A prerequisite for designing materials with low adhesion to ice is to accurately measure the ice adhesion strength of the surface. The majority of studies in this field have typically focused on manipulating and measuring the adhesion strength of different materials under shear stress. Among them, elastomers have proven to be promising ice-phobic surfaces because they enable interfacial cavitation, a tension-driven surface instability. In this work, a high throughput, low cost device is designed to measure the tensile ice adhesion strength of different surfaces. The design and construction of the tensile ice adhesion measurement system is presented, along with the reasoning for the design decisions. The performance of the setup is characterized using experimental trials varying parameters such as temperature, pull-off speed, thickness of the substrate, and ice/substrate interfacial area, to verify the precision of the measurements. Specifications table:1The accretion of ice can cause significant issues and can damage the iced structure. Regular removal of the ice can lead to further damage, mainly due to the strong adhesion between ice and most materials. To overcome this issue many scientists have successfully designed and fabricated surfaces with reduced adhesion to ice There is no standard or high throughput system readily available, and few studies have utilized devices and methods that measure the adhesion of ice by applying a tensile force Here, we discuss the design and construction of a tensile ice adhesion measurement device which exhibits many advantages over previous measurement systems. The benchtop system has a small footprint  and is simple to construct and operate. The device mainly consists of a motor, force gauge, cold stage, ice holders, chiller, and aluminum framing which provides mechanical support to the structure. A thermocouple and a PID controller regulate the temperature of the Peltier stage where measurement takes place. Various experimental test parameters such as temperature, speed of the applied force, iced area, and thickness of the test material can be varied independently.1.1Unlike a typical uniaxial tension setup, our tensile ice adhesion setup includes a Peltier stage with an area of 12 cm × 6 cm, which also serves as the measurement stage. Larger Peltier stages could be substituted to allow for a greater number of tested samples per freeze cycle. The temperature of the stage is tunable in the range of 0 °C to −25 °C using a thermocouple and a cold-water circulation system. This makes it possible to measure the tensile adhesion of ice to substrates at different temperatures. The size of the ice holder affects the number of measurements per freeze cycle. For example, the ice holder that was used to verify the operation of the setup under different temperatures, pull-off speeds, and thicknesses had an outer radius of 6 mm, allowing for 6 tensile ice adhesion measurements during each freeze cycle. Making several measurements during a single freeze cycle decreases the risk of errors due to varied testing conditions, resulting in more precise and consistent values.Unlike previous tensile measurement systems •Measures the tensile force needed to detach ice from a substrate•Is inexpensive and simple to build•Can measure several samples during a single freeze cycle•Was verified and the measurements matched known laws of fracture mechanicsThis device:1.2L = 60 mm, W = 60 mm, and t = 0.5 mm. The elastic moduli of aluminum, PLA, and ice were set to 70, 3.5, and 8.5 GPa, respectively, with Poisson ratios of 0.3, 0.33, and 0.3, respectively. The static analysis step was defined as ‘solver’ for all numerical simulations. After several iterations, the ice holder with the geometry shown in Unlike shear-based methods, the design of the ice holder was more complex as tension must be applied normal to the interfacial plane. An ice holder was designed and analyzed using finite element modeling (Abaqus) in order to minimize stress concentration and avoid any induced bending moment. In contrast to the push-off shear ice adhesion test method, where uniform compression is applied on the lateral side of the ice cube holder, a pull-off method was simulated in order to investigate ice adhesion in Mode I fracture (tensile detachment). Uniformity in the stress distribution at the interface was considered in the optimized design of the ice holder, along with a methodology for easily and repeatedly applying tension. The deformable solid parts of the ice holder, cylindrical ice, and an aluminum substrate were modeled. In these simulations, the ice holder  was placed on an aluminum sheet with dimensions Interfacial fracture was modeled by applying a cohesive zone model to the ice/aluminum interface In order to optimize the element size in the numerical simulations, mesh convergence was studied. 14,400, 9,724 and 33,086 elements with type C3D8R, consisting of an 8-node linear brick with reduced integration, were used for the sheet, ice cube, and ice holder models, respectively. No significant variation in the maximum stress was observed in the aluminum sheet for a larger number of elements, and the optimum element size of 0.5 mm was selected. Overall, the relatively uniform stress distribution at the ice/aluminum interface f,g indic22.1i.e. Part 1 refers to the component numbered as 1 in In 2.2The Designed Ice Holder.IGS file provides a 3D model of the ice holder which is designed for tensile ice adhesion testing. Users can use the Designed Ice Holder.STL to print the holder on any standard 3D printer. Two sizes are available, with radii of 5 mm and 7.5 mm.2.3This file contains the assembly model of the tensile ice adhesion testing apparatus. The assembly model provides the mounting positions for each individual part listed in 2.4This file provides an assembly model of the Peltier plate stage mounted on the stationary structure and connected to the optional water cooling system.2.5This file includes the assembly of the crosshead which allows for a uniaxial degree of freedom for the force gauge. Mounting positions for each component of the crosshead assembly are provided to users in this file.2.6The Movable Structure Assembly.IGS file contains the assembly of the movable structure built of T-slotted framing, which provides translational degrees of freedom for the mounted crosshead assembly.2.7The Stationary Structure.IGS file contains the assembly model of the base structure, which the movable structure and Peltier stage are mounted on. Users can find the mounting positions of different components with respect to each other.2.8The assembly model of the water cooling system contains a closed-loop water circulation system which connects the Peltier plate to the cooling system, including a water chiller and mini-fridge. The length of tubing shown in this file is arbitrary and may differ based on the positions of the chiller, fridge, and testing apparatus.3The total price to build the exact device comes around $2,000 USD . However3.1•Philips head and flat head screw drivers•Alan keys/hex wrenches•Soldering gun and solder•Glue/strong adhesive (3 M scotch-weld adhesive DP604NS and 3 M 9629PC double-sided tape recommended4This device consists of four major components : (1) The4.1Cut and drill the aluminum sheets (9), (13), and (26) with respect to the dimensions depicted in Phase 1 (see):1.Slide two T-nuts (5) into a T-slotted profile (2). Do the same for another T-slotted profile (2).2.Use corner concealed brackets (4) to attach the T-slotted profiles se 1 see:1.SlidPhase 2 (see):3.Attach the motor (6) to the Z axis CNC slide (7) using four steel socket head screws (8).4.Attach the Z axis CNC slide (7) from Step 3 to the aluminum sheet (9) using four pairs of steel socket head screws (10) and locknuts (11).5.Fix the force gauge (12) to the aluminum sheet (13) using two steel socket head screws (14)6.Attach the aluminum sheet (13) from Step 5 to the Z axis CNC slide (7) from Step 4 using four pairs of steel socket head screws (10) and locknuts (11).se 2 see:3.AttaPhase 3 (see):7.Slide three T-nuts (5) into the outer slot and one into the top slot of two T-slotted profiles (15).8.Slide one T-nut (5) to each of the vertical T-slotted profiles 9.Attach T-slotted profiles (15) from Step 7 to the Phase 1 structure from Step 8 using two corner brackets (16), two 90°angled flat plates (17) and twelve Flanged Button Head Socket Cap Screws (FBHSCS) (18).se 3 see:7.Slid10.Use four FBHSCS (18) and the previously inserted T-nuts in Step 1 to attach the final structure from Phase 2 to the structure from Step 9.11.Insert four corner concealed brackets (4) to the bottom slots of the T-slotted profiles (15).Note: The structure from Phase 1 should be able to slide freely on T-slotted profiles (15) at this point.Phase 4 (see):12.Begin from the bottom of the structure.13.Slide T-nuts (5) in the slots of the T-slotted profiles as illustrated in 14.Insert all corner concealed brackets (4) as illustrated in 15.Use two T-slotted profiles (2) and two T-slotted profiles (15) to form a frame around the MDF Sheet 4 (23). The MDF sheet should be inserted within the slots of the T-slotted profiles.16.Slide two horizontal T-slotted profiles (2) into the slots of the two vertically standing T-slotted profiles (19) from the bottom (let them be loose).17.Slide one T-slotted profile (2) horizontally into the two shorter vertically standing T-slotted profiles (2) from the bottom (let them be loose).18.Use two T-slotted profiles (15) and corner concealed brackets (4) to connect the tall (19) and short (2) T-slotted profiles.19.Use the corner concealed brackets (4) to fix the vertically standing T-slotted profiles (2) and (19) in the slots of the bottom structure from Step 14.20.Insert MDF Sheets 3 (22) and 5 (24) into the slots of the vertically standing T-slotted profiles as shown in 21.Insert the MDF sheets from the top with the horizontal T-slotted profiles inserted in previous steps and fix the structure.22.Insert the MDF Sheet 2 (21) into the slots of the tall T-slotted profiles (19), but below the second horizontal T-slotted profile (2).23.Fix the second horizontal T-slotted profile (2) on top of the MDF sheet by inserting the sheet into the slots.24.Slide MDF Sheet 1 (20) into the slots of the tall T-slotted profiles (19).25.Fix a T-slotted frame (2) on top MDF Sheet 1 using corner concealed brackets (4).26.Fix the whole structure from two sides usinga 90° angled flat plate (17) and FBHSCS (18).se 4 see:12.BegNote: Now all the components of this structure may now be strongly tightened and should be without any free movement.Phase 5 (see):27.Use screws (37) and (34) and nuts (39) and (35) to attach the electronics to the MDF sheets of the structure from Phase 4, as illustrated in Fig. 828.Attach PID controller (30) to MDF Sheet 5 (24).29.Attach the LCD shield for Arduino (31) to the housing (32).30.Glue the housing to the MDF Sheet 5.se 5 see:27.UseSTOP! At this point the user may want to skip to the Electronics Instructions below, as at this point there is maximum access to all mounted electronics components.Phase 6 (see):31.Attach Nylon single barbed tube adapter (28) to the Peltier plate (27).32.Use eight FBHSCS (18) and the T-nuts inserted in Step 13 to fix aluminum sheet (26) as shown in 33.Remove the 3 internal screws on the bottom of the Peltier plate (27). Fix the Peltier plate (27) on the aluminum sheet (26) by re-inserting its three internal screws into the 3 central holes of (26).34.Attach the PVC tubes (29) to the Nylon single barbed tube adapter (28).35.Attach and fix the movable structure to the stationary structure now containing the Peltier assembly, as shown in se 6 see:31.AttThough not necessary, the ends of all T-slotted profiles may be capped with End Caps (55) if there remains any sharp edges from cutting.Phase 7 (see):36.Attach PVC tube (41) to (29) using a stainless-steel hose clamp (42).37.Attach the submersible pump (44) to the stainless-steel bucket (43).38.Fill the stainless-steel bucket (43) with water and place inside the mini fridge (45).39.Connect the Peltier plate system, chiller cooling system (46) and submersible pump (44) using the PVC tubes (41) to create a closed-loop water circulation system.se 7 see:36.Att4.2The overall wiring diagram is shown in 4.3•The entire instrument is powered by a Type B power plug (52) connected to a wall power socket that has 110 V 60 Hz power. Connect the exposed positive, negative, and ground terminals to Terminal Block A (33) on separate ports using fork connectors (50).•Connect the power supply (40) to the power plug through Terminal Block A  with fork connectors (50) using the stripped wires emanating from the power supply.4.4•Carefully mount the Keypad Shield (31) on top of the Arduino UNO (53). The shield uses pins 4–9 for the display, A0 for buttons, and the +5 V and GND pins.•Connect ports GND, 13, and 3 of Arduino UNO (53) to ports , DIR+, and PUL+ of the stepper motor controller (6), respectively, using jumper wires (54). Solder these on the back of the Arduino as the Keypad Shield (31) sits on top of Arduino.•Connect the Arduino UNO to the DC-DC step down converter (49), which lowers the +24 V from the Power Supply to +5 V. Use the USB cable  included in the Arduino UNO kit. Insert the USB type B male connector into the Arduino UNO and the type A male into the USB port of the DC-DC converter.•Connect the positive and negative terminals of the DC-DC converter to the positive and negative terminals of the power supply through Terminal Block B (33) with fork connectors (50).4.5•Connect ports 1, 2, and 3 of the PID controller (30) to the positive, neutral, and negative terminals, respectively, of the power plug via Terminal Block A. Reuse the same terminals that the power plug is already using by loosening the clamp screw, adding the additional wire, and then tightening.•Connect ports 9 and 10 of the PID controller to ports 3 and 4 of the solid state relay (36), respectively, with stripped wires  and fork connectors (50).•Connect the stripped red wire  coming out of the Peltier plate (27) to port 1 of the solid state relay (36) with fork connectors (50).•Connect port 2 of the solid state relay (36) to the positive terminal of the power supply (40) through Terminal Block B using a stripped wire and fork connector.•Connect the negative terminal (black wire) of the Peltier plate (27) to the negative terminal of the power supply through Terminal Block B using a fork connector and stripped wire.•Connect the positive and negative terminals of the Type-K thermal couple (51) to ports 7 and 6 of the PID controller, respectively.•Mount the thermal couple on the Peltier plate with a screw (14).4.6•Connect the VCC+ and GND terminals of the stepper motor controller (6) to the positive and negative terminals of the power supply, respectively, through Terminal Block B using stripped wires and fork connectors. Reuse the same terminals that the power supply is already using by loosening the clamp screw, adding the additional wire, and then tightening.•The stepper motor (6) comes with black, green, red, and blues wires emanating from it. Connect the stepper motor controller ports A+ to black, A- to green, B+ to red, and B- to blue of the stepper motor.55.1•Turn the system on either by plugging the power cord into an outlet, or turning on a surge protector if using one. Set the temperature as preferred using the PID controller. Calibrate per manual instructions if using for the first time.•Set the pull-off speed (Slow Forward/Reverse) using the Arduino (see 5.2 below for details). The UNO remembers the last used configuration, so skip this step if not changing the speed from a prior measurement.•Turn off the device to place the test material and the ice holders. It is important to strongly fix the test material on the Peltier stage in order to prevent deflection and bending during tension. When the expected tensile forces are low, substrates can be fixed to the Peltier stage using 3 M 9629PC double-sided tape. If higher forces are expected, or if the materials exhibit poor tape adhesion, the materials can be glued to aluminum foil using an appropriate adhesive (3 M scotch-weld adhesive DP604NS recommended) and then the foil taped to the Peltier stage using the 3 M 9629PC double-sided tape.•Clean the surface using isopropyl alcohol, acetone, or any solvent suitable for the substrate material.•Place the ice holders on the surface with a clearance of at least 1 mm from each other and the edges of the substrate.•Inject de-ionized water into the ice holders from the openings in its upper surface using a syringe or pipette, until the cylinder is completely full. The 1 cm diameter ice holders require ~ 0.785 mL whereas the 15 cm diameter ice holders require ~ 1.766 mL. Make sure no leaking occurs from the bottom of the holders as it will affect the measured tensile force.•Turn the water circulation system (submersible pump) and the device on at the same time. The temperature of the cold stage will begin to drop to the pre-set temperature. Freezing occurs directionally from the bottom of the holder contacting the cold sample surface.•Wait until the water inside the holders is completely frozen and forms glaze . To verify that the ice has fully frozen, poke the ice through the opening on the upper side of the ice holders with a syringe tip.•Turn on the force gauge and align the hook manually, exactly above any holder. Place the looped kite string around the force gauge hook.•Use the Fast Forward/Reverse buttons on the LCD shield to position the hook end far enough from the holder such that the kite string is not under tension but there is minimal amount of slack in the cord.•Turn on the force gauge or, if already on, zero any residual measurements. Push the Slow Reverse button to pull the ice holder off of the substrate at the pre-set rate, until complete detachment occurs. Record the measured force.•Repeat the previous 3 steps for all additional ice holders.•In order to remove the material from the stage, detach it while the stage remains at sub-zero temperatures, as the tape comes off much easier.•Clean up the tape residue from the Peltier stage before running a second test.•To turn off the machine simply unplug the power cords.Before using the system, the ice holders should be 3D-printed out of a desired resin. PLA printed very well and was sufficiently hydrophobic in order to prevent leakage. For each printed ice holder, thread a ~ 20 cm long piece of kite string (56) through the hole at the top of the ice holder and tie the two ends in a knot so that a loop is formed.5.2i.e. rpm is converted to linear velocity). For part (6) this corresponds to 0.47 µm/sec, although the user should verify this amount manually after install. In the Arduino code, this value is held in the variable linConv (Line 38). The string on Line 135 should also be adjusted if this value is changed.The Arduino code allows the user to move the force gauge up and down at two different speeds: SLOW FORWARD, FAST FORWARD, SLOW REVERSE, and FAST REVERSE. The FAST speeds are set by the variable maxRate in the code (Line 34), which gives a suitably high speed to quickly move the gauge up and down. The SLOW rate is programmable using the LCD shield buttons. The linear stage moves by the rotation of the motor, which corresponds to a certain distance  and RIGHT (SLOW FORWARD) buttons. The value can be cycled using the UP (FAST FORWARD) and DOWN (FAST REVERSE) keys. Once a suitable multiple of linConv is chosen, press SELECT again to exit the menu. For example, 100.11 µm/sec corresponds to 213 as 0.47 µm/sec × 213 = 101.11 µm/sec.The four directional buttons are all binary ON/OFF. For example, to move down at the selected velocity, press SLOW FORWARD. To stop the instrument, press SLOW FORWARD again. While moving, only the currently pressed button can stop the instrument. Caution should therefore be used when pressing FAST FORWARD and FAST REVERSE, as the end of the linear stage could be reached and this could damage the instrument or substrate.6σice, of aluminum, stainless steel, and brass were first measured. Next, the performance of the device was characterized with respect to different test parameters such as temperature, pull-off speed, ice/substrate interfacial area, and thickness of the substrate. To evaluate these parameters the ice/surface fracture needs to be adhesive (rather than cohesive failure through the ice itself), so polyethylene (PE) and polypropylene (PP) sheets were used as they are known to exhibit lower ice adhesion than metals In order to validate the performance of the system, the tensile ice adhesion, 6.1Polypropylene, polyethylene, aluminum, brass, and stainless-steel sheets were purchased from McMaster-Carr.6.2σice = 1,400 ± 300 kPa, 1,300 ± 400 kPa, and 1,200 ± 200 kPa, respectively . The tensile ice adhesion of PP and PE were independent of temperature within the range tested , ν being the Poisson ratio. Accordingly, a plot of Fc against [Eγ(2a)3]1/2 should fall on a straight line. The recorded tensile ice adhesion measurements when varying the iced area for PP and PE followed the above relation well, as shown in The adhesion of a rigid stud to an incompressible elastic solid has been previously shown to depend on stud size 2 at −15 °C and pull-off speed of 100 µm/s (The reproducibility of the ice type and repeatability of the interfacial fracture process was studied by monitoring the critical force to detach ice on the 0.84 mm thick PP with an interfacial area of 1.77 cm100 µm/s e. The smThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The half-maximal inhibitory concentrations (IC50), cytotoxic concentration (CC50), and selective index (SI) values of hWJ-MSC-S were determined using Vero-E6 cells. The virucidal, anti-adsorption, and anti-replication antiviral mechanisms of hWJ-MSC-S were evaluated. The hWJ-MSC-S significantly inhibited infection of SARS-CoV-2 and IBV, without affecting the viability of cells and embryos. Interestingly, hWJ-MSC-S reduced viral infection by >90%, in vitro. The IC50 and SI of hWJ-MSC secretome against SARS-CoV-2 were 166.6 and 235.29 µg/mL, respectively, while for IBV, IC50 and SI were 439.9 and 89.11 µg/mL, respectively. The virucidal and anti-replication antiviral effects of hWJ-MSC-S were very prominent compared to the anti-adsorption effect. In the in ovo model, hWJ-MSC-S reduced IBV titer by >99%. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis of hWJ-MSC-S revealed a significant enrichment of immunomodulatory and antiviral proteins. Collectively, our results not only uncovered the antiviral potency of hWJ-MSC-S against SARS-CoV-2 and IBV, but also described the mechanism by which hWJ-MSC-S inhibits viral infection. These findings indicate that hWJ-MSC-S could be utilized in future pre-clinical and clinical studies to develop effective therapeutic approaches against human COVID-19 and avian IB respiratory diseases.Human SARS-CoV-2 and avian infectious bronchitis virus (IBV) are highly contagious and deadly coronaviruses, causing devastating respiratory diseases in humans and chickens. The lack of effective therapeutics exacerbates the impact of outbreaks associated with SARS-CoV-2 and IBV infections. Thus, novel drugs or therapeutic agents are highly in demand for controlling viral transmission and disease progression. Mesenchymal stem cells (MSC) secreted factors (secretome) are safe and efficient alternatives to stem cells in MSC-based therapies. This study aimed to investigate the antiviral potentials of human Wharton’s jelly MSC secretome (hWJ-MSC-S) against SARS-CoV-2 and IBV infections in vitro and  Nidovirales, family Coronaviridae, and sub-family Orthocoronavirinae. SARS-CoV-2 belongs to the Betacoronavirus genus, while IBV belongs to the Gammacoronavirus genus [Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and infectious bronchitis virus (IBV) are highly contagious coronaviruses and leading causes of devastating respiratory tract diseases in humans and chickens, respectively. Both SRAS-CoV-2 and IBV are enveloped viruses, with a positive single-stranded RNA genome, and classified into order us genus ,2,3. SARus genus . Like SAus genus ,5. IBV ius genus . There aus genus . The emeMesenchymal stem cells (MSCs) possess tremendous potential as therapeutic agents for various human diseases ,10,11,12Human Wharton-jelly-derived MSCs (hWJ-MSCs) have recently gained more attention for their superior proliferation rate, immune-privileged characteristics, and lower carcinogenic profile after in vivo transplantation ,28,29. h2. SARS-CoV-2, hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820) [Vero-E6 (African green monkey kidney) cells were used to study the antiviral effect of hWJ-MSC-S in vitro. Cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum (FBS) , and 1% antibiotic-antimycotic (AA) mixture  at 37 °C in a humidified atmosphere of 5% CO_430820)  and IBV,_430820) , viruses_430820) .n = 20) were obtained from the department of obstetrics and gynecology, Al-Azhar University Hospital in Assiut. This study was approved by the ethical committees of faculty of medicine and the faculty of science at Al-Azhar University in Assiut (APPROVAL NUMBER/ID:202015). Informed consent was obtained from all individuals who participated in the study. hUCs were collected under sterile conditions after normal full-term healthy pregnancy from 20 healthy mothers and transported to the laboratory in phosphate-buffered saline (PBS) supplemented with antibiotics, 100 U/mL penicillin, and 100 μg/mL streptomycin. At the laboratory, the outer surface of UC was sterilized by 70% ethanol and the cord was washed twice with PBS and serum-free DMEM  to remove excess blood. The UC was cut longitudinally with a sterile surgical scissor and Wharton’s Jelly (WJ) within the cord was scraped using a scalpel. Then, the cord blood vessels (two arteries and one vein) were removed, and the remaining cord tissue (CT) was collected. WJ and CT were minced separately into 1–2 mm pieces before digestion with the enzymatic blend for 30 min at 37 °C in a 5% CO2 incubator [hUC samples  supplemented with 10% FBS and 1% AA solution. Plates were incubated at 37 °C in a 5% COncubator . After 7g for 10 min. Then, 106 hWJ-MSCs in 100 μL volume were stained with 10 μL of Peridinium-chlorophyll-protein (Per-CP)-conjugated anti-CD105/Endoglin , Carboxyfluorescein (CFS)-conjugated-CD73 , allophycocyanin (APC) conjugated anti-CD90/Thy1 , phycoerythrin (PE) conjugated anti-CD45 , and PE-CD34  monoclonal antibodies for 30 min. The viability of cells was monitored using 7-aminoactinomycin D (7-AAD) staining. A total of 50,000 events were acquired and analyzed using the FACS Cantor flow cytometer  and Kaluza analysis software 1.5a .hUC-MSC cells were detached using 0.25% Trypsin-EDTA and collected by centrifugation at 1000× g for 10 min to remove cells residues, and stored at −80 °C until used in subsequent experiments.hWJ-MSCs were grown in a complete growth medium to reach 80% confluency before replacing the culture medium with a serum-free medium. After 48 h, Conditioned medium (CM) was harvested, centrifuged at 1000× The 3--2, 5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the cytotoxicity of hWJ-MSC-S in Vero-E6 cells as previously described . BrieflyThe plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (CC50).in ovo as previously described [Embryotoxicity of hWJ-MSC-S was evaluated using toxicity assay escribed ,38. Brie4 in 96-well plates and incubated overnight at 37 °C and 5% CO2 condition. Serial dilutions of hWJ-MSC-S were mixed with 103 PFU of virus (SRAS-CoV-2 or IBV) and incubated at room temperature (RT) for 1 h. Cell monolayers were washed with PBS and inoculated with hWJ-MSC-CM/virus mixtures, and incubated at 37 °C and 5% CO2 for 72 h. Next, cells were fixed with 100 μL of 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet in distilled water for 15 min at RT. The crystal violet dye was then dissolved using 100 μL absolute methanol per well, and the optical density was measured at 570 nm using Anthos Zenyth 200 rt plate reader  [50 of the hWJ-MSC-S is that required to reduce the virus-induced cytopathic effect (CPE) by 50%, compared to the untreated virus control. The infection assay was performed in BSL-3 facility.The IC50 value of hWJ-MSC-S was determined as previously described ,40,41. Berlands) . The IC55 in a 6-well plate and incubated overnight at 37 °C. Following this, 200 µL (103 PFU) of the virus was mixed with different non-toxic concentrations of hWJ-MSC-S and incubated at RT for 1 h before inoculation onto the monolayer of Vero-E6 cells. One hour later, the supernatant was aspirated, cells were washed, overlaid with DMEM medium supplemented with 2% agarose, 1% AA mixture, and 4% bovine serum albumin (BSA), and incubated at 37 °C. Three days later, the overlay medium was discarded, and cells were fixed for 1 h in 10% formalin solution and stained with 0.1% crystal violet working solution. The infection experiment was performed in BSL-3 facility. The plaque-forming units (PFUs) were counted, and percentages of reduction were calculated according to the following equation:Plaque-reduction assay was used to determine the antiviral activity of hWJ-MSC-S as previously described . Briefly3 PFU) of the virus was mixed with different non-toxic concentrations of hWJ-MSC-S and incubated at RT for 1 h before infecting the cell monolayer for 1 h at 37 °C. To determine the anti-adsorption effect, the cell monolayer was pre-treated with different concentrations of hWJ-MSC-S for 2 h at 4 °C before infection with 200 µL (103 PFU) of the virus for 1 h at 37 °C. In another set of experiments, the cell monolayer was infected with 200 µL (103 PFU) of the virus at first for 1 h at 37 °C and then treated with hWJ-MSC-S for 1 h at 37 °C to determine the anti-replication effect. In all protocols, the supernatant was aspirated, cells were washed, and incubated with DMEM medium supplemented with 2% agarose, 1% AA mixture, and 4% BSA at 37 °C. Three days later, the overlay medium was discarded, and cells were fixed for 1 h in 10% formalin solution and stained with 0.1% crystal violet working solution. The PFUs were counted, and the percentages of reduction were calculated as described above.The mode of antiviral activity of hWJ-MSC-S was determined by evaluation of its virucidal, anti-adsorption, and anti-replication effects in Vero-E6 cells as previously described ,43. To e5 EID50/0.1 mL) was mixed with 0.1 mL of different non-toxic concentrations of hWJ-MSC-S and incubated at RT for 1 h before inoculation into the allantoic cavity of 10-day-old SPF-ECEs (five eggs per concentration). Inoculated SPF-ECEs were incubated at 37 °C for 5 days and inspected daily by candling to check the embryo viability [IBV (10iability . SPF-ECE® probe (IBV-TM: FAM-TTGGAAGTAGAGTGACGCC-CAAACTTCA-BHQ1) specific to IBV N gene were used to determine IBV copy numbers in one step RT-qPCR [Viral RNA was extracted from allantoic fluids using the QIAamp Viral RNA mini kit  as per the manufacturer’s recommendations. The RNA concentration was measured with a NanoDrop ND-2000 spectrophotometer . The forward primer IBV (IBV-F: 5′-ATGCTCAACCTTGTCCCTAGCA-3′), reverse primer (IBV-R: 5′-TCAA-ACTGCGGATCA-TCACGT-3′), and TaqMan RT-qPCR . The mas RT-qPCR . The ream/z and 170–1500 m/z, respectively. A design of 120-min linear gradient 3–80% solution  was used. The 40 most intense ions were sequentially selected under data-dependent acquisition (DDA) mode with a charge state 2–5. For each cycle, survey full scan MS and MS/MS spectra were acquired at a resolution of 35.000 and 15.000, respectively. To ensure accuracy, external calibration was scheduled and run during sample batches to correct possible TOF deviation.Proteins were precipitated from hWJ-MSC-S samples collected with four-times chilled Acetone. After incubation at −80 °C for 30 min and at −20 °C overnight, samples were centrifuged at 10,000 rpm for 30 min. The protein extract of hWJ-MSC-S was denaturated by placing 50 μL lysis solution  with complete ultra-proteases . Samples were incubated at 37 °C for 1 h with occasional vortex, and then centrifuged at 12,000 rpm for 20 min. The protein concentration of hWJ-MSC-S was assayed using the bicinchoninic acid (BCA) method  at Å562 nm before digestion. Next, 30 µg of hWJ-MSC-S protein was subjected to in-solution digestion. In brief, protein pellets were re-suspended in an 8 M urea lysis solution and reduced with 5 mM tris 2-carboxyethyl phosphine (TCEP) for 30 min. The alkylation of cysteine residues was performed using 10 mM iodoacetamide for 30 min in a dark area. Samples were diluted to a final concentration of 2 M urea with 100 mM Tris-HCl, pH 8.5, before digestion with trypsin . For endHomosapiens  with target and decoy sequences. The search space included all fully and semi-tryptic peptide candidates with a maximum of 2 missed cleavages. Precursor mass and fragment mass were identified with an initial mass tolerance of 20 ppm and 10 ppm, respectively. The carbamidomethylation of cysteine (+57.02146 amu) was considered as a static modification and oxidation at methionine (+15.995), acetylation of protein N- terminal and K (+42.01 amu), and pyrrolidone from carbamidomethylated C (−17.03 amu) as variable modification. To assure a high-quality result, the false discovery rate (FDR) was kept at 1% at the protein level. Final assembly and emerging of sample replicates were applied to generate the final outputs of each sample using in-house software ‘ProteoSelector’ (https://www.57357.org/en/department/proteomics-unit-dept/in-house-bioinformatics-tools/ (accessed on 3 December 2021)) (Mascot generic format (mgf) files were generated from the raw file using the script supplied by AB Sciex. MS/MS spectra were searched using X Tandem in Peptide shaker (version 1.16.26) against Uniprot r 2021)) . Normalip-value of 0.05 and an odds ratio of more than 1.Gene ontology was performed on uniquely retrieved genes from the consolidated proteome profile of hWJ-MSC-S and searched against gene ontology database biological process, molecular function, cellular component, and protein class analysis was utilized with Fisher’s Exact test with significance level 0.05 and 5% FDR. Or pathway enrichment analysis, Enrichr’s web-based tool was used for pathway enrichment analysis with a COVID-19 database search ,50. The t-test were used to compare different treatments and quantify significance using Prism . p values < 0.05 were considered statistically significant. Graphics were drawn by R [Data were shown as means ± standard deviation (SD) of three independent experiments. One-way ANOVA and Student’s awn by R  and PrisThe overall isolation of UC-MSCs was successful in 15 out of 20 (75%) of the collected umbilical cords. Cells were successfully isolated from 12 out of 15 (80%) and 6 out of 15 (40%) of the collected WJ and CT samples, respectively. The isolation rate was significantly higher in WJ than in CT, as shown in 50), half-maximal cytotoxicity concentration (CC50), and selective index (SI) of hWJ-MSC-S were calculated. To determine CC50, Vero-E6 cells were treated with different concentrations of hWJ-MSC-S, and the cytotoxicity levels were measured using an MTT assay. The CC50 value of hWJ-MSC-S was 39,200 µg/mL  virucidal, (ii) inhibition of viral adsorption, and (iii) inhibition of viral replication, as illustrated in 50) was assessed by inoculating SPF-ECEs with various hWJ-MSC-S concentrations and embryo viability was checked daily. The LD50 was calculated as the concentration that causes the death of 50% of inoculated embryos. The LD50 value of hWJ-MSC-S was 4121 µg/mL . Although hWJ-MSC-S similarly inhibited SARS-CoV-2 and IBV infections, there were variations in the inhibitory concentrations between the two viruses. It showed markedly higher IC50 and SI against SARS-CoV-2 in comparison to IBV. In addition to that, IBV was less sensitive to inactivation by hWJ-MSC-S. This suggests that hWJ-MSC-S may exploit different strategies to inhibit SARS-CoV-2 and IBV infections. Further, the differences in sensitivity to hWJ-MSC-S between the two viruses could be attributed to the nature of each virion. Moreover, IBV is relatively resistant to inactivation with soluble antiviral factors compared to other coronaviruses, such as SARS-CoV [Interestingly, hWJ-MSC-S reduced plaque formed by SARS-CoV-2 and IBV in Vero-E6 cells by more than 90%. Similarly, in the SARS-CoV ,70.β4), Peptidyl-prolyl cis-trans isomerase A (PPIase A), and tissue metalloproteinase inhibitors 1 (TIMP1) in hWJ-MSC-S. Tβ4 is an anti-inflammatory and antioxidant protein that has been shown to inhibit coronavirus mouse hepatitis virus (CoV MHV) Infection in vivo [The hWJ-MSC-S inhibited SARS-CoV-2 and IBV infections using different strategies—directly by virucidal effect and indirectly via inhibiting viral replication. This variation in the mode of action could be ascribed to the wide range of molecules that have been identified in hWJ-MSC-S proteomic analysis . In gene in vivo . PPIase  in vivo ,75. The  in vivo . Mesench in vivo .β4 has been reported to significantly increase the survival of CoV-MHV-A59-infected mice by inhibiting viral replication and alleviating infection-associated immune activation and liver damage [β4 have been associated with hepatitis B virus-related liver failure, and higher Tβ4 values indicated improvement and recovery from the disease [β4, CypA is involved in the life cycles of several viruses, including SARS-CoV, CoV-229E, CoV-NL63, FCoV, HIV-1, HCV, and influenza virus [Tr damage . In addi disease . Interes disease . Like Tβza virus ,75,80,81za virus . Recentlza virus ,85,86,87β4 and CypA and TIMP1) and EVs (MVs and EXos). These findings imply that hWJ-MSC-S-based therapeutic agents can potentially be used in future studies to target SARS-CoV-2 and IBV infections and their associated diseases. The low cytotoxicity and embryotoxicity profiles of hWJ-MSC-S indicated that it could be used safely in vivo studies, particularly to treat IBV-infected poultry flocks. Although drinking water and feed medication are common practices for the treatment of poultry diseases, for hWJ-MSC-S-based anti-IBV therapies, the parenteral administration would be the main route. This is because the biologically active extracellular paracrine factors of hWJ-MSC-S could be deactivated by impurities in drinking water and feed additives. Moreover, a lyophilized form of hWJ-MSC-S could be more suitable for drug administration to poultry flocks. Further, hWJ-MSC-S could be used therapeutically to treat IBV-infected chickens, as well as prophylaxis to control IBV infection in chicken flocks.Like stem cell-derived soluble factors, extracellular vesicles (EVs) in stem cell secretome have been shown to carry out immunomodulatory and antiviral activity ,89,90,91in ovo, respectively. Our antiviral mechanistic studies suggest that the antiviral effects of hWJ-MSC-S are mainly mediated via virucidal and anti-replication mechanisms. Using proteome profiling and gene ontology, we have identified various immunomodulatory and antiviral bioactive factors in hWJ-MSC-S. This study provides a perspective on new antiviral therapeutics against SARS-CoV-2 and IBV infections, using hWJ-MSC-S-based approaches. Findings from this study may, thus, have implications for developing effective strategies to combat COVID-19, IB, and future coronavirus outbreaks. While this study aimed to evaluate the antiviral effect of hWJ-MSC-secreted factors collectively, it will be particularly meaningful to determine the role of an individual secreted factor in the hWJ-MSC-S-mediated antiviral effect.In this work, we have demonstrated the antiviral activity of hWJ-MSC-S against two medically important coronaviruses: human SARS-CoV-2 and avian IBV. The hWJ-MSC-S significantly inhibited SARS-CoV-2 and IBV infection in vitro and


Ice shelves regulate the stability of marine ice sheets. We track fractures on Pine Island Glacier, a quickly accelerating glacier in West Antarctica that contributes more to sea level rise than any other glacier. Using an on‐ice seismic network deployed from 2012 to 2014, we catalog icequakes that dominantly consist of flexural gravity waves. Icequakes occur near the rift tip and in two distinct areas of the shear margin, and TerraSAR‐X imagery shows significant fracture in each source region. Rift‐tip icequakes increase with ice speed, linking rift fracture to glaciological stresses and/or localized thinning. Using a simple flexural gravity wave model, we deconvolve wave propagation effects to estimate icequake source durations of 19.5–50.0 s and transient loads of 3.8–14.0 kPa corresponding to 4.3–15.9 m of crevasse growth per icequake. These long‐source durations suggest that water flow may limit the rate of crevasse opening. Fracture at Pine Island Glacier generates flexural gravity waves, a wave type related to interaction between a floating plate and supporting fluidRift‐tip seismicity rate increases with ice speed either due to changes in the underlying ice shelf stress state or localized thinningRecorded flexural gravity waves are consistent with a point load of ∼10 kPa applied over ∼30s, corresponding to ∼10m of vertical cracking PIG, part of the larger WAIS, contributes more to present‐day global sea level rise than any other glacier  speed time series derived from five continuous GPS stations. The GPS stations were colocated with seismometers  is the opposite of typical surface waves in the solid Earth. In the latter case, low‐frequency waves travel faster because seismic wave speeds generally increase with depth.To detect flexural gravity waves in the data set, we design a two‐stage detection scheme that identifies broadband, dispersive seismic events. Our detection approach, detailed in Text S2 in Supporting Information We next determine locations for all icequakes in our final catalog. Given the poor distribution of the stations with respect to fracture locations, we employ single‐station approaches to locating icequakes. We compute epicentral back azimuths by analyzing the polarization direction of recorded horizontal waves , and the northeast shear margin near Evan's knoll (“shear margin”), which are depicted in Figure 44.1The rift‐tip icequakes are coincident in space and time with several fracturing processes, including rift propagation, wing cracking, small‐scale calving within the rift, and calving along the southern edge of the nascent iceberg. Rift‐tip events occurred more frequently in 2013 than in 2012 Figure . The meaPeak levels of rift‐tip seismicity were observed during the calving of Iceberg B‐31 in the week of 5 November 2013. That week had 115 rift‐tip events, the highest event count of any week across all three source regions. Furthermore, elevated rift‐tip icequake activity in 2013 corresponds to a period of accelerating ice velocities Figure . While r4.2The rift/margin icequakes are coincident in space and time with the growth of ∼20 rifts formed in the northwest shear zone as well as smaller‐scale fractures and widening of the main rift itself. Rift/margin icequakes occurred more frequently in 2012 than in 2013. The mean seismicity rate was 27.7 icequakes/week in 2012 and 19.3 icequakes/week in 2013. Four weeks of 2012 equaled or exceeded the maximum 2013 seismicity rate of 70 icequakes/week. The timing of icequakes in the rift/margin group is independent of ice speed. Peak levels of rift/margin seismicity were observed during the week of 15 May 2012, which contained 109 rift/margin icequakes. Rift/margin icequakes reach peak seismicity rates in the weeks following the opening of the secondary rift branch in May 2012, suggesting that the crack opening caused aftershock‐like seismicity and/or destabilized the margin, enhancing the growth of nearby fractures.4.3The shear‐margin icequakes are coincident in space and time with the initiation of new cracks and growth of extant cracks near Evans Knoll. This area marks the transition from a primarily intact shear margin upstream of Evans Knoll to a highly fractured shear margin downstream of Evans Knoll. Imagery shows that multiple fractures longer than 1 km were initiated in this area during 2012 and 2013 Figure .5D≡EI=Ehi3/121ν2 is the flexural rigidity with the second moment of area I=∫−hi/2hi/2z2dz, E is the Young's modulus of ice, ν is the Poisson's ratio of ice, t is time, x is horizontal position, g is gravitational acceleration constant, hi is the ice thickness, ρi is the density of ice, ρw is the density of water, w is the vertical displacement of the beam, ϕ is the ocean surface velocity potential, and P is an applied point load. From left to right, the terms in Equation hi = 400 m δ(t). In Text S4 in Supporting Information G that we are able to analytically invert in the time domain and numerically invert in the frequency domain. We then derive Gm, the vertical displacement response to a point moment source.We obtain the Green's function of the floating beam equation as the impulse response of the mechanical system to a point load (force per unit length) source. Rewriting Equation G and Gm from waveform stacks to estimate the source load or moment distribution of events in each spatial group. Figure We deconvolve We examine the sensitivity of our deconvolution to the assumed value for the ice thickness by varying the ice thickness between 300 and 500 m  (Freund, What process sets the duration of the observed icequakes? The above scaling for tectonic earthquakes is based on the reasoning that the duration is set by the time required for a shear crack to propagate across a fault of length  Freund, . In our  Freund, . The cra7We detect and locate icequakes that propagate as flexural gravity waves on the Pine Island Glacier ice shelf from 2012 to 2014. When compared to satellite imagery, the back‐azimuthal distribution of the detected events suggests that the icequakes were generated by fractures at the tip of a large rift and in two distinct portions of the northern shear margin. Most of the events were generated at the shear margin near Evans Knoll in agreement with imagery that suggests significant fracture initiation. Increased fracturing at the rift tip is associated with increased ice speed and elevated basal melting in 2013 (Christianson et al., Supporting Information S1Click here for additional data file.


This study aims to investigate the risk factors for screw loosening after single-level posterior lumbar interbody fusion (PLIF) utilizing cortical bone trajectory (CBT) screw and establish a nomogram for predicting screw loosening.A total of 79 patients (316 screws) who underwent single-level PLIF with CBT screw were included in the study. Preoperative, postoperative, and final follow-up demographic data, surgical data, and radiographic parameters were documented and analyzed to identify risk factors, and a predictive nomogram was established for screw loosening. The nomogram was assessed by concordance index (C-index), calibration plot, decision curve analysis (DCA), and internal validation.P = 0.029), the coronal angle of the screw , and cortical bone contacted layers  were risk factors and incorporated in the nomogram for predicting screw loosening after single-level PLIF with a CBT screw. The C-index of the nomogram was 0.877 (95% CI 0.818–0.936), which demonstrated good predictive accuracy. The calibration plot indicated an acceptable calibration of the nomogram that also had a positive benefit in guiding treatment decisions.The incidence of screw loosening was 26.6% in 79 patients and 11.4% in 316 screws. Multifactorial regression analysis confirmed that fixed to S1 (FS1, OR = 3.82, 95% CI 1.12–12.71, FS1, CA, and CBCLs are identified to be significant risk factors for screw loosening after single-level PLIF with the CBT technique. The nomogram we have established can be used to predict screw loosening and contribute to surgical decisions. CBT screw (TPS) –4. Likewew (TPS) , 5, 6. Tew (TPS) –9. As reew (TPS) , 10, 11,ew (TPS) –14, and Screw loosening in both the TPS and CBT screw may require revision surgery due to symptomatic spinal instability and instrument failure , 15, 16;The present study aimed to detect the prevalence of screw loosening in single-level PLIF using CBT screw with a minimum of 2 years of follow-up and to establish a nomogram for predicting screw loosening individually in each vertebra.This was a retrospective study in the institution. A total of 88 consecutive patients were included in the study from November 2017 to January 2020, and 79 eligible patients were evaluated . InclusiThe patient was placed in a prone position. A 5-cm midline skin incision was performed in the lumbar area. Muscular dissection was performed until the vertebral isthmus was exposed. The facet joints were exposed, and adjacent facet joints of the fusion area were avoided. The entry point was selected as an intersection of a vertical line through the center of the inferior facet joint of the adjacent cephalic vertebra and a horizontal line 3–4 mm below the inferior facet joint of the cephalic vertebra (a notch might be identified on the isthmus). The track was drilled with a 2 mm burr into the cortical bone with an approximate 10°–15° angle from medial to lateral and 20°–25° angle from caudal to cranial. Locating pins were placed into the track, and fluoroscopy was performed to check the position. Then, decompression was performed, and a cage (PEEK) filled with autogenous bone was implanted into the intervertebral space after endplate preparation and autogenous bone insertion. After the decompression, pins were removed, and CBT screws  were inserted through the tracks with spinous process preservation. Bended rods were then positioned and tightened bilaterally after compression was performed. Finally, fluoroscopy was performed to recheck the position of the screws and cage before the skin was sutured layer by layer.Clinical, demographic, and surgical data including age, gender, body mass index (BMI), operation time, and estimated blood loss (EBL) were collected. Radiological parameters including the coronal angle of the screw (CA), sagittal angle of the screw (SA), fixed to S1 (FS1), Hounsfield unit (HU) measurement of the trabecular bone of screw location, and cortical bone contacted layers (CBCLs) were evaluated , 3. The t-test and Mann–Whitney U test for continuous data and discontinuous data, respectively, and quantitative data were listed as means ± SD with normal distribution or as medians with interquartile ranges with non-normal distribution. The chi-squared test was used for categorical data analysis.SPSS Statistics Version 23.0  and R software (version 4.1.2) were used for data analysis. Univariate analysis was performed with an independent P < 0.05). In this study, CBCLs, CA, and FS1 were put into the logistic model. A multiple logistic regression model was applied to select significant variables with a stepwise forward method, and the odds ratio (OR) and 95% confidence interval (CI) of the variables were recorded. A nomogram was established with R software. The concordance index (C-index) of the nomogram was calculated to evaluate the predictive accuracy of the nomogram utilizing the “rms” package. A calibration plot and the Hosmer–Lemeshow test were used to assess the calibration, and the nomogram was internally validated by the bootstraps of 1,000 resamples. Decision curve analysis (DCA) was calculated by the “rmda” package to evaluate the clinical usefulness of the nomogram. The reliable outcome is considered as C-index >0.75, and P value <0.05 was considered statistically significant for all data.Multivariate logistic analysis was further performed on variates that had significant differences in univariate analysis (P = 0.039), FS1 (P = 0.029), and CBCLs (P < 0.001) between the two groups (P = 0.029), CA , and CBCLs  were risk factors for screw loosening after single-level PLIF with CBT screws  were included in the study. The cohort contained 35 (44.3%) male patients with an average age of 65.14 ± 9.74 years, and the average BMI was 26.83 ± 4.49. The mean follow-up time was 25.38 ± 1.77 months . The inco groups . MultiplT screws .P = 0.755). The internal validation by bootstraps of 1,000 resamples was excellent, with a 0.880 (95% CI 0.815–0.932) C-index. Decision curve analysis was performed, as shown in The nomogram was conducted by R software (version 4.1.2) with a 0.877 (95% CI 0.818–0.936) C-index, which demonstrated good discrimination and predictive accuracy . CalibraApplication indication of the nomogram is explained in To indicate whether the screw's location was in S1, three-dimensional surface plots are shown in Screw loosening is common, as reported in PLIF, with an incidence of 1%–60% , 10, 13.The odds ratio of FS1 was the highest compared to other parameters (OR = 3.82). In our study, there were 22 screws fixed in the S1 vertebra, and 9 of them (40.9%) were found to have an obvious lucent zone in the CT scan. Grigoryan et al.  conducteWith regard to CA and CBCLs played an important role in screw loosening of CBT screw, according to the nomogram. The typical trajectory of the CBT screw contains four parts for the cortical bone to increase the stability of fixation; among these, the lateral par as the starting point is essential. The lateral par is an identifiable structure as an entry point and is less influenced by a degenerative change to provide a good bony reference in the surgery , 31. TheIn the present study, we have documented and provided a reference for the measurement of SA as an angle between the screw line and vertical line because we think that this might reduce the error for measurement of wedge-shaped vertebra in some cases, while some authors recommended a method of the measurement of the angle between the screw and vertebral endplate , 34. HowLower BMD evaluated by dual x-ray absorptiometry (DXA) was significantly associated with screw loosening by influencing the pull-out strength , 36, 37;The study had some limitations, mainly due to retrospective analysis with small sample size. The surgery with the CBT technique was performed during the learning curve of the early period, and this might contribute to the loosening of the S1 screw. Our study focused on the local view of screws to explore the potential factors of screw loosening; however, we did not include parameters related to fusion because it was hard to judge whether the fusion failure was caused by screw loosening or bone graft. Further studies with experienced surgical techniques will be performed to validate the present study.The CBT technique offers an alternative method for lumbar surgery with TPS. Although CBT screws provide good stability for fixation, we have identified significant risk factors for screw loosening. A perioperative evaluation with the nomogram can provide a reliable prediction of screw loosening with CBT screws and contribute to surgical decisions to avoid complications.


Neuroblastoma (NB) is a heterogeneous cancer of the sympathetic nervous system, which accounts for 7–10% of paediatric malignancies worldwide. Due to the lack of targetable molecular aberrations in NB, most treatment options remain relatively nonspecific. Here, we investigated the therapeutic potential of BCI, an inhibitor of DUSP1 and DUSP6, in cultured NB cells. BCI was cytotoxic in a range of NB cell lines and induced a short‐lived activation of the AKT and stress‐inducible MAP kinases, although ERK phosphorylation was unaffected. Furthermore, a phosphoproteomic screen identified significant upregulation of JNK signalling components and suppression in mTOR and R6K signalling. To assess the specificity of BCI, CRISPR‐Cas9 was employed to introduce insertions and deletions in the DUSP1 and DUSP6 genes. Surprisingly, BCI remained fully cytotoxic in NB cells with complete loss of DUSP6 and partial depletion of DUSP1, suggesting that BCI exerts cytotoxicity in NB cells through a complex mechanism that is unrelated to these phosphatases. Overall, these data highlight the risk of using an inhibitor such as BCI as supposedly specific DUSP1/6, without understanding its full range of targets in cancer cells. This study examined whether the cytotoxic effects of BCI on neuroblastoma tumour cells were exerted by its known inhibitory action on dual specificity phosphatases DUSP1 and DUSP6. We demonstrated that this was not the case and that BCI elicits cytotoxicity through off‐target effects, potentially through the activation of MTOR and JNK pathways. BCI(E/Z)‐BCI hydrochlorideDUSPdual‐specificity phosphataseindelsinsertion deletionsKSEAkinase substrate enrichment analysisMAPKmitogen‐activated protein kinaseNBneuroblastomaPTPprotein tyrosine phosphataseROSreactive oxygen speciesNeuroblastoma (NB) is the most prevalent extracranial paediatric cancer and accounts for 7–10% of childhood cancers worldwide . PatientThe dual‐specificity phosphatase family (DUSPs) represents a large group of protein tyrosine phosphatases (PTPs) that can dephosphorylate both Tyr and Ser/Thr residues . DUSPs rin vivo chemical screen, (E/Z)‐BCI hydrochloride (BCI) is a dual‐inhibitor of DUSP1 and DUSP6 . Proteins were digested into peptides using trypsin as previously described . The z‐score represents the distribution of changes in phosphorylation of substrates for given kinases relative to the distribution of all fold changes. Each row displays a kinase substrate alongside the substrates related to that kinase identified in the phosphoproteomic dataset. The z‐score is colour‐coded as blue (decreased) or red (increased) with asterisks denoting the significance of differences.Click here for additional data file.


Alopecurus myosuroides Huds.), one of the most aggressive grass weeds in Europe, is also a strong competitor of crops. This study aimed to assess the impact of environmental conditions on the competition between (1) ACC-ase and ALS herbicide-resistant (BR) and herbicide-susceptible (BS) blackgrass biotypes, (2) BR and winter wheat cv. Arkadia (W), and (3) BS and W. In the replacement series model, the experiment was conducted at seven sites across Poland during two seasons (2018/19 and 2019/20). In the BR-BS experiment, the BS biotype was in majority more competitive toward the BR biotype. However, in the regime of optimal hydrothermal conditions and at a higher sand content in the soil we observed a higher competitiveness of BR towards BS. The combined interactions between W and BR or BS were also affected by environmental conditions, i.e., soil texture and hydrothermal coefficient, as explained by PCA and k-means cluster analysis. At most sites, W was more competitive toward both BS and BR, which could result from earlier emergence of W in relation to B in majority of sites. Except for two cases, located on heavy, clay soils, during humid seasons, where B was more competitive toward W. We summarize that blackgrass competitiveness towards other biotypes and wheat depends to some extent on environmental conditions; however, the phenomenon should be explored in more detail.Blackgrass ( Alopecurus myosuroides Huds., is a winter annual, monocotyledonous plant that produces many seeds and is one of the most difficult weeds to control3. Fields with consistently dense A. myosuroides patches are characterized by high clay, nutrient, and organic matter contents with a slightly lower pH6. Blackgrass emerges in early autumn, often before the emergence of winter crops, i.e., cereals and oilseed rape8. A phenotypic variation of blackgrass and its dormancy affect its emergence patterns in a single year, bringing this weed's evolutionary advantage over crops10. During the last 50 years, blackgrass has become more prevalent and problematic to control weeds, mostly due to widespread herbicide resistance11. The first case of herbicide-resistant blackgrass was reported in 1982 in Great Britain12. Since then, the weed has evolved field resistance to a few different herbicide modes of action13, especially to post-emergence acetyl-CoA carboxylase inhibitors (aryloxyphenoxypropionate (AOPP) and cyclohexanedione (CHD) and acetolactate synthase (ALS) inhibitors14. The mechanism behind blackgrass resistance is reported to be enhanced metabolism in the case of resistance to AOPP/CHD herbicides15 and target-site resistance (TSR) in the case of ACCase-inhibiting and ALS-inhibiting herbicides17. Non-target-site-based resistance (NTSR) mechanisms in blackgrass can confer unpredictable resistance to herbicides with different chemistries or modes of action21, but no glyphosate resistance has been reported to date22. The UK's latest report showed that the herbicide resistance-prone, outcrossing A. myosuroides exhibits low genetic differentiation among field-collected populations, with some geographical  clines in genetic diversity and isolation by distance23. In Poland, herbicide-resistant blackgrass populations are mostly found in winter wheat, especially in the northeastern parts of the country26.Blackgrass, 8. At a density of 25 plants m−2, a 10% decrease in wheat yield is observed27. Studies on wheat-blackgrass competitiveness could support integrated weed management techniques, in addition to mechanical, biological, and cultural control means28. Wheat planting design, e.g., plant density, row spacing, and orientation, increases wheat competitiveness against weeds31, which is one of the methods of weed management. In the case of the herbicide-resistance trait in agrocenosis, what counts is the competitive pressure of resistant weeds on crops and the interspecies competitiveness of resistant biotypes towards susceptible ones33. Identifying this interaction type could be important, especially when herbicide resistance has developed in the field. Nevertheless, due to the introduction of sustainable weed management methods, herbicides have substantially reduced. In that situation, weeds of different susceptibilities to herbicides could compete with each other34. The available literature on that topic shows that the competition between resistant vs susceptible biotype depends on many factors, i.e., type of resistance, habitat stresses, and weed densities35. On the other hand, the cited studies were performed either in the controlled conditions or in one field site only. For these reasons, testing the resistant vs susceptible biotype in different climatic conditions is a novel aspect of our research. The methods developed to study competition among plants allow the estimation of the losses caused by weeds and the competitiveness among species36. The models for blackgrass and wheat competition may differ significantly in predicting the yield damage caused by blackgrass, and there is no optimum design for competition experiments since the aims, objectives, and practicalities vary from study to study and species to species. However, they could potentially be used in future weed management systems38. Our study focused on a replacement-series competition model39 to study the competitive abilities of herbicide-resistant blackgrass toward the susceptible biotype and toward winter wheat cv. Arkadia. A standard replacement series comprises a set of pure and mixed populations in which the combined density of the components is held constant40. The method was developed in 1960 by de Wit41. Correctly used, the approach can lead to some valid interpretations. However, this methodology has some limitations, i.e., a replacement series is unsuitable for the quantitative evaluation of interference or niche differentiation or in predicting the long-term outcome of an association between species40. Based on the replacement series design, a recent study showed that the multiple herbicide-resistant bentgrass (Apera spica-venti (L.) P. Beauv) was more competitive toward winter wheat than the susceptible bentgrass42.Blackgrass is a very competitive weed for winter wheat that can cause grain yield losses of up to 41%This study aimed to assess the impact of habitat conditions on the competition between (1) herbicide-resistant and herbicide-susceptible blackgrass biotypes, (2) resistant blackgrass and winter wheat, and (3) susceptible blackgrass and winter wheat. We therefore hypothesized that both habitat conditions, species/biotype frequencies, and herbicide resistance of blackgrass affect the competition of plants.The amplification of the blackgrass ALS sequence resulted in the generation of a 1919 bp nucleotide sequence. Mutations were identified in the analyzed nucleotide sequence fragment, where 22 changes were synonymous and seven were nonsynonymous. The analysis of the amino acid sequence to find mutations known to confer resistance to ALS inhibitors revealed the presence of an amino acid substitution at position P197. Two mutations were found: P197A and P197T  domain , which is known to be the site of action for the active substance of the herbicides belonging to group 1. Four mutations were identified in the analyzed nucleotide sequence of the CT domain, where three changes were synonymous and one was nonsynonymous. The nonsynonymous mutation resulted in the substitution of Trp to Leu at position 1999 in the amino acid sequence  and the relative grain number (BCRgn) of S and R blackgrass biotypes are presented in Table The values of BCRb (BCRgn) < 1 indicate that the R biotype is more competitive toward the S biotype in the case of biomass (grain) production. In contrast, if BCRb (BCRgn) > 1, then the S biotype is more competitive toward R.r = 0.69) between BCRb and BCRgn in 2018/19, but the correlation was nonsignificant   Fig. B,C, indi50) Fig. D,E.Figurr = 0.87) and significantly correlated  indexes had the opposite direction  as in the dry season of 2018/2019. The increasing K provoked an increase in the competitiveness of the S biotype, both in biomass and grain production , respectively  was noted 8–16 and 6–19 days after sowing in 2018 and 2019, respectively  and grain number (WBCRgn) were similar for the susceptible and resistant B biotypes in the competition with W Figs.  and 6. H1 − 0.83x2 − 0.44x3 − 0.63x4 − 0.65x5 − 0.90x6 − 0.25x7 − 0.70x8 − 0.90x9. , and it was significant for the thousand grain weight   and cluster (CA) analyses separately for the 2018/19 Fig. A,C and 21 = 0.92x1 − 0.62x2 − 0.80x3 + 0.02x4 − 0.44x − 0.92x6 − 0.11x7 − 0.55x8 − 0.91x9), and the second principal component Y2 explained 22.1% of the total variance of nine characteristics  and the Lower Silesia region49. We conducted short-term (two growing seasons) experiments in situ in the replacement series model as a tool to investigate the competitiveness between blackgrass-blackgrass and wheat-blackgrass with environmental factors and blackgrass herbicide resistance.Various modeling approaches might be employed to examine the effectiveness of integrated weed management, reducing the need for more expensive and cumbersome long-term in situ experimentation50, in different sites across Poland. The sites in this experiment were diversified in terms of soil conditions, from the lightest loamy sands , sandy loams , medium-heavy sandy clay loam (Wrocław), to heavy and compacted silt loams . Additionally, two vegetative seasons during the studies displayed contrasting weather conditions; the first season was dry in most of the sites, whereas the other season was humid.Within two years of study, the tested blackgrass biotypes, herbicide-susceptible (BS) and herbicide-resistant (BR), displayed different competitiveness with each other and with winter wheat. Based on two competitive ratios, i.e., plant biomass and the number of grains produced by the tested plants, we could draw main competitive relations, significant from biological and agronomic points of view51. In other studies, the blackgrass biotype with enhanced metabolism resistance to ALS inhibitors produced longer tillers than the susceptible plants during vegetative growth. However, there was a reproductive cost of resistance, i.e., 27% fewer seed heads per plant and a 23% reduction in total seed head length52. The worse performance of herbicide-resistant biotypes, the so-called resistance penalty, was also observed in other herbicide-resistant weed species55. Our study also showed site-specific correlations between both competitive ratios of BR and BS biotypes, especially in the humid season. We found that the content of sand affected the competitive relations between the BS-BR biotypes during the humid season. Specifically, the more sand there was in the soil, the higher the competitiveness of the BR biotype toward the BS biotype, and vice versa. In our opinion, this finding suggests the existence of internal plasticity of blackgrass biotypes that change their intraspecies competitive efforts depending on the site-specific conditions.In the BR-BS experiment, on average, the BS biotype was more competitive toward the BR biotype, which was expressed by the values of the competitive ratio greater than one. This is consistent with other authors' findings, who point out that herbicide-susceptible weed biotypes perform better than resistant ones in the absence of herbicide selective pressure. For example, under drought stress, the nontarget site-resistant biotype of blackgrass germinated less than the susceptible biotype56. In our study, wheat cv. Arkadia has been shown to suppress blackgrass at most study sites, particularly in the humid season of 2019/20 and regardless of the herbicide-susceptibility level of B. Increasing wheat competitiveness against weeds through a combination of breeding and planting design  has a strong potential to reduce weed-induced yield losses in wheat57. Sardana58 declared that cultivars possessing traits such as fast germination and growth, high biomass, and large leaf area have a competitive advantage over weeds. Lazzaro59 listed four traits linked to the competitive ability of wheat against weeds, i.e., aboveground biomass before stem elongation, tillering index, plant height, flag leaf morphology, and two production-related traits (grain yield and thousand-kernel weight). The combined effect on wheat-blackgrass interactions was also studied by Andrew and Storkey60 using the INTERCOM model parameterized for two wheat cultivars with contrasting competitive ability and simulations run across 10 years at different crop densities and two sowing dates. The authors found that sowing date, sowing density, and cultivar choice largely work in a complementary fashion, allowing enhanced competitive ability against weeds when used in combination. However, the relative benefit of choosing a more competitive cultivar decreases at later sowing dates and higher crop densities. Perhaps another important element in the competitiveness of W against B in our study that supported W competitiveness was the time of W emergence, which was in the vast majority of sites earlier than the emergence of B, or at least both species emerged on the same day. As other studies confirm, a delay in weed emergence to wheat emergence enhances wheat competitiveness toward weeds and a final wheat grain yield63.The diversified soil and weather conditions also affected the studied parameters of winter wheat (seed number and biomass), as was observed in the wheat (W) – barnyardgrass (BS/BR) experiment. The multivariate data mining at each site and BS/BR mixtures with winter wheat explained the division into two distinctive in situ groups. The first group was highly related to sandy soil and low moisture. In contrast, the second group had heavier soils of higher moisture where wheat productivity was significantly higher, as confirmed in the studies by10, which could support its competitiveness with W at both sites.The observed W-B competition pattern exception was noted only in two sites, namely, Mydlniki and Czesławice (south and south-eastern Poland), located on heavy, clay soils, where B was more competitive toward W during humid seasons. Blackgrass also takes advantage of environmental conditions, such as clay content and moisture in the soilOur study showed all the competitive replacement series models between blackgrass—blackgrass and winter wheat—blackgrass of different herbicide susceptibilities . There were site-specific changes in the blackgrass R and S competitive abilities, depending on the sand soil content, supporting the thesis about blackgrass plasticity depending on environmental conditions. Winter wheat in our study was a strong competitor for blackgrass; only in three out of a total of 28 cases was blackgrass more competitive toward wheat. One of the winter wheat characteristics that could affect this relationship was that wheat emerged 15–0 days earlier than blackgrass. A more detailed understanding of belowground competition may also be required to increase the robustness of the predictions when water or nutrients are limiting.Ethics approval and consent to participate. The collection of biotypes of blackgrass samples from plantations was permitted by local famers on survey. The plant collection and the study complied with local and national (Poland) regulations. The study was approved by the institutional research ethics committee of Institute of Plant Protection in Poznań, and written informed consent was obtained from each participant. All the methods in this manuscript were carried out in accordance with relevant guidelines and regulations.Two biotypes of blackgrass (B) with contrasting susceptibility to herbicides were used in the two-year-long pot experiments at seven sites across Poland. Both biotypes were collected in Poland in 2017. The herbicide multiresistant (BR) biotype was from western Poland, voivodship Lubuskie, from winter wheat. The susceptible biotype (BS) was from eastern Poland from the ruderal site. An initial level of herbicide resistance of both BR and BS biotypes was confirmed in two sets of greenhouse dose–response tests carried out in 2017 and is presented in Table Escherichia coli competent cells (Table E. coli cells using the NucleoSpin Plasmid (Mecherey-Nagel). Three plasmids containing inserts synthesized in each PCR were digested with BglII to confirm the presence of the insert and sequenced by Genomed . Sequencing data were analyzed using the BioEdit Sequence Alignment Editor 7.5.564.Leaves from plants of the multiresistant BR biotype in the leaf development stage (BBCH 13–14) were ground in a mortar using liquid nitrogen. Genomic DNA was isolated using a NucleoSpin Plant II Mini kit for DNA from plants . Blackgrass ALS and ACCase gene sequences were amplified by means of PCR. For the analysis of the ALS sequence, equal amounts of DNA isolated from two plants were pooled and used in PCR, while for the analysis of the ACCase sequence, DNA was isolated from one plant. The 50 µl reaction mixture contained 5.0 µl of 10X PfuUltra II reaction buffer , 0.5 µl of dNTPs mix 25 mM each), 1.0 µl of 10 µM forward primer and reverse primers  were dug into the soil at a distance of 0.5 m, and ca. A total of 2.5 cm of pots remained above the soil surface. Subsequently, they were filled with local arable soil from the 0–30 cm layer. Prior to placing it in the pots, the soil was sieved through a metal mesh to remove stones and other larger impurities. Soil samples were taken for detailed texture and chemical analyses  cv. Arkadia (breeder: HR Danko PL) at all seven sites. The proportions of winter wheat W and BR were in the ratios of W10:R0, W8:R2, W6:R4, W4:R6, W2:R8, and W0:R10. The same ratios were used for the W and BS plants: W10:S0, W8:S2, W6:S4, W4:S6, W2:S8, and W0:S10.Seeds of the W, BR, and BS biotypes were sown on the same day, optimal for W Table . The gra4NO3 m−2; 50% of the dose was applied at the beginning of vegetation, and the remaining dose was applied at wheat shooting (BBCH 31–33). Wheat and blackgrass were harvested at full-grain maturity (BBCH 97–99). At harvest, a fresh mass of plants, both W and B, was weighed. Wheat plants were measured with a ruler from the base of shoots up to the highest ear's tip. All grains of W/B plants were counted from the pot, and their fresh mass was weighed and calculated per plant for statistical analysis. Based on three samples of 100 grains, the 1000 grain weight (TGW) of W and B was calculated.In the spring, the density of W and B plants was regulated according to the layout. During that time, N fertilization was applied in 30.0 g NHK) according to the equation:P is the precipitation total and t is the sum of the daily mean air temperature values. The classification for Poland's temperate climate is K < 1.3 moderately dry seasons; 1.3–1.6 optimum; and > 1.6 humid season66. The calculated K values are presented in Table Weather data, i.e., precipitation and air temperature, were collected during the study from the nearest weather stations Tables  and S2. Two parameters, namely, fresh plant biomass and the number of seeds per plant, were measured for both winter wheat W and blackgrass B. The blackgrass competitive ratio (BCR) was calculated for the mixtures of susceptible blackgrass (BS) with resistant blackgrass (BR), representing the comparative growth based on plant fresh biomass (BCRb) and the number of grains (BCRgn). The competitive ratio between wheat and blackgrass WBCR was calculated separately for the mixtures of W and BS and W and BR.67:The BCR and WBCR were calculated according to the formula described by Hoffman and BullerIf the competitive ratio BCR < 1, the R biotype is more competitive toward the S biotype; if BCR > 1, the S biotype is more competitive toward the R biotype.If the competitive ratio WBCR < 1, B is more competitive toward W; if WBCR > 1, W is more competitive toward B.The RY and the total relative yield (TRY) for all the measured parameters of B and W were calculated according to the formulas:63. where RYW is the relative yield of W; RYB is the relative yield of B; p is the proportion of species; Wmix is the value of the W parameter analyzed for the mixture; Wmon is the value of the W parameter analyzed for the monoculture; Bmix is the value of the B parameter analyzed for the mixture; Bmon is the value of the B parameter analyzed for the monoculture; and TRY is the total relative yield. The RY values were measured for both W and B and were averaged for a single plant.The analysis was performed for the replacement series experiment68. The most explanatory models were determined by using the χ2 goodness-of-fit69, giving the best fit for all tested sites and biotypes. If RY is a straight line, it denotes no competition; a convex line shows a benefit to species; a concave line represents a loss to species. If TRY equals 1 (straight line), there is competition for the same resources between both species/biotypes. If TRY is greater than 1 (convex), there is no competition since the demand does not exceed the resources. If TRY is less than 1 (concave), an antagonism resulting in a mutual loss to the species involved becomes apparent67.The biomass and grain yield or seed number, calculated into RY and TRY, were presented as graphs and fitted into one of the five competition models according to Radosevichk-means procedure).ANOVA of two models was performed, i.e. the two-way model for the year × site of plants to calculate the BCRgn and BCRb in R/S blackgrass and the three-way model for the year × site × biotype to calculate the WBCRgn and WBCRb in wheat/blackgrass. Data from the indexes were transformed to a normal distribution. The post hoc HSD Tukey test was used for the means separation. The r-Pearson correlation coefficient was calculated for relations between BCRgn/WBCRgn and BCRb/WBCRb, the relations between the hydrothermal K coefficient and BCRgn/WBCRgn and BCRb/WBCRb and the relations between sand [%] and BCRgn/WBCRgn and BCRb/WBCRb. The exploration technique of principal component analysis (PCA) was used to explain the multidimensional diversity of W and environmental variables in terms of the first two components. Five biometrical parameters of W, i.e. mean length and fresh biomass of the plant, number of seeds per plant, yield of seeds per plant, and TGW (1000 grain weight), and four environmental parameters, i.e. The hydrothermal coefficient K and soil texture were analyzed using PCA and CA .Supplementary Figures.Supplementary Tables.