes. MOAP1 interacts with the BAX protein, one of the most important apoptosis regulators. Due to its critical role in a few of disease-associated pathways, MOAP1 is associated with many diseases such as cancers and neurological diseases. In this study, we introduced MOAP1 and its biological functions and reviewed the associations between MOAP1 and a few diseases including cancers, neurological diseases, and other diseases such as inflammation and heart diseases. We also explained possible biological mechanisms underlying the associations between MOAP1 and these diseases, and discussed a few future directions regarding MOAP1, especially its potential roles in neurodegenerative disorders. In summary, MOAP1 plays a critical role in the development and progression of cancers and neurological diseases by regulating a few genes related to cellular apoptosis such as BAX and RASSF1A and interacting with disease-associated miRNAs, including miR-25 and miR1228.Modulator of apoptosis protein1 ( MOAP1), also known as MAP1 and PNMA4, belongs to the PNMA gene family, which consists of at least 15 genes on different chromosomes [PNMA1 and PNMA4 are located in human chromosome 14; PNMA8A, PNMA8B, PNMA8C, and CCDC8  are localized to chromosome 19; PNMA2 belongs to chromosome 8; and PNMA3, PNMA5, PNMA6A, PNMA6B, PNMA6E, PNMA6F, PNMA7A, and PNMA7B are located in X chromosome [MOAP1 , and we unified them to MOAP1 from now on to avoid any confusion.The modulator of apoptosis protein1  between the NCD and CCD domains was identified. The protein sequence of MOAP1 also contains a domain rich in lysine and arginine residues, named KRs [Among the poptosis , but alspoptosis . The speFirst identified by the yeast-two-hybridization assay, MOAP1 was a BAX-associated protein that binds to BAX only during apoptosis induction . It playMOAP1 as a pattern-recognition molecule (PRM) related to inhibitors of the complement system’s lectin pathway. The C4B binding protein (C4BP) regulates both classical and lectin pathways and MOAP1/C4BP chimeras have unique complement inhibition properties, providing a new therapeutic approach for upstream and central complement activation [MOAP1 also plays a role in liver Fas signaling by promoting MTCH2-mediated tBid recruitment to mitochondria [In addition, the complement system is a tightly regulated network of proteins involved in defense against pathogens, inflammatory processes, and the coordination of innate and adaptive immune responses. Hertz et al. mentioned chondria .MOAP1 also attenuates cell apoptosis by collaborating with other molecules. For example, it is known that RASSF1A and MOAP1 intimately interact to form the RASSF1A/MOAP1 molecular pathway in cancer. One potential function of this pathway is to link death receptors with BAX conformational change to promote tumor suppression [MOAP1 also interacts with RASSF6 to attenuate RASSF6-induced cell apoptosis; however, the attenuation was not observed in a p53-negative background [Besides BAX, pression . MOAP1 ackground ,8,9.MOAP1 is regulated by ubiquitin protein degradation [Trim39, can stabilize MOAP1 [UBR5 enhances MOAP1 ubiquitination. Matsuura et al. found that UBR5 ubiquitin ligase down-regulates MOAP1 in ovarian cancer, which contributes to its role in ovarian cancer resistance to cisplatin [MOAP1 expression level is low in tumor cells, MOAP1 ubiquitin can be reduced by apoptosis stimulating processes [Moreover, radation . Ubiquitze MOAP1 ,12. Moreisplatin . Since trocesses . MOAP1 in promoting autophagy signal transduction. Autophagy goes through three different stages by phagocytotic molecules and organelles, including initiation of recruitment, nucleation, and expansion of phagocytic membrane, followed by lysosomal degradation. P62 is an autophagy receptor. The BAX-binding protein MOAP1 regulates the p62-KEAP1-NRF2 signaling pathway by disrupting the p62 corpuscles. MOAP1 is recruited to p62 corpuscles by the induction of formation stimulated by cellular stress and reducing their levels independently of the autophagy pathway [MOAP1 was found to be an LC3-binding protein, which plays a unique role in promoting autophagy by interacting with LC3 to promote efficient shutdown of autophagy during starvation [Finally, there are a few studies suggesting a unique role of  pathway . Interesarvation .MOAP1 can activate the apoptosis signaling pathway, promoting its role in many diseases, such as cancers and neurological diseases , accounting for approximately 13% of new cancer incidences worldwide and causing 1.4 million deaths each year [Lung cancer is the most prevalent cancer in China and the third most common cancer in the United States . One major issue in CRC and probably all cancers is recurrence and metastasis, which eventually leads to the death of most cancer patients. That is, the cancer cell re-grows in the same location after surgical resection or move to another body location through blood flow and proliferation [Colorectal cancer (CRC), also known as bowel cancer, is among the top three most common cancers in the world. This cancer is caused by uncontrolled cell growth in the colon or rectum; the tumors in the two body parts are genetically the same and thus are called uniformly as colorectal cancer . There are five main molecular subtypes of breast cancer: Luminal A, Luminal B, Triple-negative/basal-like, HER2-enriched, and Normal-like. HER2 is probably the most important gene for breast cancer, with HER2-positive and HER2-negative patients accounting for about 25–30% and 70–75% of all breast cancer incidences, respectively [Breast cancer is the second most common woman cancer in the United States  expression will induce cell death and thus increase chemo-sensitization. Recently, Lee et al. found that this process requires the involvement of the BH3L domain of MOAP1 [PNMA family, MOAP1 also interacts with other members in this family to promote chemo-sensitization. In MCF-7 cells, overexpression of MOAP1 and PNMA1 will promote chemo-sensitization; however, the chemo-sensitization will be greatly inhibited when co-expressing with PNMA2, indicating that PNMA2 might be an antagonist of MOAP1 and PNMA1 induced chemo-sensitization [PNMA5 and MOAP1 greatly enhances the chemo-sensitivity of MCF-7 cells to Etoposide treatment [tization . For exasistance   treatment; MOAP1−/− primary cortical neurons are stronger. In the mouse transient middle cerebral artery occlusion (tMCAO) model, cerebral ischemia causes the MOAP1/BAX association, activating the MOAP1-dependent apoptosis cascade. In the study, the authors found that 24 h after tMCAO, MOAP1−/− mice showed less neuron loss and smaller infarct volume than MOAP1+/+ mice. Similarly, MOAP1−/− mice also have better integrity in neurological functions, which was proved in their rotarod test [MOAP1 plays an important role in regulating apoptosis in cerebral ischemic injury.A few early studies suggested that upregulation of BAX proteins in neuronal cells is a risk factor for cerebral ischemia ,58. BAX rod test . TogetheStress response is closely related to one of the most important mental diseases, namely depression. Depression, also called major depressive disorder, is a serious mental illness that negatively affects people’s the feelings and actions. MOAP1 plays an important role in regulating stress response by the Dorsal raphe nucleus (DRN). Forced swimming test is one of the famous techniques in wet lab to develop depression model in mice [MOAP1−/− mice in the forced swimming test showed depressive behavior with increased stationary time, which was eliminated by acute fluoxetine treatment. In young wild-type (WT) control mice, repeated forced swimming stress resulted in upregulation of tryptophan hydroxylase 2 (TPH2) and down-regulation of brain-derived neurotrophic factor (BDNF) in the dorsal renal nucleus (DRN). In contrast, TPH2 was not upregulated in aging WT mice. Interestingly, this stress response was not present in both young and old MOAP1−/− mice [Depression and BAX apoptosis signaling are highly correlated. For example, the signal cascade composed of BAD, BAX, and Caspase 3 is sufficient for synaptic depression . In addi in mice . Compare−/− mice . Parkinson’s disease (PD) becomes the second most common neurodegenerative disease in the world after Alzheimer’s disease, affecting nearly five million people worldwide, and this number is expected to double by 2030 . In ParkMOAP1 directly binds to BAX, which suggests that MOAP1 may play a role in Parkinson’s disease. Capurro et al. used external laser capture micro-anatomical data to verify the differentially expressed genes identified by PSEA  in the dense part of substantia nigra neurons. In this study, the expression of the MOAP1 gene was found to be reduced in cortical neurons of Parkinson’s disease (PD) [MOAP1 and neuron-related traits and diseases. Besides BAX, MOAP1 also interacts with other molecules like PNMA2. For example, in immunoprecipitation studies, PNMA2 was closely related to PNMA1 and MOAP1 and functionally antagonized the pro-apoptotic of MOAP1 and PNMA1 [It is well known that ase (PD) . In Tablnd PNMA1 .MOAP1 was also related to a few other diseases. For example, MOAP1 was targeted by miR-25 to block the apoptosis of vascular smooth muscle cells, which plays a critical role in inflammation and heart disease [MOAP1 is also commonly identified differential genes of coronary artery disease, ischemic cardiomyopathy, and myocardial infarction [MOAP1 is involved in the Fas signaling for apoptosis in the liver [MOAP1 might be implicated in many diseases and its acting mechanisms are yet to be discovered. Besides cancers and neuronal diseases,  disease . MOAP1 ifarction ,74. In afarction ,23,24,25MOAP1 in a few biological processes including cell apoptosis, innate and adaptive immunity, ubiquitin protein degradation, and autophagy. We also reviewed the molecules mediating the association between MOAP1 and a few diseases like cancers and neurological diseases. In this study, we reviewed the roles and mechanisms of MOAP1 can bind with the BAX protein to activate mitochondria and death receptor-mediated cell apoptosis, which will inhibit the progression and metastasis of a few cancers like lung cancer, breast cancer, and colorectal cancer and may mediate neurological diseases. In addition, a few miRNAs can directly interact with MOAP1 in a few cancers. For example, miR-25 can directly inhibit MOAP1 in lung cancer; miR-92a-3p and miR-572 can inhibit MOAP1 in colorectal cancer, and miR-1228 can inhibit MOAP1 in breast cancer. These may explain the functions of these miRNAs in cancer etiology. Moreover, MOAP1 also interacts with other genes to perform specific functions. For example, MOAP1 and RASSF1A collaborate to form the RASSF1A/MOAP1 pathway, which is important for cell apoptosis and chemosensitivity. MOAP1 also interacts with RASSF6 and MBR to promote cell apoptosis and is co-expressed with PNMA5 and PNMA1 to enhance chemosensitivity.As an important gene in the PNMA gene family, MOAP1 in cancers is relatively well studied in the literature; however, its roles in other diseases are yet to be revealed. Other diseases driven by mitochondrial-dependent apoptosis might also have a close relationship with MOAP1 and need to be further studied. Moreover, it is worthy of mentioning that there are many other biomarkers associated with MOAP1, such as long non-coding RNA (lncRNA), DNA methylation, and histone modification, regulating the expression of a gene. For example, the regulating roles of lncRNA in cancer have been widely studied recently [SNHG5 and MOAP1 in thyroid cancer has been reported; however, the underlying mechanism is unclear [MOAP1 and other regulation molecules should be explored.Interestingly, the role of recently , and a frecently ,77. The  unclear . In the


Using field and laboratory studies, we provide evidence of adaptation to local salinity regimes in the progenies of US Gulf of Mexico eastern oysters from estuaries differing substantially in annual mean salinity. Determining specific adaptations of oysters to local environmental conditions is important for conservation, restoration and the expanding aquaculture industry. Crassostrea virginica, is a foundation species within US Gulf of Mexico (GoM) estuaries that has experienced substantial population declines. As changes from management and climate are expected to continue to impact estuarine salinity, understanding how local oyster populations might respond and identifying populations with adaptations to more extreme changes in salinity could inform resource management, including restoration and aquaculture programs. Wild oysters were collected from four estuarine sites from Texas  and Louisiana  and spawned. The progeny were compared in field and laboratory studies under different salinity regimes. For the field study, F1 oysters were deployed at low (6.4) and intermediate (16.5) salinity sites in Alabama. Growth and mortality were measured monthly. Condition index and Perkinsus marinus infection intensity were measured quarterly. For the laboratory studies, mortality was recorded in F1 oysters that were exposed to salinities of 2.0, 4.0, 20.0/22.0, 38.0 and 44.0 with and without acclimation. The results of the field study and laboratory study with acclimation indicated that PC oysters are adapted to high-salinity conditions and do not tolerate very low salinities. The AB stock had the highest plasticity as it performed as well as the PC stock at high salinities and as well as Louisiana stocks at the lowest salinity. Louisiana stocks did not perform as well as the Texas stocks at high salinities. Results from the laboratory studies without salinity acclimation showed that all F1 stocks experiencing rapid mortality at low salinities when 3-month oysters collected at a salinity of 24 were used and at both low and high salinities when 7-month oysters collected at a salinity of 14.5 were used.The eastern oyster,  Crassostrea virginica , is a foundation species that provides critical ecosystem services and supports an economically valuable fishery and aquaculture industry within US Gulf of Mexico (GoM) estuaries  and Vermilion Bay . The oysters were placed in bags on an adjustable long line system  at the Louisiana Sea Grant Oyster Research and Demonstration Farm in Grand Isle, Louisiana  to ensure gonad development for spawning  . These tperature , 2. The perature .N = 10) of 35.5 ± 5.1 and is within the Upper Laguna Madre adjacent to PC, a channel that links Laguna Madre  with the GoM. AB has a mean salinity of 23.0 ± 6.9. Hydrological data associated with Texas Parks and Wildlife Department fisheries-independent monitoring program were used to calculate annual salinity and temperature within ~ 4 km of collection site  and Aransas Bay  . The PC ion site , 2. The In August 2018, each of the four broodstock groups was naturally induced to spawn at the AUSL hatchery by increasing water temperature, with individual oysters kept in separate 3-l containers to allow control of fertilization . As a stPerkinsus marinus, the protistan parasite causing dermo disease in diploid eastern oysters based on past findings (−1) were calculated by subtracting mean shell height from each bag at selected sampling time from the mean shell height of the same bag from the previous selected sampling time and standardizing to a 30-day month. Growth rates of each stock were obtained for time intervals ending in the April, July and October sampling dates when oysters were collected to measure condition index (CI) and P. marinus infection intensity as described below  and intermediate-salinity site  over 10 months  collecteed below . These m−1) were collected and transported to the Louisiana State University Agricultural Center Animal and Food Sciences laboratory building (LSU AFL), in Baton Rouge, to measure P. marinus infection intensity and the CI by dry weight (CIDW). Perkinsus marinus infection intensity (i.e. the number of parasites per gram of oyster wet tissue) was determined using the whole-oyster procedure described by −1 instead of alternative Ray’s fluid thioglycollate medium (ARFTM) during the homogenization step and 1 ml of the homogenate was then added to 9 ml of ARFTM  multiplied by 100  over the study period. Daily salinity and temperature data for Grand Bay were obtained from Point Aux Chenes station  10 km west of the Grand Bay field site. Mean daily salinity and temperature measurements at the Grand Bay field site were 0.6 ± 2.0 above, and 1.2 ± 0.3°C below mean PAC measurements on the days oysters were sampled. Measurements at PAC and Grand Bay field site on sampling days were highly correlated  over the study period. Mean salinity and temperature at the Mobile Bay and Grand Bay sites for the December –April, April–July and July–October intervals were calculated using only data when present at both sites on the same days.Salinity and temperature were measured on oyster sampling days at the Mobile Bay and Grand Bay field sites using YSI Pro30 conductivity meter . Daily temperature and salinity data for Mobile Bay were obtained from a location  less than 2 km south of the Mobile Bay field site. Mean daily salinity and temperature measurements at the Mobile Bay field site were 0.9 ± 1.3 above, and <0.1°C above mean measurements from the more southern site on the days oysters were sampled. Measurements at the Mobile Bay field and the more southern site on sampling days were highly correlated . Shell height and P. marinus infection intensity data were log10-transformed to achieve normality and homogeneity of variance. All ANOVA tests were followed by Tukey’s multiple comparison tests as appropriate when significant differences (P < 0.05) were found. All statistical analyses were performed using R 3.6.0 .Differences in salinity and temperature among sites and sampling intervals  were determined using a two-factor analysis of variance (ANOVA) test. Differences in mortalities among stocks and between sites at sampling intervals were compared using two-factor ANOVA tests; the random effect bag did not improve the model fit. Shell heights among stocks and between sites at the time of deployment in December 2018 were compared using a two-factor ANOVA test. Because significant differences in shell height were found between stocks at the time of deployment, growth rates calculated for the December–April, April–July and July–October intervals were used instead of shell height, to compare among stocks and between sites. The December–April and April–July interval growth rates among stocks and between sites were compared using two-factor ANOVA tests. The July–October interval growth rate among stocks was compared using a one-factor ANOVA test (4 GB stocks +3 MB stocks) as no PC oysters were left in Mobile Bay after July. The CIDW. Initial mean shell height and CI (± SD) for each stock were as follows: PC, 28.6 ± 2.9 mm, 5.9 ± 0.7; AB, 27.5 ± 4.7 mm, 7.7 ± 1.1; CL, 22.9 ± 2.9 mm, 6.6 ± 1.3; and VB, 25.8 ± 2.2, 7.3 ± 2.0. Fifty oysters of each stock were placed in ten 400-l tanks filled with aerated artificial seawater  with a salinity of 20.0 and maintained at a temperature of 20.0°C.In mid-November 2018, oysters (~3 months old) from each stock were transferred from Bayou Sullivan  to the static systems at LSU AFL. A subset of 15 oysters from each stock was haphazardly sampled to measure shell height and determine initial CISalinity in each tank was gradually adjusted at a rate of 3 units every 2–3 days until the target salinities of 2.0, 4.0, 22.0, 38.0 and 44.0 were reached . The treatments were replicated twice . Temperature was also gradually adjusted at a rate of 1.0°C every 2 days until the experimental temperature of 25°C was reached (i.e. after 10 days). Oysters were fed daily at ~5% of their dry meat weight with Shellfish Diet 1800®  when visual observation indicated that all algae added the day before had been cleared from the water. Every other day, the number of live and dead oysters was counted and dead oysters were removed from each tank. Oysters were considered dead when they were unable to effect shell closure when squeezed at least five times. Cumulative mortalities were calculated as described by g for 10 min . The oysters were not replaced. The supernatant, or plasma, was collected and its osmolality measured twice with a vapor pressure osmometer . At the end of the study, the ash-free dry weight (AFDW)-based CI (CIAFDW) of 15 oysters of each stock at each salinity was determined by dividing the AFDW by the weight of the whole wet oyster minus its shell wet weight and multiplying by 100 using a modification of the formula of After 1 month in the tanks, hemolymph was collected from three oysters per stock at each salinity (1 or 2 oysters per tank) and centrifuged at 400 × We tested for effects of stock, salinity and number of days of exposure on mortality via logistic regression. We constructed all possible candidate models from combination of the three predictor variables and their interaction terms and then tested these candidate models using Akaike’s information criterion AICc . We consAFDW at the end of the experiment were compared using a two-factor  ANOVA test, followed by Tukey’s multiple comparison test.Differences in cumulative mortality (%) among stocks within each salinity at the end of the study were determined using a series of chi-square analyses with Bonferroni correction. The osmolalities of plasma collected from each stock after one month were compared using a one-factor ANOVA test for each salinity treatment. CIN = 25) for each stock in November 2018 were as follows: PC, 29.4 ± 3.8; AB, 26.4 ± 3.5; CL, 23.6 ± 2.9; and VB, 26.0 ± 3.6 mm. The mean shell heights for each stock in March 2019 were as follows: PC, 33.1 ± 4.8; AB, 35.9 ± 4.2; CL, 38.4 ± 5.6; and VB, 37.0 ± 4.4.The salinity tolerance experiment without acclimation was conducted in November–December 2018  and March–April 2019  at Texas A&M University-Corpus Christi using oysters transferred from Bayou Sullivan. Salinities and temperatures at the time of collection at Bayou Sullivan were 24.0 and 12.3°C in November and 14.5 and 19.7°C in March. The mean shell heights  with aerated artificial seawater  at 25°C and salinities of 2.0, 4.0, 20.0, 38.0 and 44.0; each salinity treatment was replicated three times . Temperature and salinity from each tank were recorded daily. Oysters were fed daily with Shellfish Diet 1800® as in the previous laboratory study. Every other day, the numbers of live and dead oysters of each stock in each tank were counted over a 3-week period and the dead oysters were removed.ecotox’, 50) with 95% confidence intervals was determined for each stock and salinity treatment. LT50 are considered statistically different when confidence intervals do not overlap.Mortality data were compared among stocks and salinity treatments using probit analysis . Overall, salinities were significantly lower at the Mobile Bay site  than at the Grand Bay site  for each time interval  and site (P < 0.001). Temperature increased from December–April (15.4 ± 3.4°C) to April–July (26.7 ± 3.4°C) and again from April–July to July–October (29.8 ± 1.4°C) intervals and was significantly greater at the Grand Bay site (24.4 ± 6.6°C) than at Mobile Bay site (22.7 ± 7.2°C)  .P < 0.001) stock–site interaction in oyster interval mortality for December–April. The interval mortality of all stocks at the Grand Bay site was significantly less (P < 0.001) than all stocks at the Mobile Bay site (There was a significant (Bay site . MoreoveP < 0.001) stock–site interaction in the April–July interval mortality, with PC oysters (26.1 ± 6.7%) at Mobile Bay having significantly greater mortality than all other stocks  at either site.Similar to the Decemeber–April analysis, there was a significant , and all stocks maintained mortalities of <13%. Monthly changes in cumulative mortality for each stock at each site are shown in For the July–October interval, there were no significant differences in interval mortality between stocks  stock–site interaction in the initial shell height of oysters at the time of deployment. PC oysters deployed at the Mobile Bay (33.6 ± 4.5 mm) and Grand Bay (32.6 ± 4.7 mm) sites were significantly larger than all other stocks  stock–site interaction in oyster interval growth rate for December–April. The growth rates of all stocks at the Mobile Bay site were significantly less (P < 0.001) than all stocks at the Grand Bay site (There was a significant (Bay site . In addiBay site .P = 0.016) with growth rate at the Grand Bay site (4.0 ± 0.9 mm month−1) being significantly greater than at the Mobile Bay site (2.7 ± 0.8 mm month−1).For the April–July interval, growth rate significantly differed between sites (−1) was significantly greater than the AB stock from Mobile Bay (5.0 ± 0.6 mm month−1). Interval growth rates for each stock at each site are provided in For the July–October one-factor ANOVA test (4 GB + 3 MB), only the PC stock from the Grand Bay site (6.7 ± 0.6 mm monthDW in April was significantly affected by site (P < 0.001) with significantly greater CIDW at the Mobile Bay site (14.7 ± 3.0) than at the Grand Bay site (13.4 ± 1.8) (The CI4 ± 1.8) .DW was significantly affected by site (P < 0.001) and stock (P < 0.001). The CIDW continued to be significantly greater at the Mobile Bay site (13.5 ± 1.5) than at the Grand Bay Site (9.0 ± 1.4). In addition, the CIDW of the PC stock (9.8 ± 2.4) was significantly less than all other stocks .In July, the CIDW of all stocks at the Mobile Bay site  were significantly greater than those of all stocks at the Grand Bay site . Additionally, the CIDW of PCGB was significantly less than that of ABGB and CLGB , but not VBGB (P = 0.111).In October, the one-factor ANOVA test (4 GB + 3 MB) indicated the CIPerkinsus marinus infection intensity in April was significantly affected by site (P < 0.001) and stock (P = 0.017) (P. marinus infection intensity (log10 parasites g−1 wet tissue) was significantly greater at the Grand Bay site (2.79 ± 0.51) than the Mobile Bay site (2.05 ± 0.75) and was significantly greater (P = 0.013) in CL oysters (2.66 ± 0.42) than in VB oysters (2.24 ± 0.86).= 0.017) . The P. P < 0.001) with significantly greater infection intensity at the Grand Bay site (2.27 ± 0.57) than at the Mobile Bay site (1.51 ± 0.83).In July, the infection intensity was significantly affected by site (P. marinus infection intensity among stocks (P = 0.008), but no significant differences between stocks could be found using Tukey’s Multiple Comparison of Means.In October, the one-factor ANOVA test (4 GB + 3 MB) indicated significant differences in −1  to 1340 ± 4 mOsm kg−1 , with osmolality increasing with salinity (P > 0.1), except for marginally lower osmolalities for VB (1147 ± 1 mOsm kg−1) versus PC (1153 ± 2 mOsm kg−1) stocks at salinity 38 (P = 0.03).Osmolality ranged from 80 ± 5 mOsm kgsalinity . There wUnder both low  and high  salinity, the best supported logistic regression models all included effects of stock, salinity and number of days of exposure on mortality .P < 0.001), 4.0 (P < 0.001), 38.0 (P = 0.026) and 44.0 . At salinity 4.0, the cumulative mortality of PC oysters (7.3%) was significantly higher than all other stocks (P = 0.042), which experienced no mortality. There were no significant differences in mortality among stocks at salinity 22.0 (P = 0.187). At salinity 38.0, Louisiana stocks  had significantly higher cumulative mortality than PC , but were not different from AB . At salinity 44.0, cumulative mortality of Louisiana stocks  was significantly higher than the PC stock , and the VB stock was significantly higher than the AB stock .At the end of the experiment, significant differences in cumulative mortality were found among stocks at salinity 2.0 (P < 0.001) stock–salinity treatment interaction for CIAFDW at the end of the experiment. In general, the CIs of Louisiana stocks were higher at the lower salinity treatments, while CIAFDW of Texas stocks were higher (PC) or tended to be higher (AB) at the higher salinity treatments at the end of the experiment  was significantly higher than at 38.0  and 44.0 . Similarly, the CIAFDW of VB at salinity 2.0 (2.69 ± 0.66) was significantly higher than at 22.0 , 38.0  and 44.0 . In contrast, the CIAFDW of PC at salinity 2.0 (1.61 ± 0.64) was significantly lower than at salinities of 38.0  and 44.0 .There was a significant  and CIAFDW of PC and AB at salinity 4.0 . Similarly, CIAFDW of VB at salinity 2.0 was significantly higher than the CI of PC at salinity 2.0 and 4.0 (P < 0.001). At the higher salinities, only the CIAFDW of PC at salinity 38.0 (2.51 ± 0.55) was significantly higher than CIAFDW of VB at salinity 22.0  and 44.0 . However, the CIAFDW of Texas oysters tended to be higher than the CIAFDW of Louisiana oysters.Significant differences in CIg stocks . Specifi50 were found among stocks  than all other stocks . At salinity 4.0, CL stock had significantly lower LT50  than all other stocks . In Trial 1, LT50 values could not be calculated for any stocks exposed to salinities of 20 or higher because mortality rates were too low (<3%).Mortality of oysters from each stock exposed to treatment salinities differed between Trials 1 and 2. At the end of Trial 1 (23 days), mortalities of oysters exposed to salinity 2.0 and 4.0 were high , whereas mortalities of oysters exposed to salinity 20.0, 38.0 and 44.0 were low  than all other stocks . At salinity 4.0, AB had lower mortality than all other stocks, but 95% confidence intervals could not be calculated for the other stocks. No LT50 values could be calculated for any stock at salinity 20.0. At salinity 38.0, VB had a significantly lower LT50 (15.4) than all other stocks . At salinity 44.0, the mortalities went from 0% on Day 1 to >89% on Day 5 for AB, CL and VB stocks or >75% on Day 7 for PC stock. The LT50 of the AB (3.6), CL (3.6) and VB (3.6) stocks seemed lower than that of the PC stock (7.5) but no 95% confidence intervals could be obtained using Probit analysis because too few data points were collected (i.e. every 2 days) when most mortalities occurred.At the end of Trial 2 (25 days), mortality was greatest at the highest and lowest salinities , followed by the more moderate salinities tested , with lowest mortalities at salinity 20.0 (0–17%). Greater than 50% mortality was achieved in 5 days for all stocks at salinity 44.0, and in 1 week for the three lower salinity stocks  at salinity 2.0 and for the two lowest salinity stocks  at salinity 4.0 . Signifi Trial 2 . At saliUsing a combination of laboratory and field studies on F1 cohorts, we tested whether GoM populations of a key estuarine foundation species differ in their physiological responses to salinity. Salinity tolerance varied among GoM oyster stocks acclimated to extreme salinities in the laboratory. The Texas stock originating from the highest salinity estuary (PC) performed significantly worse at lower salinities  and better at higher salinities  than the Louisiana stocks from the lower salinity estuaries . The Texas stock from a moderate salinity estuary (AB) performed as well as the PC stock at high salinity and as well as the Louisiana stocks at low salinity. Field results corroborated these laboratory findings, providing evidence of local adaptation to salinity. Different laboratory results were obtained, however, when the F1 oysters were transferred directly, without acclimation, to extreme salinities.−1 and constantly hyperosmotic to the seawater at a salinity of 2  with PC stock more so than the other stocks. PC stock plasma osmolality was also more variable at a salinity of 2.0 indicating more differences among PC oysters in their ability to osmoconform at that extreme salinity. This is in agreement with past studies that found a minimum plasma osmolality limit of about 80 mOsm kg−1  in Louisiana oysters at a low salinity field site  or exposed to simulated freshet events (< 3) (−1) in this osmoconformer , whether following acclimation in the laboratory or in the field. PC oysters started dying after about a month at extreme low salinity and over 50% were dead within another month in contrast to the other stocks that had mortality of <5% in the field and 15% in the laboratory. It is clear that PC oysters were not as competent as the other stocks in handling low salinity. During the laboratory acclimation study, the mean plasma osmolalities of all stocks were >80 mOsm kgts (< 3) . The difonformer . This hy. In this case, PC oysters were better able to cope with higher salinities than the other stocks. The underlying cellular, biochemical and molecular mechanisms for the physiological differences in the contrasting salinity tolerance between stocks are unknown and will need to be determined in future studies. Interestingly, a past study identified differences in both tissue concentration and composition of the amino acid pools, used as osmolytes, of oysters between Chesapeake Bay and Atlantic oysters, along the mid-Atlantic United States coast  for low salinity survival in C. virginica  which is common in less than 1-year-old oysters and not expected to cause mortality  when exposed to extreme salinities than in the first trial  and were likely due to differences in the magnitude of salinity change F1 oysters were exposed to, from field collection to immersion, between trials. While most oysters in the first trial  survived direct immersion at salinities of 38.0 and 44.0, most oysters in the second trial  died when transferred to a salinity of 44, and between 25% and 50% died, depending on the stock, when transferred to a salinity of 38. It is also likely that oyster size and season contributed to some of the minor differences between trials. Oysters used in the first trial in November 2018 were 3-month-old juvenile oysters , compared to the 7-month-old oysters (33.1–38.4 mm) in the second trial in March 2019, which were larger and more mature with obvious gonad in development. Oysters that are larger and also closer to spawning have been reported to be more susceptible to stressors such as salinity and temperature changes and would explain the higher mortality rate  than the other stocks upon transfer to extreme salinities. We also found in a preliminary study that PC oysters survived under hypoxic conditions (1 mg O2 L−1) longer than the other stocks . This ability to survive longer under anaerobiosis might be related to the fact that oysters from Laguna Madre are predominantly exposed to changes in salinity at the higher end of estuarine salinity range, conditions under which amino acids plays a major role as osmolytes. It has been suggested that the synthesis of amino acids via known anaerobic biochemical pathways may facilitate high salinity acclimation  to 50% seawater . DiffereIt has long been assumed that local adaptation in marine environments was only evident in comparisons of populations on broad geographic scales due to dispersive larval or mobile adult stages . This asIn the US GoM, oyster production brings in >$100 M in average annual landings . The differences in stock performance from this study not only highlight the importance of stock selection for aquaculture and restoration in estuaries that are currently, or will be in the future, experiencing extreme salinity conditions but also raise important questions about the potential genetic impact of hatchery-propagated oysters on wild populations.The results of these field and laboratory studies indicated that PC oysters  are adapted to high-salinity conditions (>38) and do not tolerate very low salinities (<4). The AB stock (Texas) seemed to show the highest plasticity as it performed as well as the PC stock at the high salinities and as well as the Louisiana stocks at the lowest salinity. The AB stock also experiences more salinity variation from year to year as indicated by This work was supported by the National Science Foundation Biological Oceanography Program (grant number OCE 1731710) and Louisiana Sea Grant (award NA14OAR4170099).Supplemental_Material_coab065Click here for additional data file.