Photo-degradation of organic semiconductors remains as an obstacle preventing their durable practice in optoelectronics. Herein, we disclose that volume-conserving photoisomerization of a unique series of acceptor-donor-acceptor (A-D-A) non-fullerene acceptors (NFAs) acts as a surrogate towards their subsequent photochemical reaction. Among A-D-A NFAs with fused, semi-fused and non-fused backbones, fully non-fused PTIC, representing one of rare existing samples, exhibits not only excellent photochemical tolerance in aerobic condition, but also efficient performance in solar cells. Along with a series of in-depth investigations, we identify that the structural confinement to inhibit photoisomerization of these unique A-D-A NFAs from molecular level to macroscopic condensed solid helps enhancing the photochemical stabilities of molecules, as well as the corresponding OSCs. Although other reasons associating with the photostabilities of molecules and devices should not excluded, we believe this work provides helpful structure-property information toward new design of stable and efficient photovoltaic molecules and solar cells. Non-fullerene acceptors have contributed hugely to the best-performing organic solar cells, yet the photo-degradation remains a hurdle preventing the practical application. In this work, research group from Zhejiang University discovers non-fused acceptors with excellent photochemical tolerance in aerobic condition, and identify the main degradation pathway. However, the photodegradation of organic semiconductors remains to be an obstacle preventing the practice of such optoelectronics10. Typically, the desirable operational lifetime of OSCs13 is only secured under the exclusion of oxygen and UV light. It is because that the intrinsic photostabilities of organic photovoltaic materials, especially for the high-efficiency acceptor–donor–acceptor (A–D–A) non-fullerene acceptors (NFAs) are not ideal yet that could degrade under illumination in ambient16. In particular, typical A–D–A NFAs are synthesized through Knoevenagel condensation (KC) of aldehyde and active methylene19, wherein the exocyclic vinyl groups are constructed to the conjugate electron-rich core (D) with electron-deficient terminals (A). This structural design enables the excellent tuning of the optoelectronic properties, hence high photovoltaic performance of A–D–A NFAs, and also undermining their intrinsic photochemical stabilities20. It is because the exocyclic vinyl double bond is chemically more vulnerable toward photooxidation than those in aromatic units15. Besides, the vinyl bonds from KC reaction exhibit reversible reactivity and could be hydrolyzed back to starting materials21. Therefore, to prevent the side reaction of exocyclic vinyl group will be critical to enhance the intrinsic stability of A–D–A NFAs, which suffers a lack of effective approach so far.Power conversion efficiencies (PCEs) of organic solar cells (OSCs)24, which possess relatively low synthetic complexities and decent PCEs. To our surprise, among them, NFRA PTIC exhibits a steady absorption profile even after 32-h one-sun-equivalent illumination in ambient, suggesting a photostable A–D–A acceptor16. Notwithstanding all of those efforts, the understanding is quite limited, and there lacks of knowledge on how to access A–D–A NFAs with excellent resistance towards photochemical reaction. It, therefore, motivates researchers to explore organic semiconductors with inherent photochemical stabilities, as well as to understand their structure–activity relationship. These efforts should be highly valuable for eventually developing stable organics and related optoelectronics.Recently, semi-fused and non-fused ring acceptors (SFRAs and NFRAs) have been developed−4 h−1) > non-fused PTICH (2.30 × 10−3 h−1) > semi-fused HF-PCIC (1.36 ×  10−2 h−1) > fused IT-4F (1.48 × 10−2 h−1), under one-sun-equivalent illumination in ambient. Non-fused PTIC-based OSCs display about 359 times and 322 times slower decay rates than fused IT-4F and semi-fused HF-PCIC-based OSCs, respectively.In this work, we investigate the photodegradation pathway of A–D–A NFAs with the representative molecular skeletons of fused, semi-fused and non-fused backbones Fig. , and dis3 carbon-free backbones  to suppress the photoisomerization of vinyl groups by installing outward-chain in A–D–A NFAs Fig. ; (2) to nes Fig. . As resu−2 h−1 (IT-4F), 1.36 × 10−2 h−1 (HF-PCIC), and 9.00 × 10−4 h−1 (PTIC) in the first 64 h illumination. The photostability of PTIC remains distinct as well in the bulk heterojunction blend with polymer donor, PBDB-TF, wherein PTIC maintains absorption characteristics well while the fast decay of polymer  of neat films was measured , semi-fused (HF-PCIC), and non-fused-cores (PTIC), respectively Fig. . Their ples Fig. . In sharles Fig. . The phoF, semi-fies Fig. .Fig. 2In1H nuclear magnetic resonance (1H NMR) spectra. It reveals that, under illumination, the vinyl groups of IT-4F underwent photoisomerization, and then followed by photooxidation with the generation of clear epoxide species, along with the other decomposed species upon extending illumination time. The original chemical shifts of vinyl protons (Ha) are characteristics at 8.55 ppm (singlet for IT-4F), 8.85 ppm (multi peaks for HF-PCIC), and 8.95 ppm (singlet for PTIC) in dichlorobenzene-d4, respectively  raised up accompanying the decrease of vinyl protons (Ha) within the first 10 h illumination (details will be discussed hereafter) . Followed by this, a new peak at 4.51 ppm (denoted as H″a for IT-4F) appears, which is assigned as the proton signal of epoxide15. Whereas, it is interesting to note that PTIC remains stable in solution even after 220 h illumination  in solution. It is consistent with time-resolved photoluminescence (TRPL) spectroscopy , the illuminated IT-4F exhibits new signals with m/z of 1515.958 and 1531.906, suggesting that one or two additional oxygen atoms are introduced, revealing the possible epoxidation of vinyl bonds for DFIC-segments14. As shown in Fig. −1 for IT-4F and 1623 cm−1 for HF-PCIC, both of which, along with other characteristic signals, disappeared during photodegradation. It is a sharp contrast to the stable PTIC films (1601 cm−1) with negligible changes32.To reveal the influence of photostability on their chemical structures, we have monitored the structural change of NFAs by ely Fig. . The fusion Fig. . The cheion Fig. . The reaopy Fig.  of IT-4F34. The distorted C=C bonds after photoisomerization appear to be more reactive towards photooxidation that is likely a critical step opening up the photodegradation of NFAs.Together with above results from solution and film measurements, we describe the plausible photodegradation process of IT-4F in Supplementary Fig. 35. PTIC adapts the “brick-like” end-to-end packing  and outward thiophene side chain (with versus without). The molecular structure itself affects their solid stacking properties. Often, those crystalline films with densely packed molecules exhibit superior photostability due to the confinement of structural rearrangement in solid Fig. . To accuing Fig.  with π–πing Fig. . Additioing Fig. . The clo3 carbon-free planar backbones, we further examine the effect of terminal side chain on the photostability of NFAs, by comparing non-fused acceptors with the same backbone, PTIC (with hexyl chain) and PTICH (without hexyl chain)   of NFAs. TFIC with hexyl chains displays only one conformation with S…O geometry, whereas TFICH without hexyl chain shows stereoisomers with S…O geometry (accounting for 86% ratio) and H…O geometry (24% ratio)  Fig. . TFICH i36 is clearly observed for TFICH (R = H) in solution, transforming from Z-state (ZS) to E-state (ES) under illumination. The subsequent photooxidation of TFICH can be detected at the elongated illumination. However, the photoisomerization and photooxidation of TFIC can be largely suppressed by introducing the terminal chain (R = C6H13)  under illumination. The absorption profile and solution color of TFICH changed from yellow to deep red (due to isomerization of vinyl bond vary the intramolecular charge transfer of TFICH) in first 10 h illumination, and then appear to be dark upon the prolonged 220 h illumination . Interestingly, TFIC (R = C6H13) only exhibits little change of absorption profile and colors during the prolonged illumination.The distorted TFICH in loosely packed solid likely results in the exocyclic double bonds more vulnerable toward photoisomerization and photooxidation. Interestingly, the photo-induced volume-conserving isomerization of vinyl group13) Fig. –26. Figu1H NMR measurements , accompanying the shift of Hb (thiophene proton) from 7.714 to 7.730 ppm. When cooling down the solution from 120 °C back to 25 °C, only the ZS structure can be detected, indicating the full recovery of isomeric ES to ZS , suggesting that the hexyl chain in the thiophene bridge effectively suppresses the photoisomerization of the vinyl group. Whereas the recovery region of TFIC (at 25–80 °C range) shows less activation energy needed than that of TFICH (at 60–120 °C range)  upon heat treatment of both TFICH and TFIC solution, which is also consistent with the temperature varied nts Fig. –26. Upon ZS Fig. . After 1ge) Fig. –29.After the prolonged illumination, the epoxidation product (EP) can be detected with the characteristic chemical shift of 4.382 ppm in TFICH solution, which yields ~12% content after 50 h illumination. Thereafter, the content of EP-TFICH keeps increasing, accompanying the formation of more complicated signals due to the generation of other photodegraded species. Overall, this pathway of structural change involving photoisomerization and epoxidation of the vinyl group of TFICH is similar to that of IT-4F. In contrast, TFIC in solution generates little epoxidation content, and maintains mainly ZS isomers along with a very small ratio of ES isomer, after 220 h illumination. It clearly suggests that, at the molecular level, the introduction of outward-chain effectively suppresses the photoisomerization, hence the subsequent photooxidation of vinyl groups in the model compound and A–D–A NFAs.3/Ag  illumination , which are close to those with HF-PCIC  and PTIC   and HF-PCIC-based devices (from 0.969 to 0.921), whereas there is almost no influence for PTIC-based devices (from 0.952 to 0.958).Surprisingly, PBDB-TF:PTIC-based device allows maintaining about 72% of its initial PCE value for 500 h illumination, which is much better than that of PBDB-TF:IT-4F and PBDB-TF:HF-PCIC-based devices . Note that all measurements are subject to the freshly made devices without pre-illumination treatment, and therefore the burn-in loss of OSCs are accounted into performance decay. The fast decay of PBDB-TF:IT-4F and PBDB-TF:HF-PCIC-based devices occur at first 50 hVoc on the light intensity  recombination (from 1.08 to 1.28 kT/q) after photodegradation (Supplementary Fig. 41. CE and TPV curves of NFEA PTIC-based devices show nearly identical before and after photoaging. Whereas, FREA IT-4F and SFRA HF-PCIC-based devices show much longer charge extraction time after photoaging than their fresh devices in the CE curves, and charge lifetime is also declined. These results indicate that abundant charge traps are generated in the photoaged devices consisting of IT-4F and HF-PCIC, which lead to the deteriorated OSC efficiencies.We further investigate the dependence of ity Fig. , the FRE3 carbon-free skeleton and outwards thiophene sidechains embed also good photostability of PBDB-TF:Y6-based OSCs without solvent additive (maintaining ~77% PCE of their fresh devices after 300 h illumination, Supplementary Fig. The above-obtained results demonstrate that the diverse structural factors of photovoltaic molecules lead to the discrepant degradation processes under light illumination. NFRA PTIC and the derived OSCs exhibit exceptional photostabilities among the head-to-head compared with other FREA and SFRA A–D–A NFAs. We can correlate the superior photostability of NFAs and their derived OSCs to the chemical structural confinement of NFAs at the molecular level and in the solid. To investigate the applicability of this structural guidance, we extend studies into the high-performance A–D–A NFA, Y6. The identical traits of Y6 and PTIC on planar sp3 carbon-free backbones play important roles to enhance the intrinsic photostabilities of these A–D–A NFAs and their derived OSCs. Even though, there are still some other complicate reasons associating with the photostabilities of non-fullerene solar cells. We believe the structure–property correlation from these unique examples can be beneficial to the community, providing new chemistry insights for designing photostable organic semiconductors and optoelectronics.In summary, we disclose, for the first time, that the volume-conserving photoisomerization of exocyclic vinyl groups is one critical step toward the subsequent photodegradation of a unique series of A–D-A NFAs. Among the studied NFAs with the fused, semi-fused, and non-fused backbones, non-fused PTIC displays superior photostability in solution, films, and solar cells. Non-fused PTIC-based OSCs displayed about 359 times and 322 times slower decay rates than fused IT-4F and semi-fused HF-PCIC based OSCs, respectively. It is because the structural confinement prevents the photoisomerization of NFAs at the molecular level and in condensed solid. In this regard, the structural factors, including hinder outward-chain and planar spSupplementary InformationPeer Review File


In vitro cholinesterase inhibitory effects were measured by Ellman's method in 96-well microplates. Blumea lacera (Compositae) and Cyclea barbata (Menispermaceae) were found to have the highest acetylcholinesterase inhibitory  and butyrylcholinesterase inhibitory effect . Cyclea barbata demonstrated competitive inhibition, where Blumea lacera showed an uncompetitive inhibition mode for acetylcholinesterase. Smilax guianensis (Smilacaceae) and Byttneria pilosa  were also found to show moderate AChE inhibition , although no significant BChE inhibitory effect was observed for extracts from these plant species. Among others, Thunbergia grandiflora (Acanthaceae) and Mikania micrantha (Compositae) were found to display noticeable AChE  and BChE  inhibitory effects, respectively. Molecular docking experiment suggested that compounds 5-hydroxy-3,6,7,3′,4′-pentamethoxyflavone (BL4) and kaempferol-3-O-α-L-rhamnopyranosyl-(1⟶6)-β-D-glucopyranoside (BL5) from Blumea lacera bound stably to the binding groove of the AChE and BChE by hydrogen-bond interactions, respectively. Therefore, these compounds could be candidates for cholinesterase inhibitors. The present findings demonstrated that Blumea lacera and Cyclea barbata are interesting objects for further studies aiming at future therapeutics for Alzheimer's disease.Many Bangladeshi medicinal plants have been used to treat Alzheimer's disease and other neurodegenerative diseases. In the present study, the anticholinesterase effects of eight selected Bangladeshi medicinal plant species were investigated. Species were selected based on the traditional uses against CNS-related diseases. Extracts were prepared using a gentle cold extraction method.  Alzheimer's disease (AD) is a devastating neurodegenerative disorder linked with the two most common symptoms, memory dysfunction and cognition impairment. In the neuropathological symptoms of AD, cognitive deficit is consistent with the presence of cholinergic deficit, due to the degeneration or atrophy of cholinergic neurons in the basal forebrain, including senile plaques and neurofibrillary tangles . AcetylcInhibition of the cholinesterase enzymes (AChE and BChE) can boost ACh levels in several parts of the brain and symptoms associated with the progressive loss of cholinergic function in AD improve . StudiesPharmacological treatment of AD is largely based on treating symptoms and severity with advanced stages rather than targeting the etiological mechanisms. The currently approved medications for AD are mostly cholinesterase inhibitors, including donepezil, rivastigmine, galantamine, and NMDA antagonist memantine . Among tNatural products have already proven to be promising sources of useful acetylcholinesterase (AChE) inhibitors . The curBlumea lacera (Burm.f.) DC., Byttneria pilosa Roxb., Clerodendrum infortunatum L., Cyclea barbata Miers, Mikania micrantha Kunth, Smilax guianensis Vitman, Spermacoce articularis L.f., and Thunbergia grandiflora Roxb. for anticholinesterase activity in rejuvenating and improving the memory and cognitive function. A study of ethnopharmacological background of the selected species is presented in Our study is an attempt to identify and compare potential candidates from the following eight selected plant species  , acetylthiocholine iodide (ATCI), butyrylcholinesterase (BChE) from equine serum (lyophilized powder), butyrylthiocholine iodide (BTCI), and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich . Trizma hydrochloride (Tris-HCl) and bovine serum albumin (BSA) were obtained from Sigma-Aldrich . Deionized water was produced using a Milli-Q water purification system .Acetylcholinesterase (AChE) from electric eel , 125 μL of DTNB (3 mM), 50 μL of buffer B , and 25 μL of each test extract solution at the different concentrations or negative control (25% DMSO in MeOH) were mixed and incubated for 10 min at 37°C. The reaction was started by adding 25 μL of 0.05 U/mL AChE. The absorbance was measured at 405 nm, and the reaction rates were calculated by MARS Data analysis software . Estimation of BChE inhibition was performed in a similar way described above using 25 μL of 5 mM butyrylthiocholine iodide as substrate and 0.05 U/mL of BChE as an enzyme. Galantamine was used as a positive control for both enzymes. Three independent assays were performed in triplicate at different concentrations.ChE inhibitory activity was measured based on Ellman's method  as reporTo investigate the type of inhibition of the effective extracts, the enzyme activity was determined in the presence of increasing concentrations of substrate ATCI (0.16–20 mM) and the absence or presence of two/three concentrations of each extract. The analysis of the type of inhibition of AChE activity was determined by the Lineweaver-Burk (LB) plot, whereas the kinetic parameters km and Vmax were obtained by curve fitting according to the classical Michaelis-Menten equation by using GraphPad Prism version 6.The crystal structure of cholinesterase enzymes has been collected from RCSB PDB (rcsb.org) . PDB ID Blumea lacera [X = −2.0303, Y = 38.0503, and Z = 29.9752, and the dimension (angstrom) was X = 42.2467, Y = 50.5623, and Z = 41.3235. In case of BChE, the grid box center was fixed at X = 1.6818, Y = −1.3768, and Z = −10.0914 and the dimension (angstrom) was X = 39.1883, Y = 39.4042, and Z = 33.0087. The docking process was performed in triplicate for every ligand and the average of the binding affinities has been presented. Furthermore, the extracted ligand from the crystal structure was docked with the optimized protein following the same protocol, which validated our docking protocol [Nine compounds  from Blua lacera –29 were a lacera , 31. Thea lacera . The opta lacera . The comprotocol . The docn = 3). IC50 values were calculated via nonlinear regression analysis using GraphPad Prism v. 6.0 . One-way analysis of variance (ANOVA) and Tukey test were used to compare means among extracts; an observation was considered statistically significant if the p value is less than 0.05 (p < 0.05).Data are given as the mean ± SD .The extracts of eight plant species used as herbal medicine in different disorders were tested for AChE and BChE inhibitory activity using Ellman's method. The results are shown in Cyclea barbata extract showed the highest inhibition against BChE followed by Mikania micrantha, Blumea lacera, and Byttneria pilosa with their IC50 values of 124 ± 2, 216 ± 24, 297 ± 13, 358 ± 32, and 536 ± 23 µg/mL, respectively . The inhibition of BChE is also increasing corresponding to the concentration of extracts  and BChE is more abundant in the peripheral system, the potency toward both ChE found in the extracts is of paramount importance. These findings could activate mostly the central as well as peripheral cholinergic transmission to improve the mental abilities of AD patients. Among the eight screened extracts, Blumea lacera, Cyclea barbata, Smilax guianensis, and Byttneria pilosa were selected as efficacious candidates as sources of strong ChE (AChE and BChE) inhibitors. Due to the multifactorial pathogenesis of AD, multitargeted drugs will be preferred as the effective therapeutic strategy against AD.In the BChE inhibitory activity, ectively . The IC5extracts . Our resBlumea lacera, a widely distributed herb in Bangladesh, is locally known as Kukursunga or Shimura. B. lacera is used to cure spasms, fever, and bronchitis and alleviate burning sensation [B. lacera showed an IC50 value of 150 ± 11 and 297 ± 13 μg/mL for AChE and BChE inhibition, respectively; efficacy of these leaves on enhancing cognitive functions might be due to the presence of terpenoids [B. lacera displayed lower activity  compared with acetone extract.ensation . Leaves ensation , 27 and ensation . Acetonerpenoids . AcetoneCyclea barbata is a tropical indigenous plant widely distributed in Asia, especially in Bangladesh. This climber is traditionally used against various diseases such as headache, epilepsy, allergy, asthma, lipoma, tetanus, and throat sore [Cyclea barbata exhibited IC50 value of 176 ± 14 and 124 ± 2 µg/mL for AChE and BChE inhibition, respectively, which might be owing to its high alkaloid contents [oat sore . Acetonecontents .Smilax guianensis, locally known as Kumarika, is believed to possess CNS-modulating properties and is used in many herbal formulations. S. guianensis is also used in the treatment of epilepsy, venereal and skin diseases, fever, swellings, sores, and abscesses [S. guianensis methanolic extract inhibited AChE with an IC50 value of 205 ± 31 μg/mL which might be due to the presence of steroidal saponin glycosides [bscesses , 37. Youbscesses , 38. Driycosides , while nByttneria pilosa, a large woody climber, is widely distributed in Chittagong, Bangladesh. Leaves and stems of this species are usually used to treat snakebite by the traditional healers and indigenous people in Chittagong hill tracts, Bangladesh. It is also used to cure scabies, bone fracture, and elephantiasis [B. pilosa leaves revealed its AChE and BChE inhibitory potentials with an IC50 value of 221 ± 2 and 536 ± 23 μg/mL, respectively, because of the presence of favorable anticholinesterase natural products such as alkaloid, saponin, and terpenoid [antiasis , 12, 13.β-amyloid plaques, antioxidant activity, and modulation of APP processing [Both acetylcholinesterase and butyrylcholinesterase are the key enzymes in the cholinergic nervous system. Therapies designed to reverse the cholinergic deficit in AD are mostly based on inhibitors of AChE. Several studies revealed that cholinesterase inhibitors could act on multiple therapeutic targets such as preventing the formation of ocessing . Moreoveocessing . Nonetheocessing , 42. BioBlumea lacera, Byttneria pilosa, and Smilax guianensis showed uncompetitive inhibition in which inhibitor only binds to enzyme-substrate complex   . These tIn silico molecular docking is an efficient tool in recent structure-based drug design. In this anticholinesterase screening study, Blumea lacera exhibited the highest inhibition among the eight plant species. Here, the docking study was conducted to identify the responsible compounds from Blumea lacera on anticholinesterase potential. However, the binding affinity and binding pose of selected compounds  residues of AChE  does not interact with the PAS and catalytic triad residues. BL1, BL4, and BL7 displayed 16, 16, and 12 hydrophobic interactions as well as three, ten, and one hydrogen bonds with AChE, respectively -β-D-glucopyranoside (BL5) formed a hydrogen bond with Ser198 and His438 with side-chain oxygen of BChE. Kaempferol 3-O--β-D-glucopyranoside (BL6) interacted with the catalytic residues and PAS residues with side-chain oxygen and hydrogen atoms, forming hydrogen bonds with Asp70, Tyr332, Ser198, and His438 (mixed inhibition). BL8 forms hydrogen bonds with Ala277, Asn289, Ser287, Ala328, and Thr284. BL5, BL6, and BL8 formed 9, 7, and 6 hydrogen bonds with BChE, respectively  inhibition assay. The currently approved therapies for AD are based on improving cholinergic transmission via the inhibition of ChE which only provide fair improvement in memory and cognitive functions. This study shows that, out of eight tested species, the leaves of


To explore the feasibility of sacral-2-alar  screw placement by measuring the length, diameter, and angle of the screw trajectory on computed tomography (CT).This study selected 100 Han-nationality adults in northern China with a normal spine and pelvis. CT data were imported into PHILIPS software for reconstructing the 3D digital images. The optimal S2-alar screw trajectory was imitated on CT. Parameters including the length of the screw trajectory, sagittal angle, coronal angle, distance between the entry point and the spinous process, and minimum diameter of the screw trajectory were measured to evaluate the application of S2-alar screws.In total, 48 males and 52 females were included. The average length of the left screw trajectory was 47.18 ± 3.91 mm. The sagittal angle was 29.06 ± 4.00°. The coronal angle was 13.31 ± 6.95°. The distance between the entry point and the spinous process was 21.0 (3.7) mm. The minimum diameter of the screw trajectory was 17.1 (2.3) mm. The average length of the right screw trajectory was 45.46 ± 4.37 mm. The sagittal angle was 23.33 ± 4.26°. The coronal angle was 14.88 ± 6.84°. The distance between the entry point and the spinous process was 22.8 (2.9) mm. The minimum diameter of the screw trajectory was 16.9 (3.1) mm. In women, the average length of the left screw trajectory was 44.80 ± 3.66 mm. The sagittal angle was 32.14 ± 5.48°. The coronal angle was 16.04 ± 7.74°. The distance between the entry point and the spinous process was 21.8 (2.8) mm. The minimum diameter of the screw trajectory was 17.1 (5) mm. The average length of the right screw trajectory was 44.01 ± 3.72 mm. The sagittal angle was 25.12 ± 5.19. The coronal angle was 16.67 ± 8.34°. The distance between the entry point and the spinous process was 21.6 (2.7) mm. The minimum diameter of the screw trajectory was 17 (4.5) mm. As seen from the data, there were significant differences in the minimum diameter of the screw trajectory in both males and females. In females, there were also significant differences between the left and right sides in the coronal angle. Between males and females, there were statistically significant differences in the length of the screw trajectory. There were no statistically significant differences in the other parameters between males and females.The optimal screw trajectory of the S2-alar screw can be found on CT. The length and deflection angle of the screw meet the clinical requirements. This method is easy to perform and feasible for clinical application. Lumbosacral fusion procedures have been widely used in clinical practice. In the treatment of moderate-severe lumbar spondylolisthesis and degenerative scoliosis deformity, standalone sacral-1 (S1) screws have a higher rate of fixation failure. This is because (1) the stress of the distal screw is large; 2) the pedicle of the S1 vertebral arch is wide, while the screw is relatively short; and (3) the vertebra contains cancellous bone with less supporting strength. The clinical application of conventional sacral-2-alar  screws is limited due to their weaker pullout resistance. We found that the S2-alar screw trajectory became longer when the screw was placed on the sides of the sacral wings and had a larger angle in the sagittal plane. Therefore, we put forward the application of S2-alar screws. By increasing the screw angle and deflecting the head of the screw to the outside, the screw will cover a longer distance in the sacrum [ the pediWe randomly screened 100 Han-nationality adults in northern China. They included 48 men and 52 women. The mean age was 46.25 ± 14.95 years. The inclusion criteria were a basically normal spine and pelvis and no obvious scoliosis or kyphosis. The exclusion criteria were obvious radiographic scoliosis, kyphosis, transitional vertebra, or other malformations.The spine and pelvis were scanned by CT, and three-dimensional (3D) reconstruction was performed. The power was set at 120 kV and 300 mA. The rotation time was 750 ms. The slice thickness was 1 mm. CT data of lumbar-2 to the pelvis were imported into PHILIPS software  for reconstructing the 3D digital images. The entry point was selected as the midpoint between the lateral border of the S1 and S2 foramens. Directing towards the sacral wing, the screw trajectory with the maximal screw distance in the sacrum was imitated as the optimal trajectory. Through repeated imitation by CT, it was concluded that the maximal bony screw trajectory could be reached with a proper angle in the sagittal and coronal planes. The distance between the entry point and the spinous process was measured to ensure normal installation of the connecting rod. The minimum diameter of the expected screw trajectory was measured to ensure normal installation of the screws.The measurements were performed by two independent investigators. The data were collected and averaged.The arrow in Fig. The transverse diameter and sagittal diameter of the trajectory can meet the requirements of the screw Fig. . The entThe caudal trajectory angulation in the coronal plane was measured to meet the needs of screw placement in clinical practice Fig. .Fig. 4CoThe caudal trajectory angulation in the sagittal plane was measured to meet the needs of screw placement in clinical practice Fig. .Fig. 5SaThe vertical distance between the entry point and the middle line of the spinous process was measured to verify the accuracy of the entry point and to estimate the convenience of installation of the connecting rod Fig. .Fig. 6ThThe minimum diameter of the imitated screw trajectory was measured to ensure safe placement of the screw with a diameter of 4.5–6.5 mm without cutting Fig. .Fig. 7Tht test was used for data conforming to a normal distribution, and the Mann–Whitney U test was used for those not conforming to a normal distribution. For the comparison of left- and right-side parameters, the paired t test was used for data in line with the normal distribution, while the Wilcoxon signed rank sum test was used for those not in line with the normal distribution. The statistical significance was set at P < 0.05. All data were analyzed using SPSS.The Shapiro–Wilk method was used to test the normality of continuous variables. Variables that fit the normal distribution are described as the mean ± standard deviation. Variables that do not fit the normal distribution are described as medians . For the possible differences in parameters between sexes, an independent sample We measured the parameters of 100 normal subjects on CT to obtain S2-alar screw placement data. Forty-eight males and 52 females were included. In males, the average length of the left screw trajectory was 47.18 ± 3.91 mm. The sagittal angle was 29.06 ± 4.00°. The coronal angle was 13.31 ± 6.95°. The distance between the entry point and the spinous process was 21.0 (3.7) mm. The minimum diameter of the screw trajectory was 17.1 (2.3) mm. The average length of the right screw trajectory was 45.46 ± 4.37 mm. The sagittal angle was 23.33 ± 4.26°. The coronal angle was 14.88 ± 6.84°. The distance between the entry point and the spinous process was 22.8 (2.9) mm. The minimum diameter of the screw trajectory was 16.9 (3.1) mm. In women, the average length of the left screw trajectory was 44.80 ± 3.66 mm. The sagittal angle was 32.14 ± 5.48°. The coronal angle was 16.04 ± 7.74°. The distance between the entry point and the spinous process was 21.8 (2.8) mm. The minimum diameter of the screw trajectory was 17.1 (5) mm. The average length of the right screw trajectory was 44.01 ± 3.72 mm. The sagittal angle was 25.12 ± 5.19. The coronal angle was 16.67 ± 8.34°. The distance between the entry point and the spinous process was 21.6 (2.7) mm. The minimum diameter of the screw trajectory was 17 (4.5) mm  screws were developed. The latter two have high reliability of internal fixation due to tricortical fixation. S2AI screws are especially popular because of their low notch and firm fixation. Both iliac screws and S2AI screws force the sacroiliac joint or directly fix the sacroiliac joint. Degeneration and fusion of the sacroiliac joint, as well as pain, are potential risks. S2-alar screws have been clinically applied since the 1980s and have received attention from and been researched by clinical peers. However, due to the short bony screw trajectory and insufficient holding force, the clinical application of traditional S2-alar screws must be abandoned. The development of sacral screws, from the initial Galveston iliac rod, Jackson sacral rod, and Kostuik trans-iliac rod to the later spine-iliac screw and trans-S2 sacroiliac screw , has resIn this paper, the radiographic measurements of S2-alar screws were described.The entry point was selected as the midpoint between the lateral border of the S1 and S2 foramens. Directing towards the anterolateral of the sacral wing, because the anterolateral sacral wing has plenty of bone and ample space, the S2-alar screw did not pass through the sacroiliac joint. Its length and biomechanics might meet the requirements of screw placement. In this way, it could avoid stress or damage to the sacroiliac joint in the distal fixation at long segments and might effectively prevent or reduce later pain or degeneration of the sacroiliac joint and decrease postoperative complications. Our results showed that by adjusting the screw trajectory and deflecting the fixed S2 screw, the length of the screw trajectory reached 45.34 ± 3.96 mm so that there was enough holding force to improve the drawbacks of traditional S2 screws. S2-alar screw placement is relatively simple, safe, and stable.The sacrum is an irregular bone, and the S2-alar screw is a new screw placement method. 3D printing is also a method to improve the patient-doctor relationship and to avoid liabilities. Explaining the surgery to the patient using 3D models could improve his preoperative emotional status and could also help him overcome the fear of surgery.Such models could also be used in defensive medical decision-making .This study is only a preliminary work for the design and improvement of S2-alar screws. The results confirmed the feasibility of S2-alar screw placement. In the next step, we will conduct further finite element analysis and biomechanical experiments to confirm the reliability and stability of the improved S2-alar screw.Through the above-described measurements, we found that there were few individual differences in the parameters of S2-alar screw placement. Although there were significant differences in the minimum diameter and length of the screw trajectory between males and females, all individuals could obtain the optimal screw trajectory. Based on the previous data, we proposed a scheme of S2-alar screw placement. By designing the screw trajectory and measuring the parameters, it was proven that the S2-alar screw could be applied to most people, which provides the radiographic basis for further verification of its clinical effectiveness.


Neurotransmitter sodium symporters (NSS) are a subfamily of SLC6 transporters responsible for regulating neurotransmitter signalling. They are a major target for psychoactive substances including antidepressants and drugs of abuse, prompting substantial research into their modulation and structure-function dynamics. Recently, a series of allosteric transport inhibitors have been identified, which may reduce side effect profiles, compared to orthosteric inhibitors. Allosteric inhibitors are also likely to provide different clearance kinetics compared to competitive inhibitors and potentially better clinical outcomes. Crystal structures and homology models have identified several allosteric modulatory sites on NSS including the vestibule allosteric site (VAS), lipid allosteric site (LAS) and cholesterol binding site (CHOL1). Whilst the architecture of eukaryotic NSS is generally well conserved there are differences in regions that form the VAS, LAS, and CHOL1. Here, we describe ligand-protein interactions that stabilize binding in each allosteric site and explore how differences between transporters could be exploited to generate NSS specific compounds with an emphasis on GlyT2 modulation. Neurotransmitter sodium symporters (NSS) are secondary active transporters that regulate synaptic concentrations of neurotransmitters via reuptake into surrounding glial cells or presynaptic terminals. Members of the solute carrier 6 (SLC6) family act on a broad range of neurotransmitter substrates: glycine (GlyTs), dopamine (DAT), serotonin (SERT), noradrenaline (NET) and γ-aminobutyric acid  . ImpaireAquifex aeolicus bacterial leucine transporter (LeuT) and supplemented by recently solved structures of the eukaryotic Drosophila melanogaster dopamine transporter (dDAT), human serotonin (hSERT) and human glycine transporter type 1 (hGlyT1)  . Substra(hGlyT1) . Crystal(hGlyT1) . Current(hGlyT1) . This isThe structure and function of SLC6 neurotransmitter transporters has been extensively reviewed elsewhere  and thusThe substrate site (S1) of the SLC6 family is located approximately halfway across the membrane and is formed by TMs 3 and 8 and the unwound regions of TMs 1 and 6 . This hyThere has been debate over whether there is a second substrate site (S2) located in the extracellular-facing vestibule above the S1 site. In LeuT, dissociation of sodium from the second sodium site (Na2) allosterically modulates the transporter, allowing cytoplasmic release of leucine from S1 . It has + and Cl−. SERT, DAT, and GlyT1 couple the transport of substrate with the co-transport of 2Na+ and 1Cl−  that mediates this interaction. Mutating this residue in GAT1 (S331D/E), GAT4 (S340E), DAT (S375E), and GlyT1 (S339D/E) results in a loss of chloride dependence  that is analgesic in rodent models of neuropathic pain  and superfusion of lamina II neurons in the dorsal horn of rat spinal cord slices delays the decay of glycinergic dependent currents without altering their amplitude (50 = 8 μM) but achieves complete inhibition compared to the partial inhibition observed with NAGly (50= 11.9 μM) but maintains a similar level of inhibition as NAGly (50 = 340 nM) has a C18 monounsaturated acyl tail and is the most potent acylcarnitine whilst di-unsaturated or fully saturated tails do not generate substantial inhibition (50 = 880 nM) compared to NAGly but is not as potent as OLCarn (N-arachidonyl glycine (NAGly) is an endogenous lipid with a 20-carbon (C20) polyunsaturated tail conjugated to a glycine headgroup. NAGly is most concentrated in the spinal cord and has been proposed to regulate nociceptive pathways with studies showing that intrathecal administration is analgesic in rodent neuropathic and inflammatory pain models . NAGly implitude . Togetheth NAGly . N-arachas NAGly . Differias NAGly . Screenihibition . Similarhibition . The infs OLCarn . Thus, bcis configuration are active and lipids with double bonds in the trans configuration are inactive  was used for in vivo experiments and found to be analgesic in a rat model of chronic neuropathic pain, highlighting the promise of GlyT2 as a therapeutic target for analgesia  that modulate transport activity have been identified in NSS family members . High se


Due to the anatomic structure of the pelvis, free-hand placement of screws in the acetabular fracture management can be difficult. Infra-acetabular screw fixation increases acetabular stability by distal fixation of the cup. Aim of this cadaveric study is to investigate if a plate-referenced drill guide can provide save placement of an infra-acetabular screw over a precontoured suprapectineal quadrilateral buttress plate (SQBP).  We constructed a drill guide for an infra-acetabular screw based on the surface of an anatomically precontoured SQBP. A total of 12 adult cadaveric acetabular specimens were used for drill guide-assisted placement of the infra-acetabular screw. The drill guide contains a radiopaque spiral to allow longitudinal fine adjustment of the SQBP along the pelvic brim to assure correct position of the plate-drill-guide construct in relation to the Koehler's teardrop. After screw placement, we conducted a computed tomography (CT) scan of all specimens to assess the actual position of the screw in relation of the infra-acetabular corridor and the acetabular joint surface.  The position of the screw was within the infra-acetabular corridor in all cases. We did not see any intra-articular or intrapelvic screw penetration. The mean distance of the centerline of the screw to the medial border of the infra-acetabular corridor was 3.35 mm. The secure distance to the virtual surface of the femoral head to was 7.3 mm.  A plate-referenced drill guide can provide safe placement of an infra-acetabular screw for treatment of acetabular fractures. Radiographic fine adjustment is necessary to access the optimal entry point. Due to the anatomic situation of the pelvis and acetabulum, management of an acetabular fracture is a technically demanding procedure. The close relationship to the iliac vessels and the complex construction of the acetabulum make it challenging to achieve reduction and fixation of the anterior and posterior acetabular column.Demographic changes in industrial societies with overaging of the population have led to a shift in the typical fracture distribution. Large registry-based epidemiologic studies have demonstrated a rising number of acetabular fractures involving the anterior column over the last 20 years . LimitedThe infra-acetabular screw following Letournel's , 12 concThe purpose of the study is to investigate if a plate-referenced drill guide can provide save placement of an infra-acetabular screw over a precontoured suprapectineal quadrilateral buttress plate (SQBP).A total of 12 adult cadaveric acetabular specimens (6 pelvises) preserved by Thiel's method  were useThe specimens were dissected and stripped of the surrounding soft tissue. The hip joint capsule was dissected at level of the capsule-labral junction .We matched a 3D-CAD model of the anatomically precontoured plate to a mean 3D surface model of 516 pelvises . The shape of the surgical drill guide used in this study reflected the surface of the anatomically precontoured plate and had an optimal fit to the plate to assure a stable attachment of drill guide and plate. The drill guide was designed to contain the measurements determined in a recently published study on the ideal position of the infra-acetabular screw. Accordingly, the ideal entry point was 10.2 mm below and 10.4 mm medial to the highest point of the convexity of the plate reflecting the IPE. The angulation of the drill guide was in 10.4° to the axial plane and 71.4° to the sagittal plane of the pelvic inlet plane of the 3D model. The drill guide was constructed by additive manufacturing in a 3D-printing process.For referencing of the drill guide position, we used a suprapectineal quadrilateral buttress plate (SQBP) . The plate was placed aiming for an optimal fit of the anatomical precontoured shape and fixed starting with a parasymphysial screw and pressing the plate laterally to achieve an adequate fit on the pelvic brim to assure alignment with the pelvic inlet plane . Prelimihttps://otaonline.org/industry-partners/stryker/multimedia/16879679/pro-pelvis-suprapectineal-plate-placement). After secure placement of the plate, we attached the drill guide to the plate and inserted another screw in the medial part of the plate  to secure the drill guide position and to avoid mediolateral tilting of the plate. After that, a 2.5 mm hole was drilled through the drill sleeve. The drill contained a depth measuring scale which was used to determine the length of the screw.Therefore, we added a radiopaque spiral to the drill guide to assure the correct position of the plate-drill-guide construct in relation to Koehler's teardrop . A fluorAfter insertion of the screw, the plate and the screws through the plate were removed leaving only the infra-acetabular screw in place to reduce material artifacts of the computed tomography (CT) scan. Since the screw does not go through a plate hole, the plate could be removed without the risk of malplacement when reinserting the infra-acetabular screw.After drill guide-assisted placement of the infra-acetabular screw, we conducted a CT scan of all specimens to assess the actual position of the screw in relation of the infra-acetabular corridor and the acetabular joint surface. The position of the screw was measured using a picture archiving and communication system (PACS) software . The infra-acetabular corridor was evaluated in a CT reconstruction plane centered at level of the infra-acetabular corridor with an inlet angulation of 25° according to Kanezaki and Miyazaki . We charThe measurement of the relation of the femoral head and the infra-acetabular screw was identified by generating a multiplanar reformation (MPR) centered through the core of the screw and the center of the femoral head according to Egli et al.  Figure .The distance of the center of the screw to the medial wall of the infra-acetabular corridor at level of the center of the femoral head (SCD: screw-corridor distance): To measure the distance of the center of the screw to the virtual cartilage surface of the femoral head , we drew a virtual circle along the chondral border of the acetabulum to simulate the cartilage border of the head. All measurement results were recorded in mm.The cadaveric specimens were donated to the university anatomy program. Donors gave their informed written consent for the donation and anatomical dissection during lifetime. No further prehumous data like medical records were used in this study. Therefore, there was no necessity for an approval by an ethics committee.a priori sample size calculation based on a primary endpoint. Based on the low variance of values in the previous radiomorphometric study and based on other experimental studies, a sample size of 12 specimens was considered feasible and expected to have enough power [Statistical analysis was performed using the software package SPSS . Since there is no previous data, this preliminary study was designed as an exploratory pilot study without any gh power , 19. UnlThe position of the screw was within the infra-acetabular corridor in all cases. We did not see any intra-articular or intrapelvic screw penetration. The mean corridor length measured at level of the screw (IACL) was 88.4 mm (±7.4). The mean distance of the core of the screw to the medial wall of the infra-acetabular corridor (SCD) was 3.36 mm (±0.83). The secure distance between screw and the virtual femoral head cartilage surface (SFHD) was 7.30 mm (±1.71).This feasibility study demonstrated that a save placement of an infra-acetabular screw is possible by a plate-referenced drill guide over a precontoured SQBP. The entry point and angulation of the drill guide enabled save advancement of the drill directly into the infra-acetabular corridor. However, radiographic fine adjustment of the plate-drill-guide construct is indispensable to assure correct placement of the plate along the pelvic brim.The secure distance between screw and the virtual femoral head cartilage surface was 7.30 mm (SFHD).Demographic changes lead to an overaging of industrial societies and a rising number of geriatric fractures with a typical fracture pattern due to reduced bone quality in elderly , 20, 21.Based on the quadrilateral screw described by Letournel , CulemanIn cases of reduced bone quality, a simple fall can cause a fracture of the anteromedial acetabulum or the quadrilateral surface by load transmission through the major trochanter . A lack Intra-articular screw penetration can cause a cartilage damage of the femoral head. We did not see any intra-articular screw position. The CT measurements revealed a secure distance of the screw to the femoral cartilage surface of 7.30 mm (SFHD). The diameter of the infra-acetabular corridor at level of the femoral head center was 6.72 mm.Recently, a large biomorphometric CT-based study identified a viable infra-acetabular corridor with a diameter over 5 mm in 93% of specimens . Due to Instead of referencing the drill guide to the bony surface that might vary between each patient, the drill guide was referenced to a SQBP with a standardized surface. Using the plate as a reference, the interface between drill guide and plate allows an accurate fit without any deviation of the drill. Altering the level of patient-specific adjustment to the interface between plate and bone surface enlarges contact surface minimizing the risk of discrepancy of drilling direction in regard of the infra-acetabular corridor. In addition, conducting the drill process after insertion of the plate allows more room of action for the surgeon. The window of the intrapelvic approach provides a good overview and access to the anterior column and the quadrilateral surface. However, it can be difficult to manipulate with bulky instruments in the depth of the pelvis. Assembling the plate drill guide like a modular construct in situ might have an advantage over a large monobloc drill guide detecting the bone surface directly. The plate is constructed of annealed stainless steel which allows bending of the plate according to the bony surface. This implies the risk of mismatch of the drill guide and the plate when adaption by bending. His bending can also occur unintentionally by screw insertion and compression of the plate bone interface This has to be taken into account as a potential cause of mismatch of the plate-drill-guide interface and consequentially misguidance of the drill. However, most of the deflection of the plate takes place on the edges of the plate and the triangular shape of the plate at level of the drill guide prevents bending at this area. We did not observe any deflection of the plate at level of the plate-drill-guide interface leading to a mismatch or deviation of the drill. There is also a trend towards personalized medicine. Several pilot studies have proven advantages of preoperative 3D model-based plate bending for acetabulum fractures , 32, 33.There is an ongoing debate about “safe zones” and “un-safe zones” in acetabular fracture management . The entThis study has some limitations. We manufactured the drill guide based on the SQBP that is adapted to a mean pelvic model based on 516 pelvic 3D CT scans of healthy subjects. This construction might not be suitable for patients with extreme skeletal dysplasia or degeneration of the hip joint. However, the pelvises used in this cadaveric study were of geriatric subjects with degenerative changes with a deep acetabular fossa narrowing the infra-acetabular canal. Even in these cases, we did not record any intra-articular screw penetration. Another limitation is that the drill guide was applied in not fractured acetabular cadavers. Incongruence of the infra-acetabular corridor due to insufficient reduction in a fracture situation can lead to occlusion of the designated screw canal or discrepancy of the reference at level of the quadrilateral surface. The limited number of cadavers that we used in this study reduces generalizability. Intention of this preliminary pilot study was to prove the principle of SQBP-based drill guide screw placement and to gain practical experience.Detection of longitudinal displaceability of the SQBP along the pelvic brim in direction of the linea terminalis was one of the major findings that can only be detected in practical application. Although we did not observe any deformation of the plate, this could lead to a mismatch of the plate and drill guide leading to deviation of the drill.Further studies are necessary to proof reliability in a large number of cases and in clinical application.A plate-referenced drill guide can provide safe placement of an infra-acetabular screw for treatment of acetabular fractures. Radiographic fine adjustment is necessary to access the optimal entry. The secure distance between screw and the virtual femoral head cartilage surface was 7.30 mm (SFHD). Further studies are needed to proof reliability in clinics.


In vivo studies showed increased activating phosphorylation of AMPKα (Thr172) and elevated content of LC3B, a known marker of autophagy, in luteal tissue during PGF2α-induced luteolysis. In vitro, AMPK activators 1) stimulated phosphorylation of regulatory associated protein of MTOR (RPTOR) leading to decreased activity of MTOR, 2) increased phosphorylation of Unc-51-Like Kinase 1 (ULK1) and Beclin 1 (BECN1), at sites specific for AMPK and required for autophagy initiation, 3) increased levels of LC3B, and 4) enhanced colocalization of autophagosomes with lysosomes indicating elevated autophagy. In contrast, LH/PKA signaling in luteal cells 1) reduced activation of AMPKα and phosphorylation of RPTOR, 2) elevated MTOR activity, 3) stimulated phosphorylation of ULK1 at site required for ULK1 inactivation, and 4) inhibited autophagosome formation as reflected by reduced content of LC3B-II. Pretreatment with AICAR, a pharmacological activator of AMPK, inhibited LH-mediated effects on RPTOR, ULK1 and BECN1. Our results indicate that luteotrophic signaling via LH/PKA/MTOR inhibits, while luteolytic signaling via PGF2α/Ca2+/AMPK activates key signaling pathways involved in luteal cell autophagy.In the absence of pregnancy the ovarian corpus luteum undergoes regression, a process characterized by decreased production of progesterone and structural luteolysis involving apoptosis. Autophagy has been observed in the corpus luteum during luteal regression. Autophagy is a self-degradative process important for balancing sources of cellular energy at critical times in development and in response to nutrient stress, but it can also lead to apoptosis. Mechanistic target of rapamycin (MTOR) and 5′ AMP-activated protein kinase (AMPK), key players in autophagy, are known to inhibit or activate autophagy, respectively. Here, we analyzed the signaling pathways regulating the initiation of autophagy in bovine luteal cells.  The corpus luteum is a transient endocrine gland crucial for the establishment and maintenance of pregnancy. Luteinizing hormone (LH), a gonadotropin produced by the pituitary gland, regulates the formation, development, and maintenance of the corpus luteum . A main The luteolytic action of PGF2α is mediated by binding to its G-protein coupled receptor and activating Gαq/11, which ultimately evokes multiple intracellular signals such as activation of phospholipases C and A2, elevation of intracellular calcium ions, and activation of calmodulin, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) signaling cascades . These pAutophagy is the so called “self-eating” process during which cells digest damaged or old organelles in order to recycle intracellular content providing building blocks required to maintain basic cellular functions . Due to The process of autophagy is oppositely regulated by two main intracellular protein kinases i.e., 5’ AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin kinase (MTOR) . AMPK acIn the ovary, autophagy has been associated with follicle development and atresia as well as luteal formation and regression . HoweverAntibodies used for this study are listed in All animal procedures were approved by the IACUC at the University of Nebraska-Lincoln. Non-lactating crossbred beef cows between 2 and 6 years in age  from the UNL Physiology research and teaching herds were used. To synchronize animal estrous cycles PGF2α  was injected on Day 1 with a subsequent injection of GnRH 36 h later. Ovulation occurs 24 h post-GnRH injection, and on day 13.5 cows will have fully functional mid-luteal phase corpus luteum, equivalent to day 10 in the natural cycle. Cows were fasted for 12–18 h before ovariectomy but retained ad libitum access to water. At day 13.5 cows were treated with an intra-muscular injection of saline (n = 3) or PGF2α  (n = 3). Transrectal ultrasonography was performed immediately before ovariectomy to confirm the presence of corpus luteum. Ovaries were removed via a right-flank approach paralumbar fossa laparotomy, to avoid the rumen and prevent internal hemorrhaging, as previously detailed . Portion2. Two hours prior to the addition of treatments, the medium was removed, cells were washed in warm phosphate-buffered saline (PBS) and equilibrated in serum-free basal medium.Cells were isolated as previously described . OvariesCell lysis and Western blot analysis were performed as previously published . Lysates5 cells/well on cell culture imaging dishes equipped with #1.5 glass bottom slide and maintained at 37°C in an atmosphere of 95% humidified air and 5% CO2. Adhered luteal cells were equilibrated in fresh culture medium enriched with 1% BSA for 2 h prior to stimulation with LH (10 ng/ml) or AICAR (1 mM) for 3 h. Organelle specific dyes, CYTO-ID® Green Detection Reagent (autophagosomes), Mitotracker Deep Red (mitochondria), Lysotracker (lysosomes), and LipiBlue (lipid droplets) were added to each well according to manufacturer’s protocol and maintained at 37°C in an atmosphere of 95% humidified air and 5% CO2 for 30 min. Following incubation, cells were fixed for 30 min with 200 µl 4% paraformaldehyde at 4°C and rinsed three times with PBS.Confocal microscopy was used to evaluate the effects of LH and AICAR on the presence and regulation of autophagosomes in bovine luteal cells. Mixed luteal cells were seeded at a density of 3 × 10Images were collected using a Zeiss LSM800 confocal microscope with Airyscan equipped with a 20× (0.8 N.A) and ×63 oil immersion objective (1.4 N.A) and acquisition image size of 1,024 × 1,024 pixel . The appropriate filters were used to excite each fluorophore and emission of light was collected between 450 and 1,000 nm. Cells were randomly selected, and z-stacked (0.3 µm) images were generated from bottom to top of each experiment. To determine the effects of LH or AICAR on mean fluorescence intensity of CYTO-ID® Green Detection Reagent, images were converted to maximum intensity projections and processed utilizing ImageJ  analysis software. Mean fluorescence intensity was determined as previously described .p < 0.05.The data are presented as the means ± standard error of the mean (SEM) from multiple experiments . GraphPad Prism v. 8.0 software was used for statistical analysis. Data were analyzed by either Student’s t-test, or one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison tests. Statistical significance was determined as In vivo experiments showed an elevated (p < 0.05) phosphorylation of AMPKα at Thr172, a site required for AMPK activity, in luteal tissue post-PGF2α injection. Simultaneously, we observed increased (p < 0.05) content of LC3B in luteal tissue of cows injected with PGF2α in comparison to untreated animals  in phosphorylation of AMPKα Thr172 in cells incubated with PGF2α  time-dependent phosphorylation of the activation loop of AMPKα (Thr172) in comparison with control cells confirming the effective action of AMPK activity in the luteal cells. Simultaneously, AICAR rapidly increased (p < 0.01) phosphorylation of RPTOR at Ser792, a site that inhibits MTOR activation  in luteal cells. Treatment of luteal cells with AICAR (1 mM) induced  and then treated with AICAR. We confirmed enhanced phosphorylation of AMPK, RPTOR, ULK1 and BECN1 in cells treated with AICAR . Pretreap < 0.001) activating phosphorylation of AMPKα (Thr172) by 70%. Simultaneously, the LH reduced (p < 0.001) phosphorylation of the AMPK substrate RPTOR (Ser792) by 67%. Increasing concentrations of LH provoked concentration-dependent elevations (p < 0.01) in the phosphorylation of MTOR (Ser2448) and two MTOR substrates, ULK1 (Ser757) and p70S6K (Thr389) in small luteal cells  phosphorylation of AMPKα (Thr172) and RPTOR (Ser792), a site phosphorylated by AMPK. Forskolin elevated (p < 0.05) phosphorylation of AMPK (Ser485), a site specific for PKA and associated with reduced AMPK activity. FSK also enhanced phosphorylation of MTOR (Ser2448) and its substrates ULK1 (Ser757) and p70S6K (Thr389) , an activator of adenylyl cyclase. Similar to LH, treatment with FSK reduced ((Thr389) .p < 0.01) phosphorylation of AMPKα at Thr172 and RPTOR at Ser792, but elevated (p < 0.01) phosphorylation of ULK1 at Ser757. In contrast, treatment with the PKA inhibitor H89 abrogated effects of LH on phosphorylation of AMPKα, RPTOR and ULK1 , and then treated with LH for 10 min. Treatment with LH decreased (and ULK1  confirmip < 0.05) phosphorylation of ULK1 (Ser757), but pretreatment with rapamycin prevented LH-mediated effects on phosphorylation of ULK1  content of LC3B-II in comparison to untreated cells and cells pretreated with chloroquine , whereas LH decreased (p < 0.05), amount of autophagosomes in luteal cells as evidenced by Cyto-ID staining . In contrast, in cells treated with AICAR there was strong co-localization of lysosomes (lysotracker) and autophagosomes (Cyto-ID), but not mitochondria (Mitotracker) and autophagosomes (Cyto-ID), reflecting enhanced autophagy in cells incubated in the presence of the AMPK activator .p < 0.05) phosphorylation on AMPKα, RPTOR, ULK1 and BECN1. Additionally, pretreatment with AICAR abolished LH-mediated effects on AMPKα (Thr172), RPTOR (Ser792), ULK1 (Ser317), and BECN1 (Ser93)   demonstrIn vivo studies revealed evidence for induction of autophagy in the corpus luteum after injection of a luteolytic concentration of PGF2α, e.g., the rapid phosphorylation of AMPKα and elevation of the autophagosome marker LC3B. In vitro studies supported previous reports that PGF2α triggers AMPK via the CAMKK2 pathway in luteal cells . AMP binds to the catalytic subunit and prevents deactivation by phosphatases and may also recruit upstream kinases that activate AMPK. AMPK is activated by kinases such as serine/threonine kinase 11  and CAMKK2, which phosphorylate AMPK at Thr172, and it is conversely deactivated by PKA-mediated phosphorylation at Ser485, as well as phosphatases . Here, wra lutea , suggestra lutea . Althougra lutea . Choi etra lutea  demonstrThe protein kinases, AMPK and MTOR, regulate activity of autophagy related proteins in opposite ways . AMPK caAutophagy is a dynamically progressing process, which includes initiation, expansion, formation of autophagosomes, and fusion of autophagosomes to the lysosome. The ULK1/ATG13/FIP200 complex is a prominent initiator of autophagy in mammalian cells . As a seThe MTOR1 complex (MTORC1) comprises MTOR, mLST8 (or GβL), PRAS40, and RPTOR. Previous studies demonstrate that gonadotropins can activate MTOR . In this2+/AMPK signaling mediate opposite and dynamic posttranslational modifications of proteins crucial for autophagy initiation in bovine luteal cells (In summary, the findings support the conclusion that LH/PKA/MTOR signaling and PGF2α/Caal cells . Activat


Based on additive manufacturing of wood flour and polyhydroxyalkanoates composites using micro-screw extrusion, device and process parameters were evaluated to achieve a reliable printing. The results show that the anisotropy of samples printed by micro-screw extrusion is less obvious than that of filament extrusion fused deposition modeling. The type of micro-screw, printing speed, layer thickness, and nozzle diameter have significant effects on the performance of printed samples. The linear relationship between the influencing parameters and the screw speed is established, therefore, the performance of printed products can be controlled by the extrusion flow rate related to screw speed. Additive manufacturing (AM) has been widely used in engineering and industry fields in recent years owing to processing of complex parts and customization, such as aircraft, dental restorations, medical implants, and automotive products ,2,3. AmoThe difference of FDM based on filament extrusion and micro-screw extrusion is analyzed to elaborate the importance of forming process. In feeding part, filament extrusion requires a specific filament diameter (1.75 or 3 mm), which restricts the diameter of the nozzle. Secondly, only a pair of gears is used to convey the filament, and the unmelted filament is used to push the molten filament to extrude. This may cause the filament to produce scrapings, break, and even slip in the fast feeding. For flexible materials, the gear clamping cannot push the molten filament and may cause the filament to bend , which rFor the heating system, the filament extrusion has only one heating zone, the filament may not keep molten during high-speed printing, which results in poor print quality. By contrast, the materials can be completely melted through three heating zones during micro-screw extrusion, thereby improving the forming quality ,13. TherMicro-screw extrusion gradually obtained a stable pressure along the extrusion direction , while fFor the deposition effect, filament extrusion deposition will produce a lot of voids between the filament , which aBased on the above differences, it is necessary to conduct further study on WF/PHA micro-screw extrusion AM to characterize the influence of various parameters.The main influencing parameters of filament extrusion FDM are raster angle, layer thickness, nozzle diameter, feed rate, infill density, and printing speed, etc. The significance of several process parameter is obtained through the literature evaluation of filament extrusion FDM: raster angle > layer thickness > printing speed > nozzle temperature ,23,24,25In this study, the forming parameters of micro-screw extrusion are divided into device and process parameters. The effects of device parameters including screw type and nozzle diameter and process parameters including raster angle, layer thickness, printing speed, and nozzle temperature on mechanical and thermal properties are evaluated. For this purpose, one-way analysis of variance (ANOVA) is used to assess the effects of various parameters on micro-screw extrusion AM, and the tensile strength is used as the evaluation index. The paper is arranged as follows. Firstly, the experimental methodology carried out in this study is briefly summarized with emphasis on specimen preparation, extrusion AM parameters and the experimental set-up. Then, the effect of the device and process parameters on printed products are summarized. Finally, conclusions of this work are outlined.The WF/PHA composite with WF content of 30 wt% used in this study were prepared by a co-rotating twin-screw extruder  with a length and diameter ratio of 40. The specific composites preparation has been described in detail in previous studies . WF/PHA Based on the parameters of previous work listed in Extrusion flow rate stability testing. The quality of the material extruded through the micro-screw extruder was tested at various temperatures and micro-screw speeds within 1 min, and each kind of sample was measured five times and the average value was taken.Melt pressure measurement of the micro-screw extruder. The melt pressure was measured by the SEN-PT series of a mini high temperature melt pressure sensor  at various temperatures and micro-screw speeds. It should be noted that ordinary filament FDM-3D printers cannot be equipped with a melt pressure sensor, while the micro-screw extrusion system designed in this study was equipped with a customized heating block for testing . The micMechanical property testing. The micro-screw extrusion 3D printer was used to print various standard samples. The mechanical properties of samples were tested according to standard testing methods. Specifically, the tensile tests and flexural tests were conducted on a universal tester  with a load cell of 5 kN. The tensile strengths of samples were measured according to GB/T 1040.2–2006 with a stretching rate of 10 mm/min. The three-point flexural tests were conducted according to GB/T 9341–2008 with a cross-head speed of 10 mm/min. Unnotched impact strength tests were conducted on an impact tester  according to GB/T 1843–2008. For each data point, at least 5 to 10 samples were tested.3) were tested according to GB/T1634–2004. The span of 64 mm, the load of 0.45 MPa, and the heating rate of 120 °C/h is used in HDT test procedure; in addition, the heat transfer medium is methyl silicone oil. Each datum is the average value obtained by testing 3 samples.Heat deflection temperature (HDT). Heat deflection temperature was tested by an HDT test equipment . Based on the test principle of three-point simply supported beam structure, the printed standard samples (80 × 10 × 4 mmScanning electron microscopy (SEM). The morphology of the impact fracture surfaces of the composites was observed by a SEM at room temperature. A SEM of Crossbeam 550  with a field emission gun and accelerating voltage of 10 kV was used to collect SEM images for the composite specimens. A gold coating was placed on the impact fracture surfaces to improve the conductivity of the specimen, and the samples were viewed perpendicularly to the fractured surface. m1 of the sample was tested first in the air, and then the sample was suspended in water to measure its mass m2. The apparent density (ρ) of the sample was calculated according to the Equation (1), and the test was repeated 5 times to get the average value.ρ is the apparent density of the sample (g/cm3), m1 is the mass of the sample in the air (g), m2 is the mass of the sample suspended in water (g), wρ is the density of water, and its value is 1 g/cm3.Density testing. A multifunctional density tester  was used to obtain the apparent density of the sample at a room temperature. During the test, the mass p), and nozzle diameter (DN), respectively, and the linear relationship is obtained by linear fitting, L = 0.08S − 0.0653 with goodness of fit (R2) of 0.9923, Vp = 3.43S + 0.135 with R2 = 0.99932, and DN = 0.235S − 0.08 with R2 = 0.93495. In this way, the forming parameters can be determined by the relationship between extrusion flow rate and screw speed. In addition, it can be seen from the Since the forming parameters based on micro-screw AM directly or indirectly affect the performance by extrusion flow rate, the printing parameters can be determined by establishing the relationship between forming parameters and extrusion flow rate. A previous study  has showThe structures of two micro-screws are shown in The two micro-screws exhibit many differences in the length of feeding section, compression section and metering section, and the extrusion flow rate (Q) depends on metering section. The Q of two micro-screws at various nozzle temperatures and screw speeds is shown in A = 0.0334S – 0.0742 with R2 = 0.95898 and QG = 0.0218S + 0.0729 with R2 = 0.94429. The Q-S curve slope of the abrupt screw is higher than that of gradient screw, therefore, the Q response of the abrupt screw is better than that of gradient screw.The reason of the pressure growth for the micro-screw extrusion is that the groove depth of the screw gradually becomes shallow, and the resistance comes from the nozzle. The peak pressure appears at the front of the metering section or the back of the compression section. As shown in Although the melt-conveying efficiency of the abrupt screw is lower than that of the gradient screw, the pressure build-up ability of the former is better. The melt pressure curves of two micro-screws at various nozzle temperatures and screw speeds are shown in The mechanical strength and modulus of samples printed by two micro-screws at the same printing parameters are shown in The heat deflection resistance of samples printed by two kinds of screws is shown in The nozzle diameter directly affects the extrusion flow rate by extrusion pulse . The mecRaster angle. The properties of abrupt screw extrusion 3D printed samples with various raster angles are shown in SEM images of the fractured cross-section of samples printed at various raster angles are shown in Layer thickness. Layer thickness has a wide range of adjustable screw speed (4–11 r/min) from Notably, when the layer thickness is 0.8 mm, the extrusion multiplier is reduced from 0.9 to 0.7 to complete the sample printing due to serious over-extrusion d. Even iIt can be concluded that the micro-screw extrusion AM is not limited to the nozzle diameter , allowinPrinting speed. The effect of printing speed on the mechanical properties are shown in Nozzle temperature. Although the temperature is a key parameter affecting the material properties, it can be seen from HDT of crystalline polymers is sensitive to thermal history . The AM One-way ANOVA is used to evaluate the effect of various parameters on the micro-screw extrusion AM, and the tensile strength is used as the evaluation index. The significance of each parameter can be obtained from the data in Printing time of a tensile sample was further evaluated to characterize the printing efficiency. The printing time of raster angle, nozzle temperature and screw type are the same (50 min). The curves of printing time at various layer thickness, printing speed and nozzle diameter of samples are shown in Overall, choosing the appropriate micro-screw extrusion AM parameters can obtain better product performance. (1)The anisotropy of micro-screw extrusion FDM was not as obvious as that of filament extrusion, which reflected in raster angle;(2)The printing speed is the most significant factor for micro-screw extrusion AM, followed by the layer thickness. Therefore, the printing speed and layer thickness should be primarily considered in the printing parameters of micro-screw extrusion FDM to obtain better surface quality and performance;(3)The structure of two kinds of micro-screw has different effect on the extrusion flow rate and melt pressure, resulting in the mechanical properties of samples printed by gradient screw are significantly higher than that of abrupt screw;(4)Although the increase in printing path can improve the surface quality, it will increase more interfaces between the melted filaments (interlayers) and reduce the mechanical properties;(5)Compared with filament extrusion FDM, higher thickness can be obtained by micro-screw extrusion. The excellent mechanical properties  were obtained with layer thickness of 0.4 mm, while the better surface quality was obtained with layer thickness of 0.3 mm. Therefore, the layer thickness should be weighed between strength and surface quality;(6)The effect of nozzle temperature on tensile strength is not significant compared with flexural strength. Specifically, the printed sample even appears delamination of deposition layers at low temperature in flexural test, however, it will lead to the degradation of WF/PHA bio-based composite at too high temperature and affecting the performance of products;(7)The influence of nozzle temperature on thermal performance is more significant than that of other parameters due to internal stress;(8)Micro-screw extrusion FDM can control the mechanical properties of products by the extrusion flow rate.In this study, the forming parameters affecting the micro-screw extrusion AM include device parameters of the screw type and nozzle diameter, and process parameters of the rater angle, layer thickness, printing speed, and nozzle temperature. These parameters are directly or indirectly related to the screw speed. The linear relationship between screw speed and forming parameters was established. The mechanical and thermal properties of various parameters for micro-screw extrusion AM were evaluated, and the differences between filament extrusion and micro-screw extrusion FDM were compared and summarized as follows:In conclusion, the effects of device and process parameters on mechanical and thermal properties of samples printed using micro-screw extrusion were comprehensively studied, which provided conditions for the AM of bio-based composites. The relationship of process-structure-property of micro-screw extrusion AM was further clarified.


Passive icephobic surfaces can provide a cost and energy efficient solution to many icing problems that are currently handled with expensive active strategies. Water-repellent surface treatments are promising candidates for this goal, but commonly studied systems, such as superhydrophobic surfaces and Slippery Liquid Infused Porous Surfaces (SLIPS), still face challenges in the stability and durability of their properties in icing environments. In this work, environmental icing conditions are simulated using an Icing Wind Tunnel, and ice adhesion is evaluated with a Centrifugal Adhesion Test. We show that superhydrophobic coral-like Silicone Nanofilament (SNF) coatings exhibit extremely low ice adhesion, to the point of spontaneous ice detachment, and good durability against successive icing cycles. Moreover, SNFs-based SLIPS show stably low ice adhesion for the whole duration of the icing test. Stability of surface properties in a cold environment is further investigated with water wettability at sub-zero surface temperature, highlighting the effect of surface chemistry on superhydrophobicity under icing conditions. Addressing the resultant loss in efficiency or outright interruption of function is costly and energy intensive, and often involves significant safety risks. For this reason, the development of passive icephobic surface treatment, i.e. treatments that can reduce ice accumulation and decrease its adhesion to the substrate without external stimuli other than gravity, wind or surface tension6, has been the subject of extensive research9. However, how ice is accreted on a surface influences significantly its ice adhesion10, which in turn is strongly dependent on the mode of ice removal13, and considerable challenges still exist on the standardization of ice adhesion testing13.Every year, icing affects several sectors of human activity, such as air15. Initial reports stated that a hydrophobic surface chemistry is beneficial against ice adhesion14, although it should be appropriately coupled with other material parameters, such as roughness and mechanical properties15. Moreover, the existence of a quasi-liquid layer on the ice surface, which confers its molecularly slippery character16, suggests that water mobility on the material surface might play a direct role on ice adhesion17. Ultimately, it has been demonstrated that ice adhesion correlates well with the practical work of adhesion γ is the surface tension of water, and 18. However, this correlation is strictly valid only for nominally smooth surfaces and relatively high ice adhesion strength (> 160 kPa)20, and a more intricate interplay of material properties is at the basis of the extremely low ice adhesion of more complex systems.Since the search for an ideal icephobic surface began in the late 1930s, it’s been debated whether water wettability plays a role on ice adhesionCassie-Baxter wetting state of the biomimetic Lotus effect21, are seen as promising anti-icing systems25. The air cushion trapped within their surface texture contributes to reducing ice accumulation by delaying freezing of impacting water droplets thanks to its low heat transfer coefficient28, and, once ice has started to accrete, facilitates its removal by promoting interfacial crack initiation30. However, many SHS are extremely sensitive to environmental conditions, such as pressure and humidity, and failed to show icephobic behavior, with the exact surface texture geometry being critical for ice adhesion32.Superhydrophobic surfaces (SHS), that owe their extreme water repellency to the 36. The presence of a stabilized liquid lubricant layer, inspired by the slipperiness of the Nepenthes pitcher plant38, grants SLIPS a water repellency that is much more stable to pressure and humidity compared to SHS, as well as a very mobile ice-substrate interface with extremely low ice adhesion strength. However, long term stability of these surfaces towards lubricant depletion under flow and frost exposure remains a significant challenge40.To overcome these shortcomings, slippery liquid infused porous surfaces (SLIPS) have been tested in anti-icing applicationsA key issue in transferring any material from fundamental research to a potential application is ensuring its ability to maintain the required function under continuous operation. Icing is no exception to this statement: a passive icephobic surface will be subjected to multiple ice accretion/ice detachment cycles during the course of a single icing event. Therefore, it’s necessary to prove not only that the ice adhesion strength to a given surface is low enough for the specific application, but also that its icephobic performance doesn’t significantly degrade over consecutive icing cycles.43. This process allows the growth of different polysilsesquioxane micro- and nanostructures with controllable shape on a variety of different substrates42, and is well suited for industrial upscaling44. The obtained coatings show excellent chemical durability42, can be easily subjected to further functionalization, and are resistant to outdoor weathering and UV exposition45. Moreover, the small scale porosity of the coatings makes them suitable to fabricate Slippery Liquid Infused Porous Surfaces (SLIPS) by infusing them with lubricants46.In the present work, superhydrophobic hierarchical coral-like Silicone Nanofilaments (SNFs) were synthesized on anodized aluminum through the vapor-phase Droplet Assisted Growth and Shaping (DAGS) routeImpacted ice was accreted from supercooled water droplets on top of the coral-like SNFs and SNFs-based SLIPS by using an icing wind tunnel (IWiT) to simulate environmental icing conditions. Ice adhesion strength on the surfaces was then evaluated with a Centrifugal Adhesion Test (CAT) throughout 5 consecutive ice accretion—ice detachment cycles, while also monitoring the evolution of their wetting behavior and, for the non-infused surfaces, their microstructure and surface chemistry. Moreover, wettability of the surfaces at low temperatures (− 10 °C) was investigated, and its relationship with ice adhesion was examined.Tested surfaces and their respective sample names are summarized in Table Figure Comparison of the EDX spectra of the samples before and after SNF coating Fig. j confirm47. Moreover, EDX mapping of F on f-SNFs, shown in Fig. EDX element mapping of the coated samples further supports the presence of a homogeneous polysilesquioxane coating Fig. e,f that Owing to the hierarchical structure and hydrophobic surface chemistry of the coral-like SNFs, the coated samples, both fluorinated (F) and non-fluorinated (S), are perfectly superhydrophobic, showing water contact angles greater than 170° and sliding angles lower than 2°. Moreover, when infused with a lubricating oil, coral-like SNFs are able to retain the lubricant and form an efficient SLIPS. The obtained slippery surfaces display water sliding angles lower than 3° for both silicone oil (LS) and perfluorinated oil (LF) infusion.48. For this reason, in general, no direct comparison between results obtained with different testing techniques can be carried out without previous calibration12. Therefore, such threshold necessarily needs to be defined for each specific testing setup based on its experimental history. For the Tampere University’s icing facilities, a first threshold for low ice adhesion materials was defined in previous work at an adhesion strength value of 50 kPa, while extremely icephobic materials were defined as those possessing ice adhesion strength values below 10 kPa48.There is no consensus on a general threshold value of ice adhesion strength for extremely icephobic materials: this stems from the fact that the measured ice adhesion is highly dependent on the specific testing conditions, such as ice type, accretion method and the ice adhesion test itself49 and of nanostructured silicone rubber measured in similar conditions (38 kPa)50. In addition to that, the values measured for SLIPS (LS and LF) lay between 10 and 20 kPa, and fall within the range observed for slippery and lubricated surfaces tested using the same IWiT and testing method51. Notably, although a direct comparison is not possible, LS and LF show values close to those observed for SLIPS with other characterization methods relative to reference surfaces47.Results of the ice adhesion testing are shown in Fig. 8. To the best of our knowledge, such behavior has not been previously reported for superhydrophobic surfaces with ice accreted in similar conditions. A similar behavior was also observed for all specimens of sample LF tested in the first and second icing cycle, but a finite value of ice adhesion strength was measured starting from the third cycle, implying a decrease in the surface icephobicity.Moreover, when testing sample F, ice detached spontaneously during handling of all the iced specimens while transitioning from the ice accretion to the ice adhesion testing phase; Supporting Fig. 51), which has been reported to cause critical failure of the anti-icing behavior of superhydrophobic surfaces53.It is also evident from Fig. 47: while, similarly to the present work, SNFs were out-performed by f-SNFs and liquid-infused SNFs, the dramatic spontaneous ice detachment observed here for coral-like f-SNFs did not occur. A number of reasons could explain this different behavior. Both works studied DAGS-derived coating, but the actual morphology of the nanostructures differed greatly, with the notable feature that the coral-like SNFs shown here possess intrinsically multi-scale hierarchical features, which should improve impalement resistance against impacting droplets and favor the stability of a superhydrophobic state, as will be discussed later54. Moreover, the two studies employed substrates with different mechanical properties, and, most of all, used different ice adhesion testing protocols. The combination of these facts makes so that the results cannot be directly compared, and ongoing effort is directed at bridging this gap. This further highlights the need for inter-laboratory cooperation to develop standardized ways of comparing different ice adhesion tests.A recent work also investigated ice adhesion on superhydrophobic and liquid-infused SNFsFurther understanding of the different mechanisms at the basis of the surfaces ice adhesion behavior can be gained from their durability, by observing how ice accretion and ice removal affected their properties. Figure 46. A corresponding increase in apparent water contact angle was observed for the same surfaces, as shown in Fig. 56. Nevertheless, while this approach does not give a full picture of the complex interplay of the interfacial tensions between the four phases present, it can still yield meaningful information on the filling state of the nanostructure, with an increase in apparent contact angle being indicative of lubricant depletion46. Indeed, this is due to the reduction in the wetting ridge height accompanying a lubricant layer height reduction56, which in turn causes the sessile water droplet to assume a more spherical shape.From Fig. 57. Assuming as a first approximation that the infused lubricant acts as an infinite liquid reservoir and that the capillary rise in the accreted ice follows Washburn’s law58, the height z reached in a given time t is given by Eq. silane , Trimethoxy(methyl)silane , Trichlorosilane , and Silicone oil  were acquired from Sigma-Aldrich (US). Sodium Hydroxide  was acquired from Merck Millipore (DE). Krytox GPL105 (viscosity 522 cSt at 20 °C) was acquired from DuPont (US). Anodized aluminum sheets  were acquired from Allega GmbH (CH). Whatman qualitative filter paper grade 1 was purchased from Sigma-Aldrich (US).3 on their surface. After this, samples were immersed for 10 s in deionized water and then copiously rinsed with running deionized water. Samples were dried with a dry N2 stream and placed in a custom 6.6 L glass desiccator42, where they were exposed for 1 h to a N2 atmosphere with controlled relative humidity RH = (36 ± 2)% at room temperature T = (22 ± 1) °C. Control over the desiccator atmosphere was achieved by flushing it with a mixture of dry and water-saturated N2, while temperature and relative humidity were measured with an EE23 sensor  at the desiccator inlet. The desiccator was then sealed, and a mixture of TMMS (2.25 mmol) and TCMS (0.75 mmol) was injected through a septum into the desiccator. The silane mixture was let react for 2 h with the humidity present in the desiccator according to the DAGS mechanism43. This process produced a coating of Coral-like Silicone Nanofilaments (SNFs) on the aluminum surface, and the obtained samples are referred to as S.Aluminum samples were etched via immersion in a 1 M NaOH aqueous solution for 90 s to form Al(OH)2 plasma chamber  at 50 W for 5 min. This step introduced OH functionalities on the SNFs surface, allowing for further modification. Samples were then placed in a custom glass desiccator, as done for the S sample preparation, and exposed for 1 h to a N2 atmosphere with controlled relative humidity RH = (25 ± 3)% at room temperature T = (22 ± 1) °C. The desiccator was then sealed and TCPFOS (0.67 mmol) was injected and let react for 16 h. This process resulted in the modification of SNFs with fluorine-containing functionalities (f-SNFs), and the obtained samples are referred to as F.S samples were activated in an OApproximately 1 µL of Silicone oil (100 cSt at 25 °C) was pipetted on the surface of S samples, which were placed vertically on grade 1 filter paper to drain excess oil for 2 days. The obtained SLIPS are referred to as LS. The same procedure was repeated with Krytox (390 cSt at 25 °C) instead of silicone oil to obtain fluorinated slippery surfaces, referred to as LF.Water static contact angle and sliding angle (roll-off angle) measurements were conducted on a Krüss DSA100 goniometer equipped with a PA3220 tilting device  with 10 μL of ultrapure water . Room temperature was (23 ± 1) °C and relative humidity was (60 ± 3)%. Measurements were carried out on at least 5 different spots on each sample, and the standard deviation of the measurements was used as uncertainty. A sliding angle of 90° was used to represent complete droplet pinning on the surface. When it was not possible to firmly deposit a droplet on the surface due to excessively low contact angle hysteresis, a conservative value of 1° was used for the sliding angle.2. The sample surface was then cooled to − 10 °C and it was let achieve thermal equilibrium for 10 min. Contact angle and sliding angle measurements were then carried out as in the room temperature wetting analysis. At least 3 different spots were analyzed for each sample.A temperature control chamber equipped with a Peltier element  was installed on the Krüss DSA100 goniometer. Sample surface temperature was monitored by a Fluke 51II thermometer  with a K-type thermocouple in contact with the sample surface, while air temperature at about 1 cm above the sample was measured with a Pt-100 temperature sensor . In order to prevent frosting on the sample surface upon cooling, a dry atmosphere was attained by continuously flushing the chamber with dry NProfile roughness measurements were performed with an optical profilometer , according to the standard ISO 4288. A 50 × magnification objective was used to image the coating areas on three different locations of the sample. The average roughness (Ra) and the mean peak to valley height (Rz) values of the roughness profile were obtained by an average of 9 measurements in different locations of the coated surface.Samples were cut to shape and fixed onto Al stubs with carbon glue . Samples were then coated with a 10 nm thick Pt layer with a Safematic CCU-010 sputter coater  using a rotating planetary stage. High-resolution field emission SEM imaging was carried out with a Zeiss GeminiSEM 450 , using an electron acceleration voltage of 5 kV. Brightness and contrast of images were subjected to minor and uniform adjustments after acquisition. Additionally, Energy Dispersive X-Ray spectroscopy (EDX) and element mapping were performed using an AZTec Advanced X-MAX80 detector  with a 5 kV voltage.T: 25 °C and 50 °C) and Krytox GPL105 (T: 50 °C and 70 °C) was determined with a capillary Ubbelohde viscometer  according to ISO 3105:1994. Temperature-kinematic viscosity relationships of the lubricants were calculated from the two experimental points following the ASTM D341-17 standard.Kinematic viscosity of silicone oil  mm2 area of the samples surface using a customized Icing Wind Tunnel (IWiT), whose operating parameters control the type of ice obtained . In the present work, mixed glaze ice was accreted for the ice adhesion test. Detailed icing wind tunnel parameters used for ice accretion can be found in previous work69, and are summarized in Table Icing testing was carried out at the icing research facilities of Tampere Universityted on a 0 × 30 mm70, where a specimen, attached to one end of a balanced glass-fiber reinforced composite beam, is spun at constant angular acceleration of 300 rpm s−1 with a radial spinning length r. Ice adhesion shear strength is calculated as in Eq. At least four parallel specimens were tested for each sample, and the average of their ice adhesion strengths was taken as the value for the sample. Uncertainty was quantified as one standard deviation. Ice adhesion properties for each ice accretion event was monitored using smooth Teflon tape  as a reference surface.Supplementary Information.