Our results suggest that the balance between the outflow of polar water from the Arctic, and input of Atlantic water from the Irminger Current into the West Greenland Current is a key factor in controlling sea‐ice conditions, and both diatom phenology and production in northeastern Baffin Bay. Our proxy record notably shows that changes in sea‐surface conditions initially forced by Neoglacial cooling were dynamically amplified by the shift in the dominant phase of the Arctic Oscillation (AO) mode that occurred at c. 3,000 yr BP, and caused drastic changes in community composition and a decline in diatom production at the study site. In the future, with projected dominant‐positive AO conditions favored by Arctic warming, increased water column stratification may counteract the positive effect of a longer open‐water growth season and negatively impact diatom production.Climate warming is rapidly reshaping the Arctic cryosphere and ocean conditions, with consequences for sea ice and pelagic productivity patterns affecting the entire marine food web. To predict how ongoing changes will impact Arctic marine ecosystems, concerted effort from various disciplines is required. Here, we contribute multi‐decadal reconstructions of changes in diatom production and sea‐ice conditions in relation to Holocene climate and ocean conditions off northwest Greenland. Our multiproxy study includes diatoms, sea‐ice biomarkers (IP We present the first continuous and high‐resolution diatom record from northeastern Baffin Bay spanning the last c. 9,000 years. Our data show that the variability in sea ice and primary production on the northwest Greenland shelf is linked to modulations in millennial‐scale Arctic Oscillation. The phenology and diatom community structure is largely controlled by water‐column stratification and sea‐ice conditions. HCl was evaporated by heating the samples for 12 hr, TOC was then measured with a LECO C‐S 125 analyzer.15N is constant through time, the nitrogen isotopic (δ15N) signal in marine sediments reflects nitrate (NO3–) supply and use in the euphotic zone. Light nitrate (14NO3–) is preferentially used by phytoplankton, resulting in higher δ15N values when phytoplankton production is elevated. Alternatively, a change in the nitrate inventory of the water masses, linked to changes in nutrient supplies, stratification of the water column or ocean circulation can also alter the sedimentary δ15N signal. At constant primary production rates, higher input of nitrate to the surface water can result in lower δ15N values. Additionally, the organic matter TOC:TN  ratio and δ13C can be used to discriminate between terrestrial, sympagic, and marine sources of sedimentary organic matter. For isotopic measurements of carbon, samples were treated with HCl followed by two distilled water rinses and were oven‐dried prior to measurements with an IRMS (IsoPrime GV). Nitrogen content and isotopic ratios were measured on untreated freeze‐dried samples with an Elemental Analyzer  coupled with the IRMS  with sediment densities derived from computed tomography (CT) numbers  and Coscinodiscus spp. , whereas the “pack‐ice/drift‐ice assemblage” includes Actinocyclus curvatulus and Melosira arctica  analysis were processed according to the protocol described by Belt et al. . The seasonal sea‐ice biomarkers were analyzed at a 1 to 10‐cm sampling interval, which corresponds to an effective age resolution ranging from c. 12 to 165 years (mean: ~33 years).Sediment samples for sea‐ice biomarker  at 8.5 kyr BP, and then decrease until 8 kyr BP. δ15N values subsequently rise until 7 kyr BP, from which point they decrease progressively until 5 kyr BP , Thalassiosira nordenskioeldii (7%), and presence of Thalassiosira bulbosa (<2%). Around 7.2 kyr BP, an ephemeral peak in the abundance of A. curvatulus is observed, while the contribution of the marginal ice zone group decreases. Total diatom fluxes remain relatively low until 6 kyr BP and then progressively increase to reach maximal values at around 4.4 kyr BP. Increasing diatom fluxes are associated with an increase in the abundance of T. antarctica var. borealis (resting spore), which largely dominates the assemblages. After 4.4 kyr BP, a stepwise decrease in total diatom fluxes is observed. At the same time, the assemblages show a decrease in the relative abundances of T. antarctica var. borealis (resting spore) and a strong increase in the marginal ice zone taxa. At 3 kyr BP, an abrupt and pronounced decline in the total diatom fluxes is accompanied by a rapid shift to a new regime of diatom productivity: a sharp decline in the marginal ice zone assemblage concurs with a marked increase in the share of the “drift‐ice/pack‐ice” and “summer subsurface” taxa. Although still a dominant contributor to the assemblages, T. antarctica var. borealis (resting spores) shows lower relative abundances from 3 kyr BP. Relatively low diatom fluxes, with slight oscillations, prevail for the rest of the core record. However, fluxes were particularly low between 3 and 2 kyr BP. From 2 kyr BP, a progressive increase in Rhizosolenia spp. is observed, whereas the contribution of T. longissima, occupying the same ecological niche, decreases to moderate values. The share of both the “drift‐ice/pack‐ice” and “summer subsurface” assemblages remains high for the rest of the core.Changes in the diatom fluxes are accompanied by clear compositional shifts in the assemblages Figure . The bot Figures  and 6. T3.325 was detected throughout the entire record, attesting to the presence of a seasonal sea‐ice cover in the study area over the past 9.2 kyr BP. Fluxes of IP25 are moderately high at the bottom of the core, increase starting from 7.8 kyr BP, peak at around 7.4 kyr BP, and then progressively decrease from 6.6 until to 4.4 kyr BP . This interval notably shows a high contribution of the marginal ice zone group  additionally suggest cold, open‐water conditions during summer. The production of Chaetoceros spores generally represents the last stage of the diatom growth season and is initiated by nutrient exhaustion in the surface water , with low contributions of the marginal ice zone group . Additionally, while diatom production progressively increases, δ15N values decline from the beginning of this interval until they reach a plateau at 4.8 cal kyr BP. This signal can suggest increasing nitrate supplies to the surface water due to reduced stratification. A reduced stratification would be consistent with reduced meltwater input from the Greenland Ice Sheet following the early Holocene  decline to minimum values at the beginning of this interval. A steep decline in the contribution of the marginal ice zone group is shortly followed by a pronounced increase in the “drift‐ice/pack‐ice” and “summer subsurface” assemblages  are an important constituent of the subsurface chlorophyll maximum  during this interval, is further associated with relatively warm summer open‐water conditions . Since it is generally assumed that climate warming in the Arctic will cause an earlier vernal bloom and extend the open‐water growth season, it can be tempting to predict a future increase in primary production, similar to that observed between 6 and 4.4 cal kyr BP in our study area. During this interval, phytoplankton production (diatoms and dinoflagellates) appears to have been promoted by the enhanced entrainment of Atlantic water by the WGC and earlier sea‐ice breakup during a predominantly negative AO phase. However, since studies suggest that the Arctic warming and the Arctic Amplification (e.g., Manabe & Wetherald, 5Our multiproxy record supports the hypothesis of a pervasive effect of the dominant AO phase on sea‐ice conditions and diatom production on the northwest Greenland shelf. Most notably, we show that an important and rapid decline in primary production starting around 3 cal kyr BP was coeval with a shift from low to high AO polarity values.Extensive seasonal sea‐ice cover from 9.2 to 7.7 cal kyr BP associated with low to moderate diatom production during an interval of significant meltwater influence and deglacial opening of the Nares Strait gateway.Polar water outflow event centered at c. 6.6 cal kyr BP associated with increased HBI III fluxes and a moderately high diatom production.Progressive decline in seasonal sea‐ice concentrations from 6 cal kyr BP culminating with primary production optimum.Onset of the Neoglacial cooling at c. 4.4 cal kyr BP accompanied by progressively increasing sea‐ice concentrations and decreasing phytoplankton production.Major shift in diatom productivity at 3 cal kyr BP reflecting a major oceanographic reorganization presumably related to a swing from dominantly negative to positive AO. Resulting decrease in the Atlantic‐sourced influence of the WGC and increase in Arctic throughflows likely led to a shorter open‐water growth season and generally reduced primary production. These conditions were exacerbated from 2.3 to 2 cal kyr BP during an interval of record‐high positive AO.From 1.2 to 0.2 cal kyr BP, only slight oscillations in diatom production and generally extensive seasonal sea‐ice cover occurred during an interval of dominantly negative AO.Changes in sea ice and diatom productivity over the past 9,000 years on the northwest Greenland shelf can be summarized as follows:Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


Surface engineering promotes possibilities to develop sustainable solutions to icing challenges. Durable icephobic solutions are under high interest because the functionality of many surfaces can be limited both over time and in icing conditions. To solve this, one potential approach is to use thermally sprayed polymer or composite coatings with multifunctional properties as a novel surface design method. In thermal spraying, coating materials and structures can be tailored in order to achieve different surface properties, e.g., wetting performance, roughness and protection against several weathering and wearing conditions. These, in turn, are beneficial for excellent icephobic performance and surface durability. The icephobicity of several different surfaces are tested in our icing wind tunnel (IWiT). Here, mixed-glaze ice is accreted from supercooled water droplets and the ice adhesion is measured using a centrifugal adhesion tester (CAT). The present study focuses on the icephobicity of thermally sprayed coatings. In addition, surface-related properties are evaluated in order to illustrate the correlation between the icephobic performance and the surface properties of differently tailored thermally sprayed coatings as well as compared those to other coatings and surfaces. Thermal spraying is used in various application fields for the production of protective coatings. ln this technology, almost all materials, e.g., metals, metal alloys, ceramics, hard metals, composites and polymers, can be used as a coating material as well as a substrate or base materials. Thermal spraying consists of different spray techniques such as flame, arc, high-velocity flame, plasma and cold gas dynamic spray processes . The basThermal spray coating solutions are under development to tackle the icing issues. These inflict serious problems for various industrial operations such as offshore industry, transport and cargo, ship industry, renewable energy production and aviation ,9,10,11.It is important to find reliable solutions for these icing challenges. Active de-icing methods have been used to remove the ice, e.g., by using heaters, which can be produced with thermal spraying of metallic materials ,15. We hGenerally, polymer materials have high chemical and environmental resistance . EspeciaIn this study, we produce polymer coating by using thermal spraying and evaluate them based on their icephobic performance. Investigations are focusing on the icephobicity and wettability of thermally sprayed polymeric coatings and several reference materials and surfaces. In the icing tests, ice is accreted in an icing wind tunnel (IWiT) and ice adhesion measured with a centrifugal ice adhesion tester (CAT). One interesting focus point was the durable icephobic slippery liquid infused porous surfaces (SLIPS) designed and manufactured by using flame spraying (FS), utilizing polymeric materials, and further, impregnated with oil. These surfaces had low ice adhesions, which express their high icephobicity.Thermally sprayed icephobic surfaces were produced by using the flame spray (FS) process. Dense FS coatings were sprayed with relatively low gas flow rates in order to prevent overheating and burning of the powder with the flame. After spraying, a few post-heating passes with the flame were done without feeding powder to densify the structure and smoothen the surface. Commercially available and thermally sprayable polyethylene , PE mixed with fluoropolymer perfluoroethylene propylene , ultra-high molecular weight polyethylene , polyether ether ketone  and polypropylene  powders were used in these experiments. More information about flame spraying of PE, PE + FEB and UHMWPE coatings can be found from our previous study . FurtherOther materials and coatings were studied for comparison. Bulk metals were mirror-polished aluminum  and stainless steel (EN 1.4301/2B). Commercial paints tested included BladeRep , wind turbine paint , Nanomyte®  and NeverWet® . UltraEverDry® film  was representing a superhydrophobic surface. Bulk polymers were polyethylene , polypropylene , polytetrafluoroethylene  and ultra-high molecular weight polyethylene . Other references were PTFE tape , low-density polyethylene sheet  and SLIXY-plane and the results are presented as surface roughness, Sa, values.Structures of FS coatings were analyzed with an optical microscope  from the cross-sectional coating samples. Wetting behavior and water contact angle measurements were done with a drop shape analyzer . Static contact angle (CA), advancing contact angle (ACA) and receding contact angle (RCA), as well as contact angle hysteresis (CAH), were studied. The experiments were achieved by dispersing 5 µL water droplets of ultra-high purity water  onto surfaces. In addition, roll-off/sliding angles were performed for SLIPS by tilting the surfaces with a 10 µm water droplet. The roughness of the solid and dry surfaces was analyzed by an optical profilometry  using a 20× objective magnification, achieving a vertical resolution of 50 nm. The area of the measurements was 0.81 mm × 0.81 mm in the Icing tests were done at Tampere University (TAU). The mixed-glaze ice type was accreted in the icing wind tunnel (IWiT) and the ice adhesion strength was examined with a centrifugal ice adhesion tester (CAT) . Icing tm and contact area A is spun along a radial length r, which spins with a constant angular acceleration α of 300 rpm/s. From this equation, the ice adhesion strength (T) via shear stress is calculated at the time of detachment t.These test facilities are located in the cold climate room, where temperature can be set from room temperature to as low as −40 °C. All tested samples were 60 mm × 30 mm in size and the ice accreted area was 30 mm × 30 mm. Laboratory grade II+ water  was used for accreting the ice. The samples were let to cool down in the cold room prior to the ice accretion process. The most important parameters for the ice accumulation procedure for mixed-glaze ice used in this study were the ambient temperature of the cold room (−10 °C), the wind speed (25 m/s), and the supercooled water droplet size . In this study, the maximum water flow rate was 0.3 L/min. The accreted glaze ice refers to ice that has characteristics from both rime and glaze ice types, having more glaze-like features without icicles or runback ice. The mixed-glaze ice type forms a clean-cut rectangular structure. An example of the accreted ice is presented in Thermal spraying is a versatile coating production technique. Traditionally, it is used, e.g., for corrosion and wear protection, thermal conductivity and insulation ,31,32,33Flame-sprayed PE, PEEK and PP polymer coatings own dense coating structures as presented in The second coating design approach relies on the SLIPS strategy. A porous coating structure was produced by flame spraying, and afterward, lubricant was impregnated to the structure. Flame-sprayed porous PE coating impregnated with silicone oil has shown excellent icephobicity . Figure In this study, FS PP and PE coatings, as well as FS-SLIPS, were hydrophobic or very close to that, In addition to contact angle (CA) values, advancing (ACA) and receding (RCA) contact angle values, as well as contact angle hysteresis (CAH), indicate the wetting performance of the surfaces. CAH can be derived from the difference between ACA and RCA, demonstrating the overall droplet mobility. High CAH indicates differences between the ACA and RCA, which results from surface properties, such as local roughness variations, surface free energy, surface chemistry leading to altering droplet behavior. Flame-sprayed dense coatings have reasonable high contact angle hysteresis, but it can be reduced with FS-SLIPS surfaces. FS-SLIPS have low contact angle hysteresis and, therefore, water droplet mobility is high. This, in turn, is beneficial for slipperiness and the slippery properties of the surfaces.Wettability of the different surfaces and coatings is presented in Roughness of the surface has influenced the wettability ,38 togetIce adhesion of the surfaces can be measured with different techniques and this affects strongly the given values . TherefoIce adhesion is one of the indicators for icephobicity in such a way that low ice adhesion reflects good icephobicity and the ice is easily removed from the surface. If ice adhesion is high, then the ice is strongly adhered to the surface and icephobicity is low. We have used this definition for the ice adhesion strengths below 50 kPa, medium-low below 100 kPa, medium below 150 kPa and when high ice adhesion strengths are higher than 150 kPa. Ice adhesion is kept as extremely low when ice the value is below 10 kPa . Figure Based on our previous studies ,22,36 anContact angles (CA) showed only local wetting behavior of the surface and better indicator could be water contact angle hysteresis (CAH) measured with dynamic contact angle or sliding/roll-off contact angle measurements. This could indicate droplet mobility and can be related to icing as well with some materials and surfaces. The CAH of the surfaces is presented in Roughness can influence ice adhesion although it is not a dominant factor itself. For example, mechanical interlocking as the adhesion mechanism between ice and surface can have an influence on the ice adhesion of textured surfaces . ChemicaThermal spraying has shown its potential to produce multifunctional polymer coatings for icing conditions. Flame-sprayed polymer coatings had medium-low ice adhesions, indicating their good icephobicity. Furthermore, most FS coatings were hydrophobic, which, in turn, showed their potential for anti-wettability conditions. Structurally dense and well-adhered PE, PEEK and PP coatings were produced by using flame spraying. This is advantageous for environments where protection is needed. Flame spraying is the robust and fast coating manufacturing method, which is important in several industrial applications. Furthermore, the polymers used in this study are cheap materials, showing the potential of this processing-material combination for anti-icing and anti-wetting purposes.Furthermore, SLIPS are interesting options as icephobic solutions. The lowest ice adhesion values were achieved with traditional SLIPS as well as with FS-SLIPS as a novel surface engineering solution by combining flame spraying of porous structure with impregnation of the oil. This way, we can have low ice adhesion surfaces as SLIPS, together with a durable structure, as thermally sprayed coatings. FS-SLIPS are shown their potential to act as slippery and icephobic surfaces.Future work will focus on the widening of material selection for icephobic thermally sprayed coatings and testing of their durability under different environmental conditions with combining icephobicity. Laboratory scale icing testing is a good way to make the ranking of the surfaces, but application-related testing is needed for further development towards specific requirements in different conditions.


Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the “lock and key” mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors. In this equation, Io represents the physiological concentration of the inhibitor and kass is an approximation of the association rate constant. The stability time of reversible inhibitors is defined as the minimal time in which the EI  complex remains undissociated (t(s) = ln 2/kdiss), where kdiss represents the dissociation rate constant. This suggest that inhibitors were of physiological relevance when the delay time was below 1 s and, for reversible inhibitors, when the stability time was above 10 min [ass and Kdiss are measured are seldomly performed. To gain insight into the relative differences between various protease inhibitor interactions, we rely predominantly on their ratio, Ki. This link between biochemical principles of inhibition and physiology does not require an update, however, the determination of a number of new structures of cysteine protease inhibitors indicates that canonical mechanisms of inhibition should be updated [Previously, we reviewed cysteine protease protein inhibitors and their role in regulation of proteolysis . In the e 10 min . Emergen updated . Because updated ,12,13,14i for their interaction with target proteases to provide an experimental basis for their classification. Due to the differences in Ki values, the same inhibitor can belong to several types in respect to the protease they inhibit. For example, cystatins differentiate among exo- and endo-peptidases, and the inhibitory fragment of the p41 form of the invariant chain associated with the major histocompatibility class II molecule (the p41 fragment) can be, in respect to the target, emergency and buffer inhibitors, and also the delay type and pro-inhibitor (a detailed explanation and references are provided below). For our review of the mechanisms of inhibition of cysteine proteases, we selected crystal structures of diverse types of inhibitors, which either target large groups of related proteases, such as papain-like proteases and caspases, or others including calpastatin and securin, with unique mechanism(s) of inhibition.Cysteine proteases use the reactive site cysteine as the catalytic nucleophile and the histidine to perform peptide bond hydrolysis. In MEROPS , an onliPapain-like cysteine proteases are the largest family (C1 according to MEROPS) among the cysteine proteases, and likely the most studied. The subgroup of cysteine cathepsins is involved in a myriad of physiological functions from protein turnover to processing of antigens, hormones, and bone remodeling . MoreoveCystatins were the first discovered and are the best studied endogenous inhibitors of cysteine cathepsins . Their mPlasmodium spp., as demonstrated by the crystal structure of a falcipain 2 complex  from complex  class II-associated p41 invariant chain fragment. The p41 fragment is homologous to sequential repeats, called thryoglobulin type-1 domains, due to their numerous occurrences in thyrogobulin ,53. The Clitocybe nebularis and Macrolepiota procera, respectively, are primarily inhibiting papain-like cysteine proteases, although inhibition of legumain and serine proteases has also been observed [Clitocypins and macrocypins from mushroom species (basidiomycetes) observed . This probserved , PDB codobserved ). The crobserved ,70.Staphylococcus aureus, staphopains A and B, which are remotely related to cysteine cathepsins. Each staphostatin specifically inhibits its target staphopain. Staphostatins have a β-barrel fold, which is similar to that of lipopains and different from that of cystatins [Staphostatins A and B are endogenous inhibitors of the secreted cysteine proteases from ystatins . The cryystatins , PDB codystatins , PDB codystatins  further Serpins obtained their name from their ability to inhibit serine proteases, however, they are cross-class inhibitors that also inhibit cysteine proteases such as cathepsins, calpains, and caspases. With over 1500 representatives in Archaea, Prokarya, and Eukarya, in addition to a number of viruses, serpins are the largest and most widely dispersed family of peptidase inhibitors , and incAmong cathepsins, cathepsin L is specifically targeted with another cross-class serpin, hurpin (serpin B13) . AnotherEscherichia coli effector protein Nlef). Cell apoptosis is an important mechanism during embryogenesis and organism growth to make place for new cells and tissues. Later in life, apoptosis is required for removal of defected, infected, and malicious cells, and is crucial for organism survival. Caspases take a central role in the apoptosis initiation and execution phases. They are cysteine proteases belonging to their own C14 family (MEROPS: ). BecausXIAP belongs to the protein family of inhibitor of apoptosis proteins (IAPs) present in viruses ,88 and eXIAP has a short helical region the authors called a “hook” followed by a long linker, which runs along the active site cleft of the caspase and the terminal BIR2 domain with the “sinker” interacting with the S4 pocket.The “hook” and the “sinker” with the BIR2 domain attach to the caspase surface and stretch the linker. The linker runs along the active site cleft in the direction opposite to the substrate binding, thereby precluding its cleavage. The concept is reminiscent of cysteine cathepsin inhibition by their propeptides in their zymogen form , in partEscherichia coli effector protein Nlef inhibits caspases-4, -8, and -9 [Erwinia chrisantemi, which fills half of the active site with its N-terminus and thereby blocks access to substrates [Escherichia coli effector proteins causing delay and inhibition of apoptosis. Its role appears minor due to its low expression profile, however, when over expressed it can severely impact apoptosis [, and -9 ,117,118., and -9 , PDB cod, and -9 . The simbstrates . Interespoptosis .Baculovirus protein p35 is a broad-spectrum caspase inhibitor. It has a flexible reactive loop with the caspase recognition sequence DQMD ; PDB cod shows hoi does not apply. For simple comparison of inhibition rates of serpins, we advise the use the kinetic constant kass of the first step only.The cowpox virus CrmA structure was chosen to represent serpins as inhibitors of caspases. Its crystal structure was determined in the cleaved form d, with tPropeptides of papain-like cysteine proteases are in their essence inhibitors attached to the framework of the mature protease structure ,135. TheIts N-terminal domain is composed of a short peptide only, for which the reactive site attachment is strengthened by a disulfide formed between the reactive site C31 and propeptide C10P. In contrast, the propeptide of cathepsin L has the largest N-terminal domain, composed of 96 residues. The role of N-terminal domains appears to be the same; they anchor the propetide in the primed side of the structure to enable its stretched binding along the active site cleft to the enzyme’s N-terminus.Cystatin E and macrocypins, such as macrocypins 1 and 3, can, in addition to papain-like proteases, also inhibit legumain  and macrocypin 4, and even the serine protease trypsin . Among t2+-dependent multidomain cysteine proteases with the catalytic domain that shares some resemblance to the papain fold. Calpains are involved in a number of processes including cell migration, cell death, insulin secretion to synaptic function, and muscle homeostasis [Calpastatin is a highly selective inhibitor of calpains, which are Caeostasis ,144,145,eostasis , neuronaeostasis ,146, heaeostasis ,146, typeostasis ,146,147,eostasis ,146,147.The crystal structure of the complex between m-calpain and the first repeat of calpastatin truncated to the residues from 119 to 238 , PDB cod reveals However, the chain then turns away from the catalytic residues, forming a cross-over of the reactive site with the IKEGT sequence called “loop out”, colored red in Saccharomyces cerevisiae securin (S. cervisiae separase with an root mean square deviation (RMSD) of 3.1 Å and 8% sequence identities. Hence, folds are superimposable. In addition, upon visual inspection, a high similarity of the folds is clearly recognizable. However, a number of secondary structure elements do not share deviations within the 3 Å of RMSD. Moreover, separase substrate specificity differs from the specificity of caspases. In contrast to caspases, separase cleave substrates after an arginine at P1, which is, however, characteristic of metacaspases, and glutamate at P4 [Securin inhibits separase, a protease involved in separation of sister chromatids during chromosome segregation during somatic cell division at mitosis and meiosis ,160,161. securin d. Separate at P4 ,161,163.te at P4 . The strPlasmodium, staphostatin, chagasin, clitocypins and macrocypins, and the exclusion domain of cathepsin C [The brief analysis presented here suggests that the protein fold does not present a major obstacle for the evolution of a protease inhibitor. Numerous folds are adopted to inhibit proteases. Simple and small folds exist, such as cysteine cathepsins’ propeptide domains based on the α-helical folds, cystatins and p41 fragments using a β-sheet combined with an α-helix, as well as ICPs from hepsin C  using β-hepsin C , equistahepsin C , and calhepsin C ). In addhepsin C . In the hepsin C . More rehepsin C . From thOur survey of the structures of the majority of cysteine protease protein inhibitors in complexes with cysteine proteases shows that the diversity of inhibition mechanisms appears to be unlimited. It appears as if nature has found numerous means of successively overcome almost any starting fold. Thus, it is to be expected that the folds first observed in protein inhibitors, such as cystatins and serpins, may be used in proteins exhibiting other physiological roles and functions, such as monellin, a sweet-tasting protein with the cystatin fold (reviewed in ), or theTo provide insight, we revisited the review of Bode and Huber of the interactions of natural protein protease inhibitors . This seOverall, the division of proteases according to those with the S1 binding site shaped as a pocket, and those with the S1 binding site loosely formed at the surface, enables identification of the families of proteases that can be targeted with protein inhibitors that bind in the active site cleft in a substrate-like manner. Among the proteases inspected here, only papain-like cysteine proteases do not possess an S1 binding pocket. This explains why protein inhibitors apply combinations of loops or chain-loop constructs to occlude the reactive site, while binding into the active site cleft of their targets. However, when the concept of spanning the active site by a peptidyl chain is applied, as in the case of propeptides, the chain can only run in the direction opposite to the substrate binding.For inhibition of the families of proteases that possess S1 shaped as a specific binding pocket, there appear to be fewer restraints in the concept. It appears that the S1 binding pocket implies smaller dynamics of a peptidyl substrate, and thus inhibitors can implement a single chain that spans the active site and still bypass the catalytic site. Among these are solutions such as “loop out” in calpastatin d and spehttps://www.who.int/ictrp/en/; ClinicalTrials.gov: https://clinicaltrials.gov/ct2/results?cond=COVID-19). Of interest, however, Novartis developed a small molecule inhibitor, LCL-161, a second mitochondria-derived activator of caspase (SMAC) mimetic, which binds to XIAP and loosens the binding of XIAP to caspase-9, thereby promoting cell apoptosis [In the age of protein engineering, we would like to go beyond repurposing natural design. We would like to apply our knowledge and understanding to the design of protein inhibitors that regulate, mark, or block the activity of proteases in biological systems. We found no inhibitors in clinical trials or in use as a drug (WHO International Clinical Trials Registry Platform ICTRP: poptosis . LCL-161poptosis , and buipoptosis . Moreovepoptosis  and to gpoptosis ,176. Thepoptosis . Howeverpoptosis . One of poptosis . It appepoptosis , systema


Continued reports of infections with infectious bronchitis virus (IBV) variants have occurred since its first isolation in the 1930s. Currently, QX-like IBVs are the predominant circulating genotype around the world. Here, the pathogenicity of QX-like IBV strain SD was characterized in chickens at different ages of exposure to the virus, and the protection efficacy of available vaccine combinations against IBV was evaluated. The results revealed that QX-like IBV strain SD was severely pathogenic in chickens, causing respiratory, urinary and reproductive infections, irrespective of age, based on clinical observations, viral distribution in tissues and a ciliostasis study. Severe respiratory signs, tracheal cilia injury, nephritis and abnormal development of the oviduct and ovarian follicles were evident throughout the experiment. A challenge experiment demonstrated that the homologous QX vaccine showed superior protection efficacy compared with other available vaccines, confirming the importance of IBV vaccine seed homology against the circulating IBV strains. Our findings aid an understanding of the pathogenicity of QX-like IBVs that may help to further control the infection. Coronaviruses (CoVs) are enveloped viruses with a positive sense, single-stranded RNA genome that can be divided into four groups: α-CoVs, β-CoVs, γ-CoVs and δ-CoVs based on genetic and antigenic criteria . CoVs prInfectious bronchitis virus (IBV) belongs to the γ-CoVs that cause avian infectious bronchitis (IB), a highly contagious disease of chickens . IB occuAmong the numerous genotypes, QX-like IBVs have emerged and gradually evolved into the predominant circulating genotype worldwide since the 1990s , 23–26. In this study, the pathogenic characteristics of QX-like IBV strain SD in 2-, 3-, 8-, 18- and 22-week-old specific-pathogen-free (SPF) chickens were characterized. The efficacy of a combination of vaccines based on Massachusetts (Ma5 and H120) and variant (LDT3-A and QX-like SZ160) strains against QX-like IBV strain SD was also assessed. Our findings offer new insight into the pathogenicity of QX-like IBVs in chickens exposed at different ages, and may aid the development of novel infection control strategies.50) was determined as described [The QX-like IBV strain SD (GenBank accession no.: KY421673) was isolated from chicken flocks vaccinated with Mass-type IBV vaccines in 2013 that showed obvious respiratory symptoms and renal disease . The virescribed .All SPF embryonated chicken eggs and SPF chickens used in this study were purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co. Ltd. . The protocols and procedures used in these experiments, including the possibility of animal death without euthanasia, were specifically considered and approved by the Animal Welfare and Ethical Censor Committee at China Agricultural University .6.0 EID50 of IBV strain SD at the ages of 2-, 3-, 8-, 18- and 22-weeks old, respectively. In each virus-infected age, another 10 chickens were not challenged as controls (group 6).To evaluate the pathogenicity of QX-like IBV strain SD in chickens at different ages of exposure to the virus, a total of 162 SPF chickens were divided into six groups. Chickens in groups 1 (n = 30), 2 (n = 30), 3 (n = 22), 4 (n = 15) and 5 (n = 15) were inoculated via the oculo-nasal route with 200 µl of medium containing 10The birds were monitored daily for clinical signs. Each group was sampled at 5, 7, 9 and 14 days post-inoculation (dpi) to measure the ciliary activity in the trachea, and to observe the gross lesions in the larynx trachea, kidney, oviduct and ovarian follicle, and to detect the level of virus RNA in the trachea, kidney, bursa, proventriculus and oviduct by RT-PCR. Two birds from each group were randomly selected and humanly euthanized by cervical dislocation at each sampling date. The body weight of chickens from each group was measured at 0 and 14 dpi. Blood was also taken for antibody detection at 9–16 dpi, prior to euthanization.6.0 EID50/bird) at 21 days post vaccination (21 days of age). Birds D–G and I were challenged via the ocular-nasal route with the QX-like IBV strain SD (106.0 EID50/bird) at 14 days post vaccination after the booster vaccination (34 days of age). After 7 days post-challenge (dpc), three birds from each group were necropsied to observe gross lesions and tracheal samples were collected and examined by ciliostasis tests.Two commercial IBV vaccines belong to Mass serotype Ma5 and H120) and another two local variant vaccines of China belong to GI-19 genotype (LDT3-A and QX-like SZ160) were used in this study. The vaccines were inoculated at the manufacturer’s recommended dose. The experimental design for the immunization and challenge tests is shown in Table 20 and anTo evaluate tracheal ciliostasis, three sections of the upper, middle and lower part of the trachea  were analysed and the average ciliostasis score was calculated as previously described . A scoreThe total RNA of the tissues  was extracted using Trizol reagent  according to the manufacturer’s instructions. Reverse transcription was conducted at 37 °C for 1 h, with 3 μg of total RNA, 1 μL of random hexamer primers (500 μg/mL)  and 0.5 μL of M-MLV reverse transcriptase (200 U/μL) (Promega). PCR was performed at 94 °C for 3 min, followed by 30 cycles of denaturation , annealing  and polymerization , and the postpolymerization step was performed at 72 °C for 7 min. Amplified sequences were analysed by 1.2% agarose gel electrophoresis. PCR was performed using a pair of primers , which amplify and detect a 740-bp fragment of the S1 gene of IBV.To detect IBV antibodies, serum samples were tested with a commercial IBV enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions. Sera with sample/positive (S/P) ratios above the cut-off level of 0.20 (titre ≥ 834) were considered positive.Ciliary activity and antibody level were statistically evaluated by one-way or two-way ANOVA tests adjusted for post hoc analysis, followed by Bonferroni’s multiple comparison tests. For all tests, the following notations are used to indicate significant differences between groups: *p < 0.05; **p < 0.01; ***p < 0.001. All data were analysed using GraphPad Prism 6.01 .Chickens infected at different ages with QX-like IBV strain SD showed obvious clinical signs, whereas the chickens in the control group remained healthy throughout the study. The most prominent clinical signs were characterized by depression, sneezing, nasal discharge, tracheal rales and difficulty in breathing, and respiratory clinical signs were predominant in all of the inoculated groups Table . ClinicaSimilar gross lesions in the larynx, trachea, kidney, oviduct and ovarian follicle were observed in the different groups inoculated with QX-like IBV strain SD Table . These gTracheal ciliary activity was observed from all groups at 5, 7, 9 and 14 dpi. The severity of the ciliostasis in chickens at different ages of exposure to the QX-like IBV strain SD proved to be similar, sometimes leading to severe ciliostasis, compared with the control group . The highest inhibitory activity was observed between 5 and 9 dpi, while partial recovery of ciliary activity was observed at 14 dpi in the infected chickens, with the exception of group 3  Figure  A. In allThe clinical signs, gross lesions and mortality rates of all groups are summarized in Table The QX-like IBV strain SD caused 100% damage to the cilia in the non-vaccinated group at 7 dpc. The immune groups administered a single vaccination with vaccine Ma5 or LDT3-A that were also primed with Ma5 or LDT3-A and boosted with H120, also showed higher ciliostasis scores. However, the average ciliostasis scores in the groups immunized with a single QX vaccine or primed with QX vaccine and boosted with H120 or QX vaccine, were significantly lower compared with other groups and with the control group (p < 0.05 or 0.001) Figure  B.Vaccine immunization is one of the critical measures needed in the prevention of avian IB. It is difficult to control this disease because of the continuous emergence of new IBV variants, resulting in different genotypes, serotypes and pathotypes , 36. It Mycoplasma galliscepticum, Escherichia coli and avian influenza H9N2 virus, would be more likely to occur when the integrity of the respiratory mucosa is compromised [The respiratory pathogenicity of QX-like IBV strain SD induced in susceptible chickens was observed in all inoculation groups of different ages, and included sneezing, tracheal rales and difficulty breathing, which indicated injury to the respiratory system. This result was further confirmed by the ciliostasis scores, with QX-like IBV strain SD causing almost 100% damage to the cilia in all of the infected groups of different ages, between 5 and 9 dpi. It is believed that secondary infection by other pathogens, such as promised , 40.Despite the QX strain being initially isolated from a case of proventriculitis, similar viruses were recovered from cases associated with a drop in egg production and renal damage in subsequent studies , 41, 42.Serious consequences of IB outbreaks are obviously decreased egg shell quality and production in layers and breeders with reduced fertility, and severe, irreversible damage in the immature ovary and oviduct, especially in the early IBV infection of young pullets , 44. In A homologous strain can induce the best cross-protection against IBV infection of field strains , 35. TheIn conclusion, this study showed that QX-like IBV strains induce severe pathogenicity in chickens. The viruses are able to simultaneously infect the respiratory system, urinary system and reproductive system of chickens at different ages, which results in poor production performance. To combat QX-like IBV infections, it is critical to develop the appropriate matching vaccine strains and determine the most appropriate vaccination regime.


Through literature curation and public database integration, here we reported an updated database of phosphorylation sites (p-sites) in prokaryotes (dbPSP 2.0) that contains 19,296 experimentally identified p-sites in 8,586 proteins from 200 prokaryotic organisms, which belong to 12 phyla of two kingdoms, bacteria and archaea. To carefully annotate these phosphoproteins and p-sites, we integrated the knowledge from 88 publicly available resources that covers 9 aspects, namely, taxonomy annotation, genome annotation, function annotation, transcriptional regulation, sequence and structure information, family and domain annotation, interaction, orthologous information and biological pathway. In contrast to version 1.0 (~30 MB), dbPSP 2.0 contains ~9 GB of data, with a 300-fold increased volume. We anticipate that dbPSP 2.0 can serve as a useful data resource for further investigating phosphorylation events in prokaryotes. dbPSP 2.0 is free for all users to access at:  This reversibly dynamic process is precisely modulated by protein kinases (PKs) and protein phosphatases (PPs), which are involved in linking or removing a phosphate group at specific residues of protein substrates5. The first eukaryotic phosphoprotein was discovered in 1883 by Olof Hammarsten, a Swedish biochemist, who detected phosphorous in a secreted protein, casein, from milk6. Although later studies demonstrated that many proteins can be phosphorylated in eukaryotes, it was long debated whether protein phosphorylation also exists in prokaryotes until the discovery of isocitrate dehydrogenase in Escherichia coli, the first identified prokaryotic phosphoprotein, in 19798. In contrast with eukaryotic phosphorylation, which occurs mainly at specific serine (S), threonine (T) and tyrosine (Y) residues of proteins5, prokaryotic protein phosphorylation can occur at additional types of amino acids, such as arginine (R), aspartic acid (D), histidine (H) and cysteine (C)13. Given the importance of phosphorylation in the regulation of protein functions13, the identification of novel phosphorylation sites (p-sites) in proteins is fundamental for understanding the molecular mechanism and regulatory roles of prokaryotic phosphorylation.As one of the most well-characterized and important post-translational modifications (PTMs), protein phosphorylation plays an essential role in almost all signalling pathways and biological processes, from eukaryotes to prokaryotes14, in vitro kinase assay (IKA) to identify potential kinase-specific p-sites15, detection of p-sites in purified proteins with LTP mass spectrometry (LTP-MS)16, and N-terminal sequencing of phosphopeptides (NSP)17. The quality of p-sites identified in LTP studies is higher, because usually multiple assays were performed, and the biological functions of p-sites were also carefully analyzed. Recently, advances in the development of proteomic techniques using high-throughput MS (HTP-MS) have enabled the large-scale phosphoproteomic identification of p-sites in prokaryotic proteins21. For example, Macek et al. conducted phosphoproteomic profiling to detect 54 phosphoserine (pS), 16 phosphothreonine (pT) and 8 phosphotyrosine (pY) residues of 78 proteins in Bacillus subtilis, as well as 81 pS/pT/pY sites of 79 E. coli phosphoproteins19. For arginine phosphorylation, Elsholz et al. systematically identified 121 phosphoarginine (pR) residues in 87 B. subtilis proteins20, whereas Schmidt et al. later quantitatively characterized 134 phosphoproteins with 217 pR sites in B. subtilis21. More recently, Lai et al. detected 159 phosphohistidine (pH) and 69 phosphoaspartic acid (pD) sites of 197 phosphopeptides in nine prokaryotic organisms13. Because an increasing number of LTP and HTP p-site investigations have been reported, the collection, curation, integration and annotation of known phosphoproteins and p-sites in prokaryotes will provide invaluable information for better understanding the host-pathogen interaction and development of antimicrobial agents.Previously, experimental identification of p-sites with conventional biochemical assays was usually labour intensive, time consuming and expensive and was accomplished in a low-throughput (LTP) manner. The LTP methods mainly included site-directed mutagenesis (SDM) of candidate p-sites22. Compared with the second largest resource, the Phosphorylation Site Database, which curated approximately 1,400 prokaryotic p-sites23, dbPSP 1.0 had a > 4-fold greater data volume. At that time, few annotations were provided, except limited information on p-sites. Due to the large number of prokaryotic p-sites found in recent studies, here we created dbPSP 2.0, which contains 19,296 known p-sites in 8,586 proteins from 200 prokaryotic organisms, through literature curation and public database integration  1.0, which contained 7,391 experimentally identified p-sites, including 2,709 pS, 2,174 pT, 2,187 pY, 142 pR, 84 pD, 90 pH and 5 phosphocysteine (pC) sites, in 3,750 phosphoproteins of 96 prokaryotes23, UniProt24, dbPTM 201925, SysPTM 2.026 and PHOSIDA27, and our database contained a much higher number of known phosphoproteins and p-sites in prokaryotes  and annually causes 1.5 million deaths29. Due to the high virulence of M. tuberculosis, two related species including the slow-growing Mycobacterium bovis30 and the fast-growing Mycobacterium smegmatis30 were established as models to study mycobacterial physiology. Additionally, we analyzed the distribution of p-sites on different types of amino acid residues and found that pS, pT and pY sites appear more frequently than other types of residues and occupy proportions of 39.67%, 31.55% and 19.87%, respectively  (https://www.uniprot.org/proteomes/?query=taxonomy:330779). Then the proportion of phosphoproteins against all protein products were counted, and top 10 species with higher coverage values were shown. From the results, we found that the coverage values of the 10 prokaryotes ranged from 8.47% (Staphylococcus aureus) to 36.06%   Fig. . Previou24, whereas corresponding accession numbers were integrated from UniProt24, Ensembl33, EMBL34, KEGG35 and NCBI GenBank36. Moreover, functional descriptions, protein/nucleotide sequences, and keywords were derived from UniProt24 to provide the basic information for each phosphoprotein entry, while the primary references with PMIDs were provided for each p-site. The gene ontology (GO) annotations in the Gene Ontology resource37 were also included if available. Furthermore, the knowledge from 88 additional public resources, such as ChEMBL38, BacDive39, PDB40, IUPred2A41, InterPro42, BioGRID43, EggNOG 5.044 and Reactome45, was integrated to comprehensively annotate the prokaryotic phosphoproteins. These resources covered 9 aspects, namely, taxonomy annotation, genome annotation, function annotation, transcriptional regulation, sequence and structure information, family and domain annotation, interaction, orthologous information and biological pathway , class II (LP ≤ 0.75 and >0.5), class III (LP ≤ 0.5 and ≥0.25), and class IV (LP < 0.25), as previously described46. In most of these HTP studies, different reference databases, distinct search engines and/or diverse parameter configurations were adopted for phosphopeptide detection in different organisms. Thus, the aggregation of false positive identifications might result in a considerable higher FPR value in the cumulative dataset. A re-analysis of all raw MS datasets under a unified platform will generate phosphopeptides with much higher quality, although such an effort is not within the scope of dbPSP 2.0, which directly collected known p-sites from published literature.In phosphoproteomic studies, phosphopeptides were derived from mass spectrometry spectral datasets, usually with a false discovery rate (FDR) of 0.01 at the peptide-spectrum match (PSM), peptide and protein level for quality control. To pinpoint an exact p-site in a phosphopeptide, a localization probability (LP) score could be calculated by a variety of tools, such as MaxQuant47. For each orthologous group, all protein sequences were multi-aligned using MUSCLE48, and a conservation ratio was calculated for the sequences containing the same types of phosphorylatable residues against all sequences in the group. The distribution of the conservation ratio ranged from 0 to 1 was illustrated for all p-sites in the orthologous groups ups Fig. , and we ups Fig. . These hB. subtilis ClpP, an ATP-dependent Clp protease proteolytic subunit, as an example to introduce the usage of dbPSP 2.0. Two browse options, ‘Browse by phyla’ ’ to retrieve a list of phosphoproteins in a tabular format with ‘dbPSP ID’, ‘UniProt Accession’, ‘Gene Name’, ‘Protein Name’ and ‘Organism’  residues most frequently occur at the +1 position, with a lesser extent at the +2 position  in E. coli53. Additionally, Lin et al. utilized p-site information in dbPSP to analyze phosphoproteomic data and dissected the dynamic alteration of phosphorylation in various phosphoproteins during antibiotic treatment and resistance54. Moreover, Hasan et al. adopted pS and pT sites in dbPSP as training datasets and developed a useful tool, Microbial Phosphorylation Site predictor (MPSite), for predicting microbial p-sites55. In addition, the phosphorylation data of representative prokaryotes from dbPSP was utilized for kinase motif enrichment analysis, and the results demonstrated that most eukaryotic phosphorylation motifs could not be recovered in prokaryotes56.After the publication of dbPSP 1.0, it has been visited more than 180,000 times and has served as a highly useful resource for studying prokaryotic phosphorylationIn dbPSP 2.0, we collected and curated newly identified p-sites in prokaryotic phosphoproteins, which could present more complete information on phosphorylation in prokaryotes. Furthermore, dbPSP 2.0 has rich annotations for phosphoproteins and p-sites, which is critical for exploring the function and mechanism of phosphorylation events. In addition, the MSA results of orthologues were provided in this database and will be important for discovering conserved functional p-sites in prokaryote cells. Based on previous studies, dbPSP could work as a well-curated data resource of prokaryotic phosphoproteins to provide helpful support for phosphoproteomic analysis, tool development, and the investigation of prokaryotic phosphorylation events. We anticipate that the updated dbPSP 2.0 could be a comprehensive data resource for better understanding the importance of protein phosphorylation in prokaryotes.57. In 2015, we collected 7,391 known p-sites of 3,750 proteins in 96 prokaryotes from published literature and developed dbPSP 1.022 to contain these datasets. Due to the accumulation of phosphorylation information, here we released dbPSP 2.0 by adding 11,905 new entries to include newly discovered phosphoproteins and p-sites in prokaryotes. Furthermore, the rich annotations derived from 88 public databases were integrated. In total, dbPSP 2.0 contained 19,296 known p-sites in 8,586 phosphoproteins and occupied the size of ~9 GB, with a 300-fold increase compared to that of version 1.0.Protein phosphorylation is one of most well-studied PTMs and is reported to be involved in regulating numerous cellular processes in prokaryotic cells58. Inhibiting the function of ClpP was reported to affect the infectivity and virulence of microbial pathogens59. Moreover, the arginine phosphorylation of ClpP was essential for maintaining its function60. As shown in Fig. B. subtilis protease ClpP is annotated as a serine peptidase and participates in eliminating damaged proteins during heat shock, and its activity can be repressed by CtsR as well as by 20,697 compounds. Meanwhile, ClpP might interact with 9 partners and self-assemble in hexameric ring structures 24 24 Fig. . We alsoely Fig. . These d40 if available. A JavaScript molecular visualization library, 3Dmol.js61, was used to support the dynamic structure chart in the browser interface. In addition, the probabilities of disordered binding regions and disorder propensity values were predicted by using ANCHOR241 and IUPred241, respectively. The details are provided on the phosphoprotein page.The 3D structures of phosphoproteins for intuitive visualization were obtained from the PDBHTML, PHP and JavaScript were applied to develop the web interface as the front-end. The MySQL server was applied to manage the data as the back-end. The backlog and cache data will be cleared regularly, and the dbPSP database will be maintained and optimized continuously.Supplementary Table 1Supplementary Table 2Supplementary Table 3


The transiliac-transsacral screw placement is a clinical challenge for surgeons. This study explored a point-to-point coaxial guide apparatus assisting the transiliac-transsacral screw insertion and aimed to investigate the feasibility and accuracy of the guide apparatus in the treatment of posterior ring unstable pelvic fracture compared with a free-hand technique.A retrospective study was performed to evaluate patients treated with transiliac-transsacral screws assisted by the point-to-point coaxial guide apparatus or free-hand technique. The intraoperative data of operative time and radiation exposure times were recorded. Postoperative radiographs and CT scans were performed to scrutinize the accuracy of screws position. The quality of the postoperative fracture reduction was assessed according to Matta radiology criteria. The pelvic function was assessed according to the Majeed scoring criteria at 6 months postoperatively.P > 0.05). The average operative time of the guide apparatus group for each screw was significantly less than that in the free-hand group . The radiation exposure times were significantly lower in the guide apparatus group than that in the free-hand group . The intraosseous and juxtacortical rate of screw placement (100%) higher than in the free-hand group (94.4%).From July 2017 to December 2019, a total of 38 patients were included in this study, 20 from the point-to-point guide apparatus group and 18 from the free-hand group. There were no significant differences between the two groups in gender, age, injury causes, pelvic fracture type, screws level, and follow-up time (The point-to-point coaxial guide apparatus is feasible for assisting the transiliac-transsacral screw in the treatment of posterior unstable pelvic fractures. It has the advantages of simple operation, reasonable design and no need for expensive equipment, and provides an additional surgical strategy for the insertion of the transiliac-transsacral screw. The posterior ring unstable pelvic fracture is a common type of pelvic fracture in clinic which carries severe injuries . PercutaHowever, the transiliac-transsacral screw placement is a clinical challenge for surgeons. Its risk of extraosseous placement is theoretically increased and the operative time, the bleeding, infection, and anesthetic risk is potentially increased , 8. In aMoreover, the other intraoperative difficulty of transiliac-transsacral screw insertion is to find suitable intraosseous trajectories to avoid neural compression or vascular injury and keep the trajectory and direction of the guidewire undeflected during the insertion process. The screws are inserted from the injured hemipelvis’ ilium, through the sacral body and bilateral ala, and exit the contralateral iliac cortical bone, which results in a small available bone volume . It meanThe purpose of this study was to explore the feasibility of the point-to-point coaxial guide apparatus assisting the transiliac-transsacral screw insertion and to investigate whether the guide apparatus can improve the accuracy of transiliac-transsacral screws in the treatment of posterior ring unstable pelvic fracture.This study was designed as a retrospective study to evaluate patients treated with transiliac-transsacral screws assisted by the point-to-point coaxial guide apparatus or free-hand technique. Surgical placement of transiliac-transsacral screw was performed by the surgeon team in our department. The records of our hospital trauma database were retrospectively screened from July 2017 to December 2019 to identify all consecutive patients treated with posterior pelvic ring fractures. The clinical application of this technique and the retrospective review has been approved by the Ethics Committee of our university and conducted in accordance with the Helsinki Declaration of 1975 as revised in 2013. All patients have signed an informed consent form.Inclusion criteria were (i) unstable posterior pelvic ring injuries and (ii) treatment with transiliac-transsacral screws with the technique or free-hand, and (iii) a minimum follow-up of 6 months. Patients were excluded if (i) preexisting altered skin condition and/or infection at the surgical side, (ii) treatment with transiliac-transsacral screws by other apparatuses.Radiological assessment included pre- and postoperative radiographs  and CT scans. All pelvic injuries were categorized into the OTA/AO classification . PostopeThe intraoperative data of operative time and radiation exposure times were recorded. In this study, the operative time is from the incision to the safe placement of screw. The radiation exposure times refer to the number of C-arm fluoroscopy exposures to confirm the safety of the screw placement intraoperatively.The quality of the postoperative fracture reduction was assessed according to Matta radiology criteria: excellent (≤ 4 mm), good (5–10 mm), fair (11–20 mm), and poor (> 21 mm) . The pelThe point-to-point coaxial guide apparatus consists of two right-angled handles that can be disassembled or combined Fig. a, b. WheAn illustrative example follows with a 56-year-old man who was involved in a car accident with a complex pelvic ring injury consisting of a complete symphysis pubis disruption, fractures of superior and inferior ramus of right pubis, vertical and rotational displacement of the left hemipelvis, and left sacroiliac joint disruption Fig. . At surgFirst, a true lateral sacral view is the ideal starting image for the procedure to determine the safe zone and starting point of the guidewire of transiliac-transsacral screw Fig. d. We insNext, the point-to-point coaxial guide apparatus was installed and fixedly locked Fig. e. The twThe operation procedure was the same as traditional transiliac-transsacral screw insertion under conventional fluoroscopy with free-hand .t test was used for continuous data (presented as mean ± SD) including age, follow-up time, operation time, and radiation exposure times. The chi-square test and Fisher’s exact test was used for discrete data between two groups, including gender, age distribution, injury causes, OTA/AO classification, and screws level. The Wilcoxon rank sum test was used for the data of function outcomes and quality of reduction. P value less than 0.05 was considered as statistically significant.The data were analyzed using SPSS 20.0 software . The independent two-sample P > 0.05) (Table P > 0.05) . The radiation exposure times were significantly lower in the point-to-point guide apparatus group than that in the free-hand group    were placed in 38 patients: 20 screws  in point to point guide apparatus group and 18 screws  in free-hand group. The intraosseous and juxtacortical rate of screw placement in point-to-point guide apparatus group was 100% and higher than 94.4% in the free-hand group. No complications including nerve palsy and revisions were noted postoperatively. According to Majeed scores at 6 months and Matta radiology criteria, function outcomes and quality of reduction has no significant differences between the two groups. , that is, the dense area of anterior ilium located directly lateral to the sacroiliac joint, and the sacral alar anterior cortical anatomy. This relationship is paramount for identifying the safe zone and an appropriate starting point . On the The so-called safe zone, most restricted between the sacral ala and sacral neural tunnel, is the area within the sacrum where completely intraosseous transiliac-transsacral screws are ideally inserted , 18. SinIt has been reported that 3D fluoroscopic navigation enabled safer iliosacral screw placement . HoweverMedical robotic systems for percutaneous iliosacral screw placement have been developed . Wang etIn our study, accuracy of the point-to-point coaxial guide apparatus was superior to that of the free-hand technique. Our clinical experience with the described screw placement technique resulted in the intraosseous and juxtacortical rate of screw placement (100%) higher than in the free-hand group (94.4%). Also, the time required to place each screw and the radiation exposure times in the guide apparatus group was significantly better than in the free-hand group, and no complications were noted at the final follow-up. Although much time was spent in finding the exiting point, the duration of the operation and the number of fluoroscopy exposures were significantly reduced, and there are no expensive surgical instruments required because the manufacture of the guide apparatus is simpler and cheaper than a robot, CT machine, or 3D navigation . UltimatHowever, some limitations are inherent in this study. First, although the learning curve of this technique is small, it still requires surgeons to have rich surgical experience and correctly interpret intraoperative imaging to verify reduction and implant placement. Meanwhile, the fracture or dislocation of the sacroiliac joint may make the standard lateral view difficult to achieve; this could make it hard for us to find the exact starting point and exiting point of the guide wire. So, an accurate sacroiliac joint reduction is extremely important for this technique and it must already be achieved. Finally, this was a retrospective evaluation of our experience; a prospective study and a more sufficient sample size might give further insights in the limitations and complications of the guide apparatus.The point-to-point coaxial guide apparatus is feasible for assisting the transiliac-transsacral screw in the treatment of posterior unstable pelvic fractures. Accuracy of the point-to-point coaxial guide apparatus was superior to that of the free-hand technique. It has the advantages of simple operation, reasonable design, and no need for expensive equipment, and provides an additional surgical strategy for the insertion of the transiliac-transsacral screw.