Here we report a high-resolution cryo-EM structure of the mammalian 20S proteasome in complex with PI31. The structure shows that two copies of the intrinsically disordered carboxyl terminus of PI31 are present in the central cavity of the closed-gate conformation of the proteasome and interact with proteasome catalytic sites in a manner that blocks proteolysis of substrates but resists their own degradation. The two inhibitory polypeptide chains appear to originate from PI31 monomers that enter the catalytic chamber from opposite ends of the 20S cylinder. We present evidence that PI31 can inhibit proteasome activity in mammalian cells and may serve regulatory functions for the control of cellular proteostasis.Proteasome-catalyzed protein degradation mediates and regulates critical aspects of many cellular functions and is an important element of proteostasis in health and disease. Proteasome function is determined in part by the types of proteasome holoenzymes formed between the 20S core particle that catalyzes peptide bond hydrolysis and any of multiple regulatory proteins to which it binds. One of these regulators, PI31, was previously identified as an  PI31 haoteasome A and S1 me A and . In contnhibitor , 18. Sucapparent , 20, 21.Microsporidia . The hPI31 densities near the β2 and β5 active sites within the central chamber of 20S were especially well resolved (D and E). Portions of two hPI31 C-terminal domains were identified within a given 20S particle and featured the same C2 symmetry characteristic of the β-subunit rings. However, the C1 map was used for PI31 model building to avoid average deviations. The modeled structure is shown as a linearized hPI31 chain extending from the vicinity of the β5 active site of one β-ring and then crossing the interface of the two β-rings toward the β2 and β1 active sites of the opposing β-ring (F). Although some hPI31 densities are present near the β1 site, we were unable to build a model at this location due to the disconnection of the densities. Because the end of the modeled hPI31 is near the β1 site, we predict an interaction between this site and the C terminus of hPI31. Biochemical analyses described below support this prediction. The C-terminal domain of hPI31 (aa 152–271) is composed of 35% proline and glycine residues and is structurally disordered (G), suggesting that the interactions between hPI31 and the proteasome active sites constitute the basis for the inhibitory activity of PI31.We determined the cryo-EM structure of a mammalian PI31–20S proteasome complex after incubating recombinant human PI31 (hPI31) with the purified latent bovine 20S proteasome featuring a predominately closed-gate conformation . Human aFigs. 1resolved , D and Eg β-ring F. Althousordered . Accordisordered G, suggesA and B). Despite these similarities, there are also a number of differences. For example, in the yeast structure, Fub1 starts (N to C terminus) from the β1 active site and then extends over the center of the β-ring to the β5 active site where it dimerizes with the second copy of Fub1. Fub1 then contacts the β5 active site of the opposing β-ring and returns to the β2 active site of original β-ring. In contrast, hPI31 does not traverse the center of proteasome in the mammalian complex. Instead, it begins from the β5 active site of one β-ring and curls toward the β2 active site of the opposing β-ring (C and D). Thus, although PI31 and Fub1 have conserved active-site interacting regions, the structural arrangement of these interactions appears to be distinct (B–D). Fub1 blocks the β5 activity through a unique conformation between two separate Fub1 molecules, whereas hPI31 achieves this inhibition using two conserved sequences (aa 194–200 and aa 206–212) within the same chain near the β5 active site. Specifically, the antiparallel hydrogen bonding between Gly194-Gly195-Glu196 and Gly206-Gly207 creates a conformation that fits into the β5 binding site (E). Human PI31 mimics the substrate binding, P4-P3-P2-P1-P1′-P2′-P3′-P4′ to S4-S3-S2-S1-S1′-S2′-S3′-S4′, to inhibit the proteolytic activity of proteasome. At the β2 active site, residues 242 to 246 of hPI31 occupy the β2 pocket in an antiparallel direction with Asp244 (P1) and Arg242 (P3) inserting into the S1 and S3 binding pockets, respectively (A). The hPI31 residues Arg242, Asp244, Pro245, and Phe246 are hydrogen bonded with the surrounding residues Thr1, Gly47, Ala49, Gly128, and Ser129 of β2 subunit to stabilize the binding sequence (B). At the β5 active site, the dipeptide Asp197-Leu198 and residues 208 to 214 of hPI31 fit into the β5 binding pocket in a parallel direction with the Met208(P1) and Ile209(P3) inserting into the S1 and S3 binding pockets (C). The hPI31 residues Asp197, Met208, and Ile209 are hydrogen bonded with the surrounding residues Thr1, Thr21, Gly47, Ala49, and Ser130 of β5 subunit to sustain the unique binding conformation (D). These results show that hPI31 interacts with each of the proteasome’s catalytic sites in a manner likely to directly inhibit substrate catalysis.The structure of this mammalian PI31–20S complex is generally similar to that of a recently reported complex between Fub1, the yeast PI31 ortholog, and an α3Δ yeast mutant 20S proteasome . In eachg β-ring , C and Ddistinct , B–D. Fuing site E. Human ectively A. The hPsequence B. At the pockets C. The hPormation D. These B), which further restrains the proteolytic activity of the β2 active site. At the β5 site, Asp197 of PI31 forms a hydrogen bond with the β5 catalytic Thr1 . Like the hydrogen bonding between Asp244 and Thr1 of β2, the hydrogen bond between Asp197 and Thr1 of β5 may prevent the formation of threonine nucleophile at the β5 site. In addition, the unique antiparallel conformation formed by Gly194-Gly195-Glu196 and Gly206-Gly207 of PI31 forces Asp197 to sit at the S1′ site and residues 208 to 214 to bind to β5 pocket in a parallel direction, which is in the reverse direction of normal substrate binding. This binding profile leaves no peptide bond between P1 (Met208) and P1’ (Asp197) and renders PI31 unfavorable for hydrolysis at the β5 site.The ability of PI31 to stably inhibit the proteasome requires that PI31 itself is resistant to degradation. Several features of the hPI31 bound to the β2 active site may contribute to this resistance. First, Lys33 of the β2 subunit was proposed to promote the deprotonation of the catalytic Thr1 and to generate the oxyanion for nucleophilic attack , 26. TheFig. 3i.e., open-gate) 20S known to be promoted by certain purification conditions . As expected, both full-length wildtype hPI31 and a truncation mutant lacking the FP domain (residues 1–151) but containing the entire C-terminal domain (residues 152–271) inhibited the catalytic activity of each subunit type by >95% (B). Similarly, a mutant consisting of residues 1 to 261 inhibited the activity of each catalytic subunit type. In contrast, a mutant consisting of residues 1 to 181  had no inhibitory activity. These results confirm that PI31 amino acids responsible for inhibition of proteasome active sites reside between residues 182 and 262. Accordingly, PI31 variants 1 to 244 and 152 to 244 each inhibited β5 activity but not β1 activity, as predicted. However, neither of these latter mutants inhibited β2 activity, as expected, but instead stimulated β2-catalyzed hydrolysis. This surprising result suggests that these truncated proteins lack residues required for the stable orientation of the amino acid(s) that contact(s) the active site β2 threonine. The differential effects of these PI31 mutants on specific types of proteasome activities were also demonstrated in an orthogonal assay using the activity probe, Me4BodipyFL-Ahx3Leu3-VS (C). Me4BodipyFL-Ahx3Leu3-VS covalently binds to each of the proteasome’s catalytically active sites, and the resulting fluorescently tagged subunits can be detected and identified after SDS-PAGE . These functional results are consistent with the structural predictions for identification of interaction sites between PI31 and individual catalytic subunits that cause proteasome inhibition and indicate that despite a clear structural resolution of PI31 residues in contact with the β1 catalytic site the latter interaction occurs between PI31 residues 245 and 261 (A).The structure of the hPI31–20S complex described above predicts hPI31 residues likely to be involved in inhibition of specific catalytic sites. To test these predictions, we first expressed and purified hPI31 truncation mutants that contain or lacked various combinations of these residues and tested these mutants in activity assays specific for each of the proteasome’s three catalytic subunit types A. As exp by >95% B. Simila3Leu3-VS C. Me4Bodnit type C. These  and 261 A.Figure D197A inhibited β5-specific peptide hydrolysis 3 times less well than wildtype hPI31 but had little or no effect on the inhibition of hydrolysis of β1- or β2-specific substrates compared with wildtype hPI31 activities (A). A similar β5-specific diminution of PI31 inhibition by the hPI31D197A mutant was observed in an assay using the active-site probe, Me4BodipyFL-Ahx3Leu3-VS (B). Likewise, hPI31D244A had significantly weaker inhibitory activity against β2-specific peptidase activity but did not affect inhibition of the β5-specific substrate hydrolysis (A). This latter mutant also blocked normal PI31 inhibition of β1-specific substrate hydrolysis to a lesser extent. Although the basis of this effect is unclear, we note that the proteasome’s catalytic sites are known to have interdependent allosteric effects  at residues 197 and 244. Our structure shows that D197 and D244 interact directly with the catalytic threonine residues of β5 and β2 proteasome subunits, respectively . Becausetivities A. A simi3Leu3-VS B. Likewidrolysis A. This l effects . TherefoA). At a concentration estimated to approximate that found in cells, hPI31WT featured nearly equal amounts of monomer and dimer . Next, we expressed and purified hPI31 containing a point mutation (V6R) in the FP domain predicted to disrupt PI31 dimerization (versus 751 dimer counts) . Finally, we expressed hPI31152–271, which lacks the FP dimerization domain and is assumed to be monomeric. Unfortunately, mass photometry cannot be used to confirm the monomeric state of hPI31152–271 (a predicted 13-kDa protein) due to limitations of lower mass detection by this method. Therefore, we used chemical cross-linking followed by SDS-PAGE to further evaluate and compare the relative oligomeric states of hPI31WT, hPI31V6R, and hPI31152–271 . As expected, cross-linking of hPI31WT produced detectable protein at a size (∼60 kDa) consistent with dimeric PI31. A significantly reduced amount of cross-linking-dependent protein was detected for the hPI31V6R mutant, consistent with the predominately monomeric nature of the protein shown by mass photometry. Importantly, cross-linking of hPI31152–271 produced no detectable products, a result consistent with the conclusion that this mutant is completely monomeric. Despite their differing oligomeric states, each of these hPI31 variants was competent for proteasome inhibition. Surprisingly, however, inhibitory activities were indistinguishable when assayed at comparable [M]T (where [M]T = [monomer] + 2 × [dimer]) . Nevertheless, a direct comparison of the relative inhibitory effects of these proteins is complicated by the nonhomogenous oligomerization state of PI31WT and PI31V6R. Assuming [monomer]/[M]T ratios of 0.32, 0.54, and 1.0 for hPI31WT, hPI31V6R, and hPI31152–271, respectively, hPI31WT achieves 50% inhibitory activity at a lower amount of functional monomers than hPI31V6R and hPI31152–271 . Thus, inhibitory activity may be a function of total subunit number ([M]T) rather than oligomerization state. Although we favor a model in which the 20S–PI31 complex structure determined here results from two PI31 monomers that enter the 20S catalytic sites from opposite ends of the 20S cylinder, we cannot exclude related models including those that feature the inhibitory action of PI31 dimers . This mic sites . This meic sites , 38. Ratic sites , 39, 40.A and B). We also examined the difference map between the PI31-bound and PI31-free EM maps and observed hPI31 densities in the 20S antechambers as well as in the catalytic chambers. These findings indicate that the C-terminal regions of PI31 in the catalytic chamber are threaded from the antechambers (C). This observation is consistent with the proposed orientation of active site engagement along the primary structure of the hPI31 C terminus, as described above and discussed further below.Unlike the unstructured proline/glycine-rich C-terminal domain, the N-terminal FP domain of hPI31 is well folded , 41 and chambers C. This oMicrosporidia  from β1 to β5 in the opposing β ring and back to β2 in the original β ring. Moreover, Fub1’s interaction with and proposed inhibition of β5 employs the cooperative action of a second Fub1 molecule. In contrast, our data show a hPI31 interaction path (in the N-to-C direction) from β5 to β2 to β1. As with Fub1, hPI31 interacts with β5 on the opposing ring from that of its interactions with β1 and β2. However, unlike Fub1, hPI31 utilizes two different segments of the same polypeptide chain for its β5 interaction. This feature indicates that a single molecule of hPI31 is sufficient to engage one copy of each β-type catalytic subunit. The functional consequence of inhibition of only one of two copies of each catalytic subunit type is unknown. Interestingly, an interaction path similar to that of the mammalian proteins has been observed for a PI31-like peptide and 20S proteasome from sporidia . NeverthD, left). This feature might contribute to the observation that high molar ratios of PI31 to 20S are required for inhibition in vitro. It is also consistent with the conclusion that the two C termini residing in the catalytic chamber enter from opposite ends of the proteasome. Nevertheless, we cannot exclude models of inhibition involving PI31 dimers . In one variant of this model, transit through the substrate access pore may be limited to one of the two C termini while the HbYX motif of the other subunit promotes gate opening via α-subunit binding. Although such a mechanism is consistent with previously shown gate-opening properties of hPI31 C-terminal peptides, it cannot be obligatory because PI31 lacking HbYX residues retains inhibitory activity . Alactivity . Additioin vitro conditions that reverse PI31–proteasome binding and inhibition, such a process has been proposed as a mechanism for termination of PI31-like-mediated proteasome inhibition that allows assembly of active 20S–19S/PA700 holoenzyme complexes after germination of dormant Microsporidia spores . Site-directed mutagenesis was performed on 10 ng pET28a(+)-HisPI31wt template with the Q5 Site-Directed Mutagenesis Kit (New England BioLabs) according to manufacturer’s instructions. Based on the length of the plasmid, an extension time of 2.5 min was used for each PCR cycle. The mutagenesis PCR product was ligated using the Kinase Ligase DpnI (KLD) mix provided in the kit. Ligated plasmid (1 μl) was then transformed into One Shot BL21 Star (DE3) competent cells (Invitrogen) with outgrowth in SOC media for 1 h at 37 °C before plating on LB agar plates containing kanamycin (50 μg/ml). Sequences were confirmed by Sanger sequencing at the UT Southwestern McDermott Center Sequencing Core with Big Dye Terminator 3.1 and capillary instruments (Applied Biosystems Inc).20S proteasome was purified from bovine red blood cells as described . Recombiescribed . A pET28g for 10 min at 4 °C. Cell pellets were resuspended in 8 ml Binding Buffer  containing 0.1 mg/ml lysozyme and 1x cOmplete, EDTA-free protease inhibitor cocktail (Roche). Cells were incubated on ice for 30 min before sonication. Lysed cells were then centrifuged at 14,000 rpm, 20 min, 4 °C. The 8 ml supernatant was applied to 1 ml packed volume of Ni-NTA His-Bind Resin (Millipore Sigma). Binding was performed with gentle mixing for 1 h at 4 °C and loaded onto a Poly Prep Chromatography Column (Bio-Rad). The loaded resin was then washed 3 to 4 times with 6 ml Wash Buffer  until protein was undetectable with Bradford Reagent (Pierce). His-PI31 was eluted in four 0.5-ml fractions with 250 mM imidazole, 50 mM sodium phosphate, pH 7.6, 500 mM NaCl. Fractions containing peak protein (as assessed by Bradford assay) were pooled and dialyzed against Buffer H  containing 10% glycerol. Purification was confirmed by SDS-PAGE Coomassie staining and Western blotting with anti-His antibody.Recombinant His-PI31 mutant proteins were purified by metal affinity chromatography. BL21 Star (DE3) cells containing pET28a(+) HisPI31 mutants from monoclonal stab cultures were used to inoculate 150 ml LB broth containing 50 μg/ml kanamycin. Cells were grown with shaking overnight at 37 °C. The following morning, 100 ml of LB/Kan broth was added to cultures with 250 to 500 μM IPTG to induce expression. Cells were grown at 37 °C for an additional 3 to 6 h. Following expression, cells were pelleted by centrifugation at 10,0004BodipyFL-Ahx3Leu3-VS activity-based probe -linked fluorogenic peptide substrates specific to each of the proteasome catalytic sites: caspase-like , trypsin-like , and chymotrypsin-like . In some experiments, latent 20S proteasome was first activated with preincubation in 0.03% SDS at 37 °C for 5 min. PI31  was then incubated for an additional 15 min at 37 °C. Proteasome activity was assayed by addition of peptide substrate . Free amc produced by proteasome hydrolysis was monitored  at 45-s intervals over 21 min at 37 °C . Proteasome activity was expressed as rates of amc production, and all were linear with respect to time and proteasome concentration in these assays. All assays were performed in triplicate and repeated independently at least three times. Differences between the effects of PI31 mutants on proteasome activity were analyzed by one-way ANOVA and Tukey’s honestly significant difference (HSD) test using GraphPad Prism 9 (v9.5.0). PI31 mutant inhibitory activity was also assessed using direct fluorescent labeling of proteasome active sites with a MeWestern blotting was conducted as described . Transfewt, was obtained from Sino Biological Inc . HepG2 cells were seeded in six-well plates at 3 × 105 cells well−1 and grown overnight at 37 °C, 5% CO2 in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, penicillin, and streptomycin. On the following day, 750 ng of pCMV3-N-FlagPI31 vector was mixed with 5 μl P3000 reagent diluted in OptiMEM medium and added to 5 μl of diluted Lipofectamine3000 according to manufacturer’s instructions (Life Technologies). Vectors or vehicle-only controls were added to replicate wells and transfected for 24 h. Following transfection, cells were washed three times with PBS and harvested in a hypotonic lysis buffer in the presence of ATP . Cells were then passed through a 27½ G needle 15 times and centrifuged at 14,000 rpm, 20 min, 4 °C. Supernatants were divided into three sets of triplicate wells in a 96-well plate and assayed for proteasome activity using fluorogenic peptide substrates  as described above for pure proteins.A mammalian expression vector, pCMV3-N-FlagPI31Proteasome and PI31 amounts in HepG2 cells transfected with vehicle controls or pCMV3-N-FlagPI31 were analyzed by semiquantitative Western blotting using polyclonal antibodies against the proteasome β5 subunit and the PI31 C terminus. Within the same gel, a range of pure proteins (bovine 20S proteasome or recombinant His-PI31) was used as standards for quantification. Secondary antibodies with IRDye (LI-COR) were used and near infrared signals were quantified on Odyssey CLx imager (LI-COR). Signals were converted to protein masses based on the standard curve of pure proteins and then converted to moles assuming values of 700 kDa for proteasome and 31 kDa for PI31 . AmountsPurified His-PI31 (wildtype or V6R mutant) proteins were dialyzed against 20 mM Tris HCl (pH 7.6 at 5 °C), 20 mM NaCl for analysis of oligomerization by mass photometry . Protein concentrations were determined by BCA assay (Pierce) and converted to molar concentrations assuming 31 kDa molecular weights. PI31 was then diluted in 0.2 μm filtered PBS into a total monomer concentration of 400 nM. Cover slips were cleaned with isopropanol and MilliQ water and fitted with silicon gaskets. The mass photometer optics were focused on 16.2 μl buffer prior to the addition and mixture of 1.8 μl sample. Sample mixture into the prefocused droplet resulted in an additional one-tenth dilution , and recordings were initiated immediately after mixing. Bovine serum albumin and thyroglobulin oligomers were used as molecular weight standards to calibrate ratiometric contrast measurements to molecular weight. Automatic Gaussian curve fitting for peaks was performed by DiscoverMP software (Refeyn). which identified peaks centered at 36 to 37 and 56 to 59 kDa for monomers and dimers, respectively.WT, hPI31V6R, and hPI31152–271 proteins were dialyzed against 20 mM Hepes (pH 7.8), 20 mM NaCl to avoid interference of Tris buffers in cross-linking. Protein concentrations were determined by Bradford assay, and samples were diluted to 23 μM based on molecular weights of 31, 31, and 13 kDa for WT, V6R, and 152 to 271, respectively. Glutaraldehyde or a buffer control was added to a final concentration of 0.05% and incubated for 15 min on ice. Cross-linking was terminated by the addition of 10 mM Tris-HCl, and samples were denatured in SDS sample buffer. Proteins were then separated by 12% SDS-PAGE and blotted against PI31 as described above.Purified hPI312, 100 mM KCl, and 1 mM DTT. The sample concentration was adjusted to 3 mg/ml for EM grid preparation. Cryo-EM grids were prepared in Vitrobot Mark IV (FEI) with the chamber set to 5 °C and 100% humidity. We first glow-discharged Quantifoil R 1.2/1.3 300 mesh gold holey carbon grids with O2-air for 30 s at 30 W in a Gatan Model 950 Advanced Plasma System, applied a droplet of 3 μl sample to the EM grid, waited for 1 s, blotted the grid for 4 s using a piece of 595 filter paper with a blotting force set to 3, then vitrified the sample by plunging the grid into liquid ethane precooled with liquid nitrogen. The cryo-EM dataset was recorded in a Titan Krios (FEI) equipped with a Quantum 967 energy Filter plus a post GIF K3 summit direct electron detector (A.K.A. BioQuantum). Movies were recorded using SerialEM software in super-resolution counting mode with a defocus range of −1.3 to −1.8 μm and a pixel size of 0.414 Å/pixel. The dose rate was 46 electrons/Å2/s with 1.3-s exposure time for a total dose of 60 electrons/Å2. A total of 19,264 movies were collected during a 2-day session.Cryo-EM was conducted at the Cryo-EM facility of Van Andel Institute. To obtain the human PI31–bovine proteasome complex, we mixed purified hPI31 and 20S at a molar ratio of 5:1 and incubated the mixture at 37 °C for 1 h in 20 mM Tris, pH 7.5, 5 mM MgClab initio 3D reconstruction, a total of 1,467,640 particles were selected for 3D multireference heterogenous refinement. The 3D class with extra density inside the catalytic β-ring of the 20S proteasome was chosen for further 3D heterogenous refinement using the classified maps with and without extra density as the references. The particles belonging to the two 3D classes (with or without extra density) were segregated and refined separately, first by homogeneous refinement and followed by nonuniform refinement  as the initial model. The PI31 structure was built manually in Coot after completion of the 20S proteasome modeling (Both 20S alone and the PI31–20S complex were modeled using the coordinates of bovine 20S proteasome (Protein Data Bank ID modeling . Real spmodeling .The structures of the 20S proteasome and the 20S proteasome–PI31 complex were deposited in the Protein Data Base with accession codes 8FZ5 and 8FZ6, respectively. Any data not in the article can be shared upon request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


This study demonstrates a new method for quantifying thread engagement in mechanical connections and verifies its applicability using biomedical implants under push-out tests. The focus is on orthopedic plate implants employed for bone fracture fixation, which, by design, allow off-axis screw insertion to accommodate different contact conditions. Thread engagement is crucial in determining connection strength and stability. In medical practice, off-axis screw placement is usually necessary due to bone geometries and implant plate rigidity. To address this, the study proposes a quantification method using non-destructive testing with X-ray micro-computed tomography and automated image processing, although tuning the image processing parameters is vital for accurate and reliable results. This enables detailed 3D models of screw-plate interfaces for precise thread engagement measurement. The results show that thread engagement decreases with both, increased off-axis insertion angles and higher torque during insertion. Correlation analysis reveals a strong relationship (R The purpose of this study is to explore the capabilities of the new method that enables the quantification of thread engagement in mechanical connections and to determine correlations between the actual screw-plate thread engement and strength of that interface in biomedical implants under destructive single-cycle-to-failure push-out test. The method has been primarily developed for orthopedic plate implants which are used for bone fracture fixation and allow off-axis screw insertion resulting in achieving different contact conditions. The development of angle-stable locking screw fixation was one of the most significant advances in bone plate osteosynthesis of fractures ,2,3. TheIn engineering, thread engagement is a crucial metric while selecting fasteners, as it directly affects the strength and stability of the connection ,9. WhileThe quantification of thread engagement in screw-plate interface can be performed using non-destructive testing that involves X-ray micro-computed tomography. Kaczmarek et al. developed a method which used automated image processing of 2D scans which were derived from 3D scanned model as captured by metrological micro-CT scanner. They applied that technique to determine differences in average thread engagement in polyaxial and monoaxial veterinary locking plates which were conducted to a static and cyclic loading . AccordiX-ray micro-computed tomography is a technique that is widely used in the investigation of bone-implant-related biomechanical problems ,13. The Apart from applying 2D or 3D bone models in the numerical simulations, microCT has been also used in analyzing the osseointegration as the untightening of the prosthetic screw is the most common problem in oral rehabilitation with implants. Batista et al. determined that the torque applied is crucial to the head deformation in titanium screws which fix the prostheses to the implant . In otheThe presented research addresses the problem of quantifying the thread engagement in screw-plate interface using micro-CT measurements and automated image analysis. The method was applied to study the effect of off-axis insertion on the average thread engagement. This measure was correlated with the push-out strength of the screw from the plate. This paper should contribute to a better understanding of the locking process in orthopaedic implants. The subject of our study is polyaxial locking system plate (PLS) . Both plate and screw were made of 316 stainless steel. The locking screws have a 3.5-mm thread and a 2.6-mm core diameter. This locking plate system allows abaxial locking screw insertion in various non-perpendicular directions. The manufacturer recommends a maximum deviation angle away from the perpendicular of 15 degrees. To achieve locked fixation, the screw head engages into five threaded contact regions inside the plate hole depending on the insertion angle.n = 4. In this paper we used the same dataset that was originally published in authors’ previous study [To determine the effect of different off-axis insertion of the screw, which hypothetically can lead to differences in thread engagement, the screws were locked at 0, 5, 10, 15 and 20 degrees in off-axis configurations . The addus study , which fTo quantify the amount of thread engagement we use the new possibilities offered by X-ray micro computed tomography which is a 3D imaging technique utilizing X-rays to measure the geometry of parts, slice by slice with high resolution. In this paper, metrological computed tomographic  data from measurement of screw head—plate hole interfaces 3.5 mm system PLS as described by Kaczmarek et al. . The scaThe procedure to calculate average thread engagement (ATE) is divided into 5 steps . In shorFor each analyzed image, it was assumed that there was a full engagement where there was no space between the screw and the plate. Each image received a score between 0% and 100% based on how much contact was determined by automatically examining the screw-plate interface region pixel by pixel. The average thread engagement (ATE) measure, which was calculated for all cross-sections, represents the arithmetic mean of the individual scores obtained for all analyzed images. The thread-to-thread contact regions of each screw head was visualized in a form of three-dimensional polar plot. The proposed method is used here to determine the relationship between ATE and the performance of the screw-plate interface using push-out strength. To evaluate the push-out strength of the locked screws in the two locking systems, each plate was firmly secured into a custom-made jig, and only the hole with the screw inserted was left accessible. The tip of the screw pointing toward the actuator was engaged by a concave surface. The push-out force was applied in the axial direction at a loading speed of 0.1 mm/s and was recorded at 10 Hz. Destructive static tests were performed using a servo-hydraulic testing machine  with a measuring range of up to 100 kN and fitted with a mechanical drive. The tests were performed under displacement control. p-value of <0.05.Data are reported as mean ± standard deviation. Shapiro–Wilk tests were performed to test for the normal distribution of residuals. For the studied insertion torques and angles two-way ANOVA was performed to determine whether these factors affected the average thread engagement. Post hoc Bonferroni tests were conducted to determine whether there were any statistically significant differences between the groups. Regression analysis was performed independently for each insertion torque. The coefficient of determination was calculated to indicate the strength of the correlation between the ATE and push-out strength. Statistical analyses were performed using Mathematica 12 software . Statistical significance was set at a p < 0.001). Image processing parameter—threshold is also influencing the ATE (p < 0.001). The combinations between torque, angle and threshold were not found to be significant factors (p > 0.005). The results are presented in Statistical analysis revealed that locking torque and angle are relevant parameters affecting ATE (2 > 0.6) between those two quantities can be seen for all analyzed threshold values. The highest coefficient of determination can be seen for threshold equal to 0.3 and 1.5 Nm torque (R2 > 0.817). The highest threshold produced least correlated results for the lowest torque. For torque equal to 2.5 Nm the strongest correlations can be observed at the lowest threshold (R2 > 0.809). The medium considered value of threshold equal to 0.3 appeared to be most convenient in terms of finding the strongest relations. The relations between push-out strength and ATE were presented in Forces acting on the fracture after its fixation during ambulation are a combination of bending, axial compression, torsion, and shear forces. From a clinical point of view, the push-out forces achieved in this experiment are likely to be sufficient to maintain the screw-plate connection until the bone has healed. However, the exact values of the force acting on the locking plate system in vivo have not been determined yet. Additionally, is still not known how much force is delivered to a single screw during ambulation and how much connection is needed to retain a stable fixation between the screw head and the plate hole. Another point worth mentioning is that a minimal number of locking screws per fracture segment should be two screws which fully penetrates the bone on both ends. As a result, the stress and strain will be delivered over the screws and the plate accordingly to its location and the working length. -a shorter screw can be used,-a plate hole can be left empty,-a cortical screw can be used,-a polyaxial locking plate can be used instead of a fixed-angle locking plate,-intentionally use a fixed angle locking screw in an off-axis position.Placing the locking screw in an off-axis fashion is a very tempting option especially when a juxta-articular fracture is being treated. This is because the intraoperative bending of locking plates follows the curved contour of the metaphysis which can inadvertently direct fixed-angle locking screws into the articular surface. There are several solutions to this problem:Each of those options might reduce the strength of the plate construct in this region. Our study demonstrated that a small deviation in a screw insertion angle in a fixed-angle locking system leads to a significant drop in its holding strength and ATE and therefore should be avoided. Another point worth mentioning is an increase of push-out strength when the insertion torque has been set on a higher value. Although higher ATE was obtained when locking at 2.5 Nm, this practice also cannot always be recommended because it may theoretically lead to cold welding and difficulties in later extraction of the screw from the plate .From the engineering point of view, it was shown that ATE was affected by the insertion angle and it decreased together with off-axis angle regardless of the automated image processing threshold value selected. The actual percentage of thread engagement is difficult to measure directly as both threaded parts are locked in each other. The application of metrological microCT allowed the measurement of the thread interface of screw and plate when engaged. Following the automated image processing of the images derived from 3D representation, it was possible to determine the ATE directly in the locked state.In the medical literature related to the threaded components the main focus in given into the interface between screw and bone ,24,25,26The lack of other method which can experimentally estimate ATE in threaded connections does not provide any benchmark we can refer to when validating our results. However, a strong correlation found between push-out strength and ATE proves the pertinence of the proposed method. Although the results were affected by the threshold value, still similar relations between the two aforementioned quantities can be found. Note that the three analyzed threshold values were selected arbitrarily from range between 0  and 1 . This was conducted by visual inspection of the images being produced by the algorithm and for threshold lower than 0.25 or greater than 0.35 it was either a situation in which contact was detected in regions where it did not appear to exist or some information about the apparent engagement was lost. This research does not provide an answer to the question what the most convenient threshold is because it will depend on the particular case and the grayscale representation of the microCT scans. It is authors’ intention to draw the attention that tuning of the image processing parameters is an important aspect of any study involving similar algorithms. Proper selection of the parameters is always important in order to achieve high quality of the results. -ATE is affected by the insertion angle and locking torque. Thread engagement decreases with off-axis insertion angle and increases with applied torque,-push-out strength strongly correlates with ATE but the strengths of correlations are affected by the threshold, so careful adjustment of the image processing parameters is important.Overall, this research contributes to a better understanding of the mechanics involved in screw-plate interfaces for orthopedic implants. The proposed method enables the quantification of thread engagement, providing valuable insights into the locking process and implant performance. The main conclusions of this study focused on PLS can be summarized in the following points:The findings can inform surgical practices, enhancing the design and placement of orthopedic implants for improved patient outcomes. By shedding light on the mechanical behavior of threaded connections, this study addresses a critical aspect of biomedical engineering and offers valuable guidance for future implant developments.A potential continuation of this research can involve development of the FEM-based model of the interface considering actual geometry of screw and plate threaded and off-axis insertion. That model, if capable of providing the right push-out correlation to engagement, would allow to connect numerically any practical case with the bone stress state. This can also help to analyze other loading conditions: shearing, bending or torsion.


Vespa velutina nigrithorax is considered a proliferating threat to pollinators in Europe and Asia. While the impact of this species on managed honey bees is well-documented, effects upon other pollinator populations remain poorly understood. Nonetheless, dietary analyses indicate that the hornets consume a diversity of prey, fuelling concerns for at-risk taxa. Here, we quantify the impact of V. velutina upon standardised commercially-reared colonies of the European bumblebee, Bombus terrestris terrestris. Using a landscape-scale experimental design, we deploy colonies across a gradient of local V. velutina densities, utilising automated tracking to non-invasively observe bee and hornet behaviour, and quantify subsequent effects upon colony outcomes. Our results demonstrate that hornets frequently hunt at B. terrestris colonies, being preferentially attracted to those with high foraging traffic, and engaging in repeated—yet entirely unsuccessful—predation attempts at nest entrances. Notably however, we show that B. terrestris colony weights are negatively associated with local V. velutina densities, indicating potential indirect effects upon colony growth. Taken together, these findings provide the first empirical insight into impacts on bumblebees at the colony level, and inform future mitigation efforts for wild and managed pollinators.The invasive hornet  Vespa velutina on Bombus terrestris colonies is quantified along a gradient of hornet densities, revealing negative impacts upon B. terrestris colony growth.The influence of the invasive hornet  When considering agriculture, this reliance is further intensified to a relatively constrained group of insect taxa, with bees alone visiting >90% of leading crop types3. As such, reductions in pollinator health constitute a threat to both biodiversity and food security at the landscape-scale, with consequent efforts being made to conserve a variety of at-risk species5. Among these, wild bees are of specific concern, as they do not benefit from anthropogenic management6 and comparatively little data exists to monitor natural population changes8. While substantial efforts have been made to characterise the drivers of pollinator declines—with habitat loss emerging as a primary threat9—there remains significant uncertainty regarding a plethora of additional prospective factors, indicating the need for further research10.The maintenance of pollination services is a global concern, as current estimates indicate that ~87.5% of all terrestrial angiosperms rely upon animals for reproduction7. While caution is warranted, it is important to note that a deficiency of data is not tantamount to an absence of effect, and instead underlines the need for additional investigation11. This point is especially pertinent when considering that other drivers of pollinator decline with comparatively little quantitative evidence appear to benefit substantially from the ‘precautionary principle’, combined with anthropocentric bias12. Nevertheless, there is a growing body of evidence that invasive organisms exert profound, though often poorly-visible, impacts upon wild pollinators and pollination services15. As such, basic empirical data is urgently needed to better identify and mitigate the risks posed by these organisms.Interestingly, in assessments of such factors, the impact of invasive species is frequently ranked below other threats to pollinators, ostensibly due to a paucity of dataVespa velutina nigrithorax—colloquially known as the yellow-legged or Asian hornet—the first insect to be legislatively classified as an invasive alien species of concern in Europe16. This social vespid is native to southeast Asia, but has spread rapidly through parts of east Asia and Europe, following accidental introductions in or before 2003 and 2004 respectively19. The invasion front has expanded at a rate of up to 78 km per year in some regions20, facilitated by an adaptable life-history21, rapid colony population growth18, and the ability of mated queens to travel substantial distances in a single day22. Control efforts are further hampered by the difficulty of locating nests23, and high potential for additional human-mediated dispersal events24, as is the case with many other invasive eusocial insects28. Consequently, efforts to contain the spread of these hornets have had limited success21, while climatic niche modelling predicts their continued expansion into new regions30.An emerging global threat is that of the hornet V. velutina to impact insect pollinators is evident through their highly-visible and intense predation of the honey bee Apis mellifera21. While these hornets are natural predators of the congeneric Apis cerana—which has consequently evolved a suite of defensive behaviours31—A. mellifera constitutes an evolutionarily naïve prey species, and thus has few effective defence mechanisms, enabling the hornets to predate upon colonies with near-impunity33. As such, predation pressure can become so severe that foraging is markedly curtailed, leading to the eventual starvation of colonies, with reported mortality rates exceeding 30% in some regions21.The capacity of V. velutina also preys upon a diversity of wild pollinators35. Indeed, previous work indicates that both hornet predation and foraging activity exert direct effects on plant-pollinator communities36. This is concerning, as the broader impact of V. velutina upon the survival and reproduction of wild pollinators remains largely unknown21. Consequently, given the scale of damage wrought by other invasive Vespidae37, there is a pressing need for robust data to bridge current knowledge gaps.Although managed honey bees have occupied much of the research focus, preliminary dietary analyses provide strong evidence that Bombus. This is because their colony-based lifecycle has the potential to attract V. velutina in a similar manner to that of A. mellifera; namely by offering a dense aggregation of potential prey at colony entrances. Further, the substantially smaller colony sizes of Bombus spp38., coupled with ongoing wild population declines in Europe41, arguably place bumblebees at even greater risk than managed honey bees. Currently, the potential impacts of V. velutina upon bumblebee colonies remain largely unknown, however, the presence of Bombus in the hornets’ diet highlights the need for further inquiry34.One group that may be particularly vulnerable are eusocial bumblebees of the genus V. velutina upon colonies of the native European bumblebee Bombus terrestris terrestris. Utilising a landscape-scale experimental design, we established standardised B. terrestris colonies at field sites across Pontevedra, Spain—a region with substantial heterogeneity in local V. velutina densities. Colonies were fitted with automated camera monitoring systems . Additionally, counts of trapped hornets peaked in early-August, and exhibited a decreasing trend thereafter , however, site ID had a significant random effect . Notably, all other land cover variables had little predictive influence, and thus were excluded from analyses.Hornet counts showed substantial spatiotemporal variability across sites Fig. , being sAcross the course of the study, during set sampling periods, hornet activity was recorded using an external Dragon Touch Vision 1 camera (Dragon Touch), trained on each colony at a distance of 1 m Fig. . This enF1,200 = 4.285, P = 0.040) and had a higher frequency of foraging . In contrast however, hornet presence at colonies was inversely correlated with both temperature  and relative humidity  at the time of sampling.Hornets were regularly observed hovering near bumblebee colony entrances, and attempting to predate upon departing or returning foragers. Specifically, results demonstrated that hornets were more likely to be present at colonies that were heavier . Instead, predation attempt frequency correlated with the amount of time that hornets spent foraging near colonies , and was again inversely related to temperature  and relative humidity  at the time of sampling.Notably, despite a total of 125 recorded predation attempts during video sampling spanning a period of 27 days, hornets had a 100% failure rate when attempting to prey upon bumblebees. The frequency of these attempts was not influenced by the weight ratio of individual hornets to bumblebees  positioned directly above the entrance of each colony Fig. . During F1,8 = 6.182, P = 0.036), peaking in mid-August , and colony ID had a significant random effect .The frequency of worker departures from colonies increased with the percentage of heterogeneous agriculture at sites .In a similar fashion, the frequency of worker returns to colonies decreased with relative humidity at the time of sampling , higher minimum relative humidity , and higher minimum temperature , increased the likelihood that colonies would survive. Colony ID also had a significant random effect when considering survival outcomes .Hornet counts at sites had no significant influence on bumblebee colony survival , while being positively influenced by higher minimum temperatures . When considering these interactions, colony ID had a significant random effect , indicating a degree of unexplained inter-colony variability, as would be expected.In contrast, colony weight over time was negatively correlated with hornet counts , while a higher percentage of heterogeneous agriculture  and water bodies at sites  increased the likelihood of new queens being present, independent of site ID .Hornet counts had no significant effect on the production of new queens by colonies . This relationship was independent of hornet counts , and the percentage of heterogeneous agriculture at sites , with site ID having a significant random effect . Similarly, the average weight of individual adult bumblebees in colonies was positively correlated with colony weight over time , yet unaffected by hornet counts , and the percentage of evergreen broadleaved and coniferous forests at sites . When considering average adult bee weight, site ID again had a significant random effect .Colony population size was positively correlated with colony weight across the study , each consisting of a queen and ~80 workers, and housed within waterproof polystyrene outer coatings (Biobest Group). Along with these, an additional three colonies were purchased for equipment testing and optimisation. All colonies were screened by the supplier for an extensive panel of pathogens and parasites using RT-qPCR, and the absence of infection was confirmed . To ensure standardisation, and facilitate controlled assessments, 36 coetaneous ed Table . Upon deed Table . FollowiVespa velutina nigrithorax densities and land cover types, allowing us to evaluate colony outcomes in a representative environment. To characterise relative hornet abundance and activity, we deployed two VespaCatch traps with VespaCatch attractant (Véto-pharma) at each site61, at a height of 1.5 m from the ground, and out of direct sunlight. Foam inserts were placed into all traps to absorb excess liquid attractant and prevent captured insects from drowning, thus limiting mortality and unwanted effects upon both native species and hornets62. Captured hornets were counted and released every second day for the two weeks preceding study initiation in late July and early August 2021. The resultant measure consisted of the total number of hornets caught in both traps at a site over two days—henceforth termed ‘hornet count’. This provided a profile of hornet activity over time, allowing us to select a final group of 12 field sites that best-encompassed the range of local hornet densities. Across the course of the study, we continued monitoring hornet counts at sites to ensure that our data remained temporally relevant, as activity can fluctuate substantially over time.Preceding colony setup, 15 prospective field sites were identified throughout the province of Pontevedra, Spain. These sites were selected to include a range of local ad libitum, while ensuring that water and carbohydrate availability were not limiting factors—although colonies were rarely observed utilising these. Colonies were situated in the shade amongst vegetation to avoid overheating, and in cases where sufficient natural shade was not present, were provided with shade in the form of pitched rooves of white Correx sheeting (Corplex UK). Experimental sampling was initiated on the 9th of August 2021, and continued for a period of 40 days, concluding on the 18th of September.Each site was provisioned with a water source and sucrose feeder, allowing the colonies to forage 2, and thus recording all bumblebees entering and exiting the colony during sampling  balance (RADWAG), checked for visible symptoms of disease, and their survival status was noted. To quantify individual behaviour, two Dragon Touch Vision 1 cameras (Dragon Touch) were trained on each colony. The first of these was an ‘entrance camera’ situated 110 mm above a customised transparent acrylic entrance tube, yielding a frame area of ~0.011 ming Fig. . The secing Fig. . Video rife Fig. . Spent c64 land cover dataset in ArcGIS Pro (release v. 3.0)  and relative humidity (%) measurements were made using Tinytag Plus 2 - TGP-4500 data loggers (Gemini Data Loggers Ltd). These recorded values at 15 min intervals, enabling the calculation of daily maxima and minima, along with ambient conditions at the time of video recordings. To characterise land cover, satellite data was extracted from a 1.5 km radius around each site, utilising the 2018 CORINE.0) Fig. . This prion Fig. .Further, from a single site per day, 10 bee and 10 hornet foragers were caught and weighed using HSW07 precision (linearity ±0.001 g) scales (Hoosiwee), thus comparing the weight ratio between species over time, to determine whether this influenced predation dynamics. Ratios were calculated using the following formula:Aphomia sociella) was recorded, along with those of the black soldier fly (Hermetia illucens).Upon conclusion of the study, colonies were destructively sampled with ethyl acetate to quantify the total adult population, mean weight of adult bumblebees (g), number of open and closed pupal cells, colony weight (g) and dimensions (mm), and the presence or absence of gynes, males, nectar, and pollen. Males were identified via extrusion of the endophallus, while gynes were differentiated by mapping their position relative to known queens in the overall weight (g) frequency distribution Fig. . AdditioVairimorpha ceranae, Vairimorpha apis, Vairimorpha bombi, Crithidia bombi, Crithidia mellificae, Lotmaria passim, and Apicystis bombi  AI-assisted tracking software to track individual bumblebees entering and leaving colonies through the observation entrances Fig. . The cuseos Fig. . When a eos Fig. . From th66, allowing observers to record a suite of prespecified events and behaviours urs Fig. . Specifiurs Fig. . Prior tMeasured variables were broadly grouped into those of hornet abundance and behaviour, colony growth, survival, and foraging activity, site climate, and land cover. Within these groups, we examined a combination of colony-level, individual-level, and site-level parameters.Hornet abundance and behaviour were assessed using hornet counts at sites over time, hornet presence at colonies, hornet duration at colonies, predation attempts, predation successes, predation failures, and worker weight ratio.Bumblebee colony growth and survival were evaluated using colony weight, colony survival status, total adult population, mean weight of adult bumblebees, number of open and closed pupal cells, and the presence or absence of gynes, males, wax moths, nectar, and pollen.The foraging activity of colonies was quantified using the frequency of bee departures, frequency of bee returns, and total bee foraging frequency.Local climatic conditions were measured using daily minimum and maximum temperature, daily minimum and maximum relative humidity, site temperature at the time of sampling, and site relative humidity at the time of sampling.Finally, site land cover was characterised by the percentage cover of nine land classes, including deciduous broadleaved forest, discontinuous urban fabric, evergreen broadleaved and coniferous forest, heterogeneous agriculture, natural grasslands, moors and heathland, transitional woodland shrub, vineyards, and water bodies  with repeated measures. Separate models were generated for each response variable, to maximise sample sizes while minimising model complexity. Hornet counts and colony assessments were repeated across all 20 time points, while automated tracking videos and semi-automated observation videos were recorded for 13 and 6 evenly dispersed time points, respectively. Model selection was based on AIC, beginning with the full model and interactions, and degrees of freedom were calculated using the Welch–Satterthwaite approximation. Model fit was validated via evaluation of the binned standardised or Pearson residuals.71.Inter-rater reliability in BORIS analyses was assessed using Cohen’s kappa coefficient, requiring a minimum concordance (κ) of 0.70 between observers at a resolution of 1 s. Across tests, we confirmed requisite sample sizes to provide a minimum power (1−β) of 0.80, at an alpha (α) of 0.05, using standard deviation (σ) and mean difference (δ) values from the data. Statistical analyses were performed in SPSS (release v. 28.0.1.1) and R (release v. 4.2.1)The GLMM assessing the determinants of hornet counts at sites used hornet count as a Poisson response variable with a log link, minimum temperature, minimum relative humidity, date, percentage of discontinuous urban fabric, and percentage of water bodies as fixed factor predictors, and site ID as a random factor.The GLMM assessing factors affecting hornet presence at colonies used hornet presence at colonies as a binomial response variable with a probit link, colony weight, total bee foraging frequency, site temperature at the time of sampling, and site relative humidity at the time of sampling as fixed factor predictors, and colony ID nested within site ID as random factors. The GLMM assessing factors influencing the frequency of hornet predation attempts used predation attempts as a Poisson response variable with a log link, hornet count, hornet duration at colonies, worker weight ratio, site temperature at the time of sampling, and site relative humidity at the time of sampling as fixed factor predictors, and colony ID nested within site ID as random factors.The GLMM assessing the effect of hornet counts on colony survival used colony survival status as a binomial response variable with a probit link, hornet count, minimum temperature, minimum relative humidity, and percentage of heterogeneous agriculture as fixed factor predictors, and colony ID nested with site ID as random factors. The GLMM assessing the effect of hornet counts on colony weight used colony weight as a gamma response variable with a log link, hornet count, maximum temperature, and minimum temperature as fixed factor predictors, and colony ID nested within site ID as random factors with a first-order autoregressive covariance structure to account for response autocorrelation.The GLMM assessing the effect of hornet counts on colony queen production used the presence of new queens as a binomial response variable with a logit link, hornet count, percentage of heterogeneous agriculture, and percentage of water bodies as fixed factor predictors, and site ID as a random factor. The GLMM assessing the effect of hornet counts on colony population size used the total number of adults in colonies as a negative binomial response variable with a log link, hornet count, colony weight, and percentage of heterogeneous agriculture as fixed factor predictors, and site ID as a random factor. The GLMM assessing the effect of hornet counts on adult bee weight used adult weight as a normal response variable with an identity link, hornet count, colony weight, and percentage of evergreen broadleaved and coniferous forest as fixed factor predictors, and site ID as a random factor.The GLMM assessing the effect of hornet presence at colonies on the frequency of worker departures used the frequency of bee departures as a normal response variable with an identity link, hornet presence at colonies, site relative humidity at the time of sampling, percentage of heterogeneous agriculture, and percentage of discontinuous urban fabric as fixed factor predictors, and colony ID nested within site ID as random factors. The GLMM assessing the effect of hornet presence at colonies on the frequency of worker returns used the frequency of bee returns as a gamma response variable with a log link, hornet presence at colonies and site relative humidity at the time of sampling as fixed factor predictors, and colony ID nested within site ID as random factors.B. terrestris colonies, assigned to field sites in triplicate, across a total of 12 sites. Foraging analyses utilised a sample of 135,983 individual trajectories, and weight ratio assessments were based on data from 700 individuals. Pathogen analyses were conducted on a subset of 10 colonies, each yielding a sample of 20 individuals, and final colony assessments were performed on all 36 colonies, incorporating 940 individuals. The source data underlying all figures and analyses are available within the supplementary data. Full details of statistical tests, subset sample sizes, and variable selection procedures are provided in the results and statistical analyses sections.Analyses employed a sample population of 36 Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary DataSupplementary Video 1Supplementary Video 2Reporting Summary


Duckweeds, a family of floating aquatic plants, are ideal model plants for laboratory experiments because they are small, easy to cultivate, and reproduce quickly. Duckweed cultivation, for the purposes of scientific research, requires that lineages are maintained as continuous populations of asexually propagating fronds, so research teams need to develop optimized cultivation conditions and coordinate maintenance tasks for duckweed stocks. Additionally, computational image analysis is proving to be a powerful duckweed research tool, but researchers lack software tools to assist with data collection and storage in a way that can feed into scripted data analysis. We set out to support these processes using a laboratory management software called Aquarium, an open-source application developed to manage laboratory inventory and plan experiments. We developed a suite of duckweed cultivation and experimentation operation types in Aquarium, which we then integrated with novel data analysis scripts. We then demonstrated the efficacy of our system with a series of image-based growth assays, and explored how our framework could be used to develop optimized cultivation protocols. We discuss the unexpected advantages and the limitations of this approach, suggesting areas for future software tool development. In its current state, our approach helps to bridge the gap between laboratory implementation and data analytical software for duckweed biologists and builds a foundation for future development of end-to-end computational tools in plant science.The online version contains supplementary material available at 10.1186/s13007-023-01065-3. Scientific interest in the diminutive freshwater plants of the Lemnaceae family has grown in recent years . DuckweeControlled laboratory growth assays have proven to be powerful tools to investigate duckweed physiology and evolutionary biology , 9, as wSince duckweeds rarely flower or set seed , each laPlant scientists have increasingly been designing, sharing, and making use of sophisticated, often automated, analysis tools to process large datasets composed of images of plants and using these to identify, characterize and compare phenotypes of interest . CompareWe set out to develop software tools to support duckweed research workflows that involve maintenance of genotypes and quantification of growth rates using time-series images. To do this, we built off of the existing Aquarium open-source laboratory management system previously developed in our lab to support synthetic biology workflows . AquariuThe Aquarium protocol scripts and our novel data analysis scripts combined provide solutions to two specific problems for duckweed laboratory researchers, the first of which is managing the maintenance of multiple separate lineages of asexually reproducing organisms for which long-term cold storage protocols are not available. Researchers can use Aquarium to keep track of the history and cultivation requirements of their duckweed stocks to ensure experimental consistency. Our open-source software also addresses the problem of managing datasets, including metadata, for experiments that involve the collection of large numbers of image files. Our scripts demonstrate how Python can interface with Aquarium to easily analyze large data sets of images.Lemna minor [Spirodela polyrhiza [Digital infrastructure for image processing requires physical infrastructure for sample handling and image collection, and there has been a corresponding interest in developing and disseminating automated imaging systems –28. Sevena minor , 13 and olyrhiza . While tWe addressed both of these challenges—managing duckweed stocks and large datasets—by developing and testing a suite of Aquarium types, the core database objects of Aquarium, including inventory types and workflow elements (operation types—OTs). Inventory types include sample types, which are unique categories of biological entity (e.g. a duckweed genotype) and containers, which are types of items that can be manifestations of a biological entity (e.g. dish of duckweed). OTs contain many elements, including a definition of the input and output sample types and containers as well as a protocol script written in Krill, a domain specific language derived from Ruby on Rails. We developed a suite of Aquarium OTs to provide a modular framework from which a range of different duckweed cultivation and research workflows can be constructed. Since the Aquarium system has been previously described, we focus here on the novel software written to support duckweed work, the guidelines we have developed for implementation, and examples from real world tests conducted in our lab.https://github.com/mtscott321/duckweed_data_analysis). The novel aquarium code as well can be downloaded from the supplementary material  consist of a name, text description, specific inputs and outputs, and a protocol script written in Krill. An example of Krill code and how this renders for laboratory technicians are shown in Fig. Contamination is a significant concern for those maintaining laboratory duckweed stocks. Stocks are generally maintained in axenic culture . Sugar iwww.github.com/mtscott321/duckweed_data_analysis.Duckweed growth assays typically measure the increase in mass or frond area over time under defined conditions . IncreasS. polyrhiza 7498 plants in media supplemented with 0, 50, 100, 150 or 300 mM sodium chloride. We collected images every 2 days over 10 days as well as fresh weight at the start and end of the experiment. In addition, for this experiment we activated an option within the code of the ‘Harvest’ OT  was executed by a single researcher, other personnel may take care of certain tasks within the plan such as collecting image data or transferring duckweed stocks on a particular day. All that needs to be communicated is the Plan ID and the OT name and then all other required information such as item locations and protocol instructions are provided just-in-time by Aquarium and the system logs that the work has been completed as well as when and by whom. Additionally, data collection and data analysis were carried out by separate individuals, using the Trident API see Fig.  to pull . This included the development of the ‘frond analysis’ option within “harvest and record fresh weight”  collected by growth chamber sensors with the Aquarium database so that each plant item has an associated set of growth condition data. Similarly, it would be useful to have an easy way to keep track of which machines were used within a particular workflow. A laboratory workflow management system that integrated with sensors throughout the laboratory could, for example, make it easier to troubleshoot when a protocol fails unexpectedly. Similarly, when collecting image data a human technician had to take photos using a camera and then upload the files to Aquarium, creating opportunities for incorrect data associations. A system that acts as a central hub for machines and sensors through the laboratory would better suit the needs of the modern research laboratory.Aquarium currently supports the definition of tasks and tracks basic data about when a task was carried out and by whom. Task management remains a significant challenge, particular for those wishing to use Aquarium to support cooperative, experimental work. Aquarium was primarily developed around the support of biofabrication workflows rather than experimental workflows, which tend to require planning out a specific series of tasks far in advance. When working with living organisms, the timings between work sessions may have limited flexibility. During work for this study, Aquarium was useful for defining the required tasks ahead of time, but without tools to schedule work ahead of time there was a greater risk of work not getting done at the right time or lack of clarity over who is responsible for what.https://github.com/mtscott321/duckweed_data_analysis. As computational tools and automation become more pervasive in scientific spaces, it is important that we develop the necessary tools to connect software to laboratory management. A crucial aspect of laboratory management software is its dynamism; the software needs to be highly adaptable to accommodate a variety of highly specific experimental designs. As such, this software is most powerful when it is shared open-source, where it can be continually developed and shared between researchers with similar needs. Laboratory management software has the potential to revolutionize the reliability and reproducibility of science done within and between labs, and we strongly encourage every plant scientist to consider integrating LIMS into their research.We developed an open-source framework for duckweed image analysis, consisting of the Aquarium code in the supplementary material accompanying this paper  petri dishes and containing 30 mL 1.5 g/L × Schenk and Hildebrandt media [Storage plates Fig.  were preImages of dishes of duckweed were collected at uniform distance using a Google Pixel 3a, then were uploaded as JPEG files to Aquarium. The image collection Aquarium protocol and the accompanying analysis script assume that duckweed are being cultivated in petri dishes or other circular, transparent containers, and that images consist of a single dish on a uniform background.“Frond dissection” began with random selection of three ramets from a given petri dish. The ramets were placed using tweezers onto a white plastic card, then fronds were manually separated by gently pulling apart using tweezers to rupture the stipe. Any roots were removed and discarded. Isolated fronds were photographed under the same conditions as the petri dishes, then uploaded to Aquarium.Data was accessed using the pydent API for Python  in Jupytwww.aquarium.bio. Aquarium is open-source, distributed under an MIT license and the code can be found on Github (https://github.com/aquariumbio/aquarium). The work presented in this study was performed using Aquarium release version 2 (latest version as of March 2022—Version 2.7.3). Aquarium is accessed via a web-app with a server accessible to all members of a research group provided with the URL as well as a valid username and password. The Aquarium server used in this study is available to members of the Klavins lab and users of the UW BIOFAB (http://www.uwbiofab.org/) and hosting is provided by Amazon Web Services.Aquarium was developed by researchers in the Klavins lab at the University of Washington and has been extensively described elsewhere . Detailehttps://pypi.org/project/pydent/). Trident documentation and installation instructions can be found on Github (https://aquariumbio.github.io/trident/). While dockerized installation can be used to explore the graphical user interface of Aquarium, for use to support research workflows Aquarium must be hosted and accessed via a server. This implementation is, based on our experience, non-trivial and requires a significant time commitment. We recommend that users be familiar with system administration before commencing with local implementation of Aquarium.Trident, the Python API for Aquarium, was used in this study to extract data from an Aquarium server. Trident is also available open-source under an MIT license and can be found at PyPI .Additional file 2: S2 - Aquarium Duckweed Workflow - README document.Additional file 3: Overview of Aquarium user experience.


Polyaxial locking plates rely on a specific thread-to-thread interface of the screw head and the plate hole. The objective of this study was to evaluate the mechanical performance of single screw interfaces when inserted off-axis and to establish correlations between those parameters and the engagement of the screw head and the plate hole thread. Three polyaxial locking screw systems were inserted into the corresponding plates at various angles . The screws were tested until failure. A micro-CT was performed to examine the interface between the plate hole and the screw head. The standard insertion at 0° sustained the greatest maximum bending strength without relocation in the screw hole. Screws inserted at 15° showed a significant reduction in force of up to 44%, 55% and 57%, respectively. Micro-CT analysis of the interface showed a significant loss of thread engagement for off-axis insertion. Polyaxial plates offer additional advantages for off-axis placement of screws. However, this flexibility is related to a significant decrease in both thread engagement and bending strength compared to monoaxial insertion. Regardless the insertion angle, the loss of stability is comparable when screws are placed off-axis. Surgeons are advised to consider off-axis insertion as a salvage option, providing access to better bone stock. Locking plate fixation is particularly beneficial in osteoporotic bones, yielding lower rates of implant dislocations and lower impairment rates to bone periosteum4. Unfortunately, the use of monoaxial plates requires a thorough surgical technique, preferably with the screw being locked in a perpendicular direction3. This requirement may result in positioning of the screw within poor bone stock or severely limit its length, especially in challenging periprosthetic fractures7. The application of monoaxial screws is also limited in periarticular fractures, where the requirement of inclining the screw is usually achieved with standard compression plates2. Clinical practice and biomechanical studies have demonstrated that only a precise insertion angle of the monoaxial screw can ensure proper fixation9. Kaab et al. stated that a deviation of more than 5° might lead to cross-threading of the screw head within the plate hole, significantly compromising stability8. As little as 5° to 10° off-axis insertion could reduce angular stability by 30% and 50%, respectively8.The advancement of monoaxial locking plates has substantially enhanced surgical techniques, guaranteeing stability at the reduced fracture site through the rigidity of the locking construct10. By now, a variety of polyaxial locking plates exists on the market11. Each manufacturer offers polyaxial plate systems with their own locking mechanism11. The hardware undergoes ASTM/ISO testing before reaching the market; however, manufacturers do not publicly disclose the exact stability of polyaxial locking interfaces12.Polyaxial designs theoretically guarantee a stable locking construct with off-axis screw positioning, potentially enabling surgeons to avoid osteoporotic areas, thus improving the purchase of the screw. Despite these substantial advantages over standard monoaxial constructs, several technical issues hindered its universal clinical use, as evidenced by observed clinical failures17. Former studies performed on cadavers and sawbones, comparing polyaxial systems and standard monaxial plates, showed no significant differences in strength. In the cited studies, the authors claimed that they did not adhere to any specific standard. Although it may be assumed that there is some resemblance to a standard, such details were not provided by the authors of that work. The general standard for plates is the American Society for Testing and Materials (ASTM), ASTM F382-17, and for angled devices, it is ASTM F384-1719.Furthermore, existing studies have predominantly evaluated the plates as complete constructs in fracture gap models20. However, to our best knowledge, none have comparatively tested the properties of locking interfaces of several variants within the same polyaxial plate system, using both destructive and non-destructive tests. Introducing a novel approach, microcomputed tomography (micro-CT) is a scientific method offering the establishment of overall connections and comprehensive examination of material microarchitecture21. It boasts advantages such as high resolution, effective visualization, and quantitative analysis of architecture related to the tested sample21. The three-dimensional reconstruction enables volumetric assessments and other more advanced measurements22.Only limited research focused on the mechanical properties of the screw head and plate hole interfaceThis study aimed to analyze the extent of thread engagement between the screw head and the locking plate hole in the polyaxial plate construct using a micro-CT. The second objective was to test the mechanical properties of the ChM manufacturer’s 4.0, 5.0 and 7.0 ChLP polyaxial locking systems with screws positioned at various angles from 0° to 15°. We hypothesized that the amount of engagement would exhibit a significant correlation with the mechanical parameters of the interface.24. The design allows for polyaxial screw insertion at an angle of up to 15° in all directions. The threads of the spherical screw head engage with the threaded part of the plate hole. In this study, the screws were inserted in three different configurations, using a pre-formed angulated block: at 0°, 5°, 10° and 15° (variance ± 1°) . The system consists of plates made of titanium alloy (ISO 5832-2/ASTM F67) and locking screws made of CoCrMo alloy (ISO 5832-12/ASTM F1537)26. Each construct was scanned using a metrological computed tomographic device . The X-ray beam power used for micro-CT scanning was 30.7 W (205 kV/150 μA), with a voxel size of 25.169 μm, and a per-image exposure time of 500 ms. Following the measurements, a 3D model of the threaded connection was generated, which was subsequently converted into a series of photos illustrating cross-sections taken every 20 μm orthogonal to the locking screw axis.Following the method developed by Kaczmarek et al. X-ray microcomputed tomography was utilized to measure the contact area between the threaded portions of the screw head and the plate hole27.In these 2D images, thread engagement manifested as a region devoid of recognizable space between the screw and the plate. The contact or gap was identified through automated image postprocessing. For each image, it was assumed that full engagement occurred where no space existed between the screw and the plate. Each image received a score between 0 and 100% based on how much contact was determined by automatically examining the screw-plate interface region pixel by pixel. The average thread engagement (ATE) measure, which was calculated for all cross-sections, represented the arithmetic mean of the individual scores obtained for all analyzed images. The thread-to-thread contact regions of each screw head was visualized in a form of a three-dimensional polar plot. The image processing algorithm was implemented in Mathematica software . More details about the method are described by Bartkowiak et al.19.A universal servohydraulic testing machine  was employed to determine the bending strength of the polyaxial plate constructs, with results reported in Nm. Each plate was securely positioned in a custom-made jig designed to prevent any movement or bending when subjected to a perpendicular compressive load Fig. . The loaStatistical analyses were performed using Mathematica 12 software . The data were reported as mean ± standard deviation, statistical significance was set at p < 0.05.One-way ANOVA was employed to assess the impact of the screw insertion method on both average thread engagement (ATE) and bending strength. The normality of the residuals was verified using the Shapiro–Wilk test. Post-hoc Tukey test was applied to determine whether there were any significant differences between the groups. Linear regression analysis was performed to determine the strengths of the correlation between ATE and bending strength.Micro-CT analysis revealed that the placement of screws at different angles affected the average thread engagement, with the initial contact area highest for screws inserted at 0° and lowest for samples aligned at 15° in all analyzed systems. Statistical examination with the ANOVA test demonstrated that the angle of insertion was a statistically significant factor affecting ATE  Table .Table 1TNevertheless, the post-hoc Tukey test revealed that, overall, screws inserted off-axis at angles ranging from 5° to 15° exhibited sufficient anchoring, with no statistically significant differences between each off-axis group (refer to Table The initial assessment involved a control group of 4.0 ChLP polyaxial screws inserted at 0°. The screws failed without relocation in the screw hole and rapid force loss, due to plastic deformation of the screw with macroscopic bending of the screw shaft. A stable construct could also be achieved at 5°, 10° and 15° angles, with all screws undergoing plastic deformation without a relocation in the screw hole. The 5.0 ChLP polyaxial screws inserted at 0° similarly exhibited failure marked by macroscopic bending of the screw shaft. Notably, the macroscopic failure of the screw head-plate hole interface was specific to the 5.0 ChLP system, resulting in identical failures for all screws inserted off-axis. This disfigurement was attributed to the plastic deformation of the titanium alloy used in the 5.0 ChLP plate.Similar to 4.0 and 5.0 ChLP systems, the 7.0 ChLP screws inserted at 0° experienced failure without relocation in the screw hole, with macroscopic breakage of the screw shaft observed in all cases. All 7.0 ChLP screws inserted off-axis failed at the screw-plate interface. Mechanical tests demonstrated that the most stable construct was achieved when screws were placed monoaxially, and its strength decreased when inserted off-axis (refer to Table 28. Kaab et al. described a substantial reduction in force of up to 68%, associated with improper insertion of monoaxial locking screws8. On the other hand, in the study conducted by Herbert-Davies et al., off-axis insertion of the monoaxial screw was associated with a reduction in load of up to 60%28.Polyaxial plates have been developed to assist surgeons during challenging fracture fixation. These systems aim to mitigate the negative effects of cold welding, a consequence observed in previous monoaxial systems when screws were placed off-axis31. Yet, several studies proved no statistically significant difference compared to traditional monoaxial fixations32. Investigations using synthetic bone constructs demonstrated similar stability between polyaxial and monoaxial plates30. However, composite models may not accurately replicate bone inhomogeneity, potentially affecting the performance of tested implants14. In contrast, other studies conducted on fracture gap models utilized different available cadaveric bones, including formalin-fixed and fresh frozen bone specimens with variable bone stock33. Although polyaxial locking screws offer advantageous properties, off-axis screw insertion comes with certain limitations10. Polyaxial interfaces, being non-standard threaded connections, rely heavily on the precision of screw insertion to maintain their strength10. Our study focuses on individual screw failure, diverging from the conventional approach of evaluating the entire construct. Few studies have investigated single polyaxial screw-plate interface28. However, caution is warranted when making direct comparisons of bending moments between different systems due to the varied range of performed tests, plate sizes, and the utilization of several types of locking mechanisms.Most studies focused on polyaxial plates conduct biomechanical tests utilizing fracture models or whole constructs with multiple screws inserted to mimic clinical standard clinical application26.In our study, we employed micro-CT to quantify thread-to-thread engagement, providing insights into the locking mechanism of polyaxial screws based on the varying insertion angles. Our data revealed that nearly 50% of thread-to-thread engagement was achievable when polyaxial screws were inserted perpendicularly to the plate. However, in the most critical scenario – a 15° off-axis placement of the screws – there was a noteworthy reduction of ATE, dropping to as little as 13%, 19% and 7% for the 4.0, 5.0 and 7.0 ChLP systems, respectively. The novelty of micro-CT analysis at screw-plate interface bridges a gap in the literature by expanding data to include the initial contact area between the threads. As of now, direct comparisons with other available systems used in human subjects are not possible. To our knowledge, micro-CT quantification methods have only been applied to veterinary in previous studies28. The maximal acceptable insertion of 15° was associated with 45% and 34% reduction of force, respectively28.The results of our non-destructive analysis aligned with the mechanical parameters of ChLP systems. The bending strength of all analyzed systems was affected by the insertion angle, revealing a significant reduction of 44%, 55% and 57% for the 4.0, 5.0 and 7.0 ChLP systems, respectively, when inserted at the maximum angle of 15°. A study of Herbert-Davies et al. found similar reductions in maximal bending force for Stryker VariAX 3.5 mm and the Peri-Loc 3, 5 mm, irrespective of the different locking mechanisms28. Our results differ from these findings, as a significant reduction of force was achieved beyond 10° of insertion for all analyzed systems. Thus the authors concluded that the actual safe zone is limited a 10° insertion of the screw28. Our post-hoc Tukey test revealed no significant differences in bending moment between each off-axis group of ChM manufacturer plates. Tidwell et al. demonstrated that off-axis insertion was almost linearly correlated with bending force loss10. However, those findings were limited to Synthes polyaxial plate10. Mehling et al. presented different conclusions, noting that the comparable cut-in design of polyaxial interfaces had the highest ultimate strength at 10° to 15° of screw insertion31. Meanwhile, the point-loading thread-in interface exhibited the highest strength at a maximum of 5° of screw insertion31. Hoffemeier et al. similarly found that the point-loading thread-in design had the highest bending strength at the neutral insertion of the polyaxial screws20.In contrast, Zimmer NCB plates showed enhanced maximal bending forces with off-axis insertion, albeit with large standard deviations. However, the initial engagement of the threads and end cap surface contact between the screw head at 0° and 15° were not assessed34. Previous research proved that mechanical performance of different designs can vary significantly, depending on the angle of insertion11. Historically, the failure model of monoaxial implants typically resulted from the pull-out of the bone, due to poor bone purchase35. Hardware complications at the screw plate interface were rare35. However, in cases involving the lower extremity and prior to radiographic union, certain polyaxial mechanisms could produce a lower-strength construct due to screw disengagement in the plate-screw interface.Despite the considerable utility of polyaxial systems, particularly in the management of periprosthetic or periarticular fractures, there is a scarcity of studies on the mechanical performance of these plates36. Another potential limitation of this study is the lack of consideration for the influence of the coating technology used for the hardware37.This study has several limitations, attributed primarily to the ex vivo testing, which cannot reflect conditions experienced by the plate in reduced fracture site. Moreover, our analysis focuses on individual locking mechanisms rather than the entire construct. Additionally, we did not analyze bone-screw interface, which could transmit loading different than that encountered in mechanical testsIn terms of the impact of screw locking angles, the thread-in polyaxial mechanism exhibits comparable stability when screws are placed off-axis. However, there is a particular decrease of force that influences stability when compared to monoaxial placement. Therefore, angulations of the screws in these systems should be considered as an option rather than a routine practice.