Drosophila endoribonuclease CG2145, an ortholog of mammalian EndoU that we named Age-related lipid regulator (Arlr), as a regulator of lipid homeostasis during aging. In adult adipose tissues, Arlr is necessary for maintenance of lipid storage in lipid droplets (LDs) as flies age, a phenotype that can be rescued by either high-fat or high-glucose diet. Interestingly, RNA-seq of arlr mutant adipose tissues and RIP-seq suggest that Arlr affects lipid metabolism through the degradation of the mRNAs of lipolysis genes – a model further supported by the observation that knockdown of Lsd-1, regucalcin, yip2 or CG5162, which encode genes involved in lipolysis, rescue the LD defects of arlr mutants. In addition, we characterize DendoU as a functional paralog of Arlr and show that human ENDOU can rescue arlr mutants. Altogether, our study reveals a role of ENDOU-like endonucleases as negative regulator of lipolysis.While disorders in lipid metabolism have been associated with aging and age-related diseases, how lipid metabolism is regulated during aging is poorly understood. Here, we characterize the  Fat is generally considered to have a negative effect on longevity but lipids can be beneficial for longevity. Here the authors show that fly Arlr endoribonuclease affects lipid metabolism by degrading the mRNAs of lipolysis genes. While mostly found in the adipose tissue, LDs are also present in other cells including hepatocytes, enterocytes, macrophages, and adrenocortical cells to store excess lipids. These spherical organelles are regulated by LD-associated proteins which regulate LD biogenesis and degradation, and that play a crucial role in maintaining lipid homeostasis and energy supply3.Lipid droplets (LDs) are highly dynamic organelles that contain a core of neutral lipids including triacylglycerols (TAGs) and cholesteryl esters, which are surrounded by a phospholipid monolayer7 where the curvature of ER tubules (smooth ER) catalyzes the nucleation of neutral lipids into lenses leading to nascent LD buds9. During de novo synthesis, LD size is regulated by Seipin and the Fat Storage-inducing Transmembrane (FIT) proteins, which are associated with lens formation and budding13. In addition, LD growth is regulated by TAG synthesis enzymes , which relocalize from the ER to the LD surface14. An alternative mechanism of LD growth is the fusion of existing small LDs by either coalescence or lipid exchange17.LDs originate from the bilayer endoplasmic reticulum (ER)19. LDs can also be degraded through autophagy/lipophagy20 whereby the whole LD is sequestered by autophagosomes, which is then delivered to lysosomes where it is degraded by lipases21. The lipolysis pathway prefers to hydrolyze larger-sized LDs and produces small LDs, whereas the lipophagy machinery can only engulf small LDs, inhibition of which results in accumulation of small LDs22.LD degradation is mediated by lipolysis and lipophagy pathways. During lipolysis, TAG hydrolysis is initiated by adipose triglyceride lipase (ATGL), followed by hormone-sensitive lipase (HSL) that hydrolyzes diacylglycerol (DAG) and monoacylglycerol lipase (MGL) that hydrolyzes monoacylglycerol (MAG), releasing free fatty acids for further degradation through mitochondrial or peroxisomal β-oxidation to generate ATP3. In adipocyte cell lines, FSP27/CIDEC is enriched at LD-LD contact sites and mediates directional transfer of lipids from smaller to larger LDs17. Located at the surface of LDs, PLIN1, the best characterized LD perilipin (PLIN), plays a dual role in lipid metabolism, limiting lipase access to stored TAGs in the fed state or facilitating in the fast state hormonally-stimulated lipolysis25. Phosphorylated PLIN1 serves as a scaffold for lipases to drive lipolysis24, and for proteins such as FSP27 to regulate LD growth3. Consistent with this, in Drosophila the lipid storage droplet protein 1 (Lsd-1/PLIN1) stimulates lipolysis by recruiting HSL to the LD surface28, while Lsd-2/PLIN2 promotes lipogenesis by antagonizing ATGL/Brummer (Bmm) activity30.A number of key proteins have been identified as regulators of the size and number of LDs, such as cell death-inducing DFFA-like effector c (CIDEC) proteins and perilipins32. For example, during aging a change in overall lipid metabolism has been observed, usually leading to lipid accumulation in the adipose tissue and ectopic accumulation in non-adipose tissues, which increases the risk of developing metabolic diseases33. Other studies have demonstrated that the activity of HSL is reduced in the aged adipose tissue35, which is consistent with lipid changes in aging organisms. Further, genetic studies on enzymes that regulate biosynthesis and mobilization of neutral lipids, such as diacylglycerol acyltransferase 1 (DGAT1) in mice36, or ATGL and diacylglycerol lipase (DAGL) in C. elegans and Drosophila40, support the association of increased lipolysis with longevity. On the other hand, increased TAG levels with extended lifespan are also observed along with increased fat synthesis and breakdown42, indicating that increased lipid mobilization helps extend lifespan43. In addition, lipid metabolism is regulated by highly conserved pro-longevity signaling pathways, such as insulin/insulin-like growth factor signaling (IIS) and mechanistic target of rapamycin (mTOR) signaling, as well as by dietary restriction. These pathways extend lifespan, usually through activation of lipolysis via upregulation of lipases, desaturation of fatty acids, and lipophagy18. In particular, the transcription factor FoxO, which is inhibited by IIS and activated by starvation, stimulates ATGL/Bmm and other lipases through transcription or indirect modifications to promote lipolysis46. Despite these studies, how LDs are regulated during aging is poorly understood.Understanding LD dynamics and metabolism is of great significance for lipid metabolism-associated disorders, aging, and lifespan52. We find that high expression levels of Arlr during aging are essential for lipid accumulation in LDs and that loss of arlr results in rapid lipid consumption. Mechanistically, we show that Arlr controls the homeostasis of LDs by affecting the stability of mRNAs encoding proteins involved in lipolysis. In addition, we demonstrate that both a paralog of Arlr, DendoU, and human ENDOU also act as negative regulators of lipolysis, as they can recue arlr mutants.Here, we identify the endoribonuclease (EndoU) Arlr as an age-related lipid regulator. The EndoU family proteins have a conserved RNA binding domain that cleaves single-stranded RNA harboring U-rich sequencesarlr in vivo, we analyzed the expression levels of arlr at different developmental stages. Interestingly, although arlr is expressed at all stages, expression of arlr was much higher during adult stages, which is consistent with a previous report50. Further, arlr mRNA expression was high at all adult stages examined (1 week to 5 weeks) . Further, an HA-tagged form of Arlr (Arlr-HA) also co-localized with the ER marker  Fig. . Consistarlr in adipose tissues, we generated two null mutant alleles, arlr262 and arlr364, using CRISPR/Cas9 mediated genome editing. arlr262 and arlr364 were associated with a deletion of 262 bp and 364 bp in the coding sequence region, respectively  arlr262 mutant adults showed no obvious phenotypes in body size and lipid storage  exhibited smaller LDs in both females  exhibited shorter lifespan (about 80 days) compared to the control (about 100 days)  Fig. . The medppl > UAS-arlr-HA) rescued the LD storage defects, i.e., lipid size and TAG levels  showed larger LDs . Expression of arlrN-HA in the fat body (ppl > UAS-arlrN-HA) of arlr mutants could not rescue the LD defects or TAG levels  protein with four distinct domains, a putative signal peptide, a proline-rich domain, a glycine-rich domain, and an EndoU-like domain . Under these conditions, LD sizes and numbers in ols Fig.  and TAG ols Fig. . These rarlr mutants is related to reduced nutrition by feeding flies with either a low or a high nutrition diet. arlr262 mutant female flies fed with a poor nutrition diet had a shorter lifespan (40 days compared to 50 days for the control). The median lifespan was 29 days and 40 days, respectively. In contrast, the lifespan was similar to the control  , is a Ca2+ binding protein, and in mice hepatocytes SMP30-deficiency show an accumulation of LDs56. Yip2 (yippee interacting protein 2) is a homolog of mammalian acetyl-CoA acyltransferase 2 (ACAA2), which catalyzes mitochondrial fatty acid β-oxidation57. Finally, CG5162 is a homolog of human lipase C (LIPC)58, which regulates the hydrolysis of triglycerides and phospholipids59. These four genes were upregulated in both young and old adults  flies to immunoprecipitate (IP) mRNAs using antibodies against GFP for cross-linking-based RIP-seq. In the IP group, Arlr showed RNA-binding affinity compared to the input group. Peak distribution analysis showed that 88.9% of the peaks mapped to the coding sequence (CDS) regions, which is significant higher than the expected value. Peaks mapped to mostly 5’ untranslated region (UTR), 3’UTR and intronic regions were lower than the expected value. These data suggest that Arlr preferentially binds to processed mRNAs . Both genes have the highly conserved EndoU-like domain but differ at the N terminus as DendoU does not contain a signal peptide and the proline-rich and glycine-rich domains , LDs were normal in size and their number was slightly decreased   had no effects on LDs, including LD size and TAG levels , which negatively affects translation of C/EBP homologous protein (CHOP)62. As a transcription factor, CHOP is upregulated by lipid accumulation-induced ER stress and interacts with FoxO to promote hepatic lipogenesis through activation of peroxisome proliferator-activated receptor γ (PPARγ) expression63. In addition, lipids are reduced in CHOP knockdown cell lines64. However, severe impairment of ER activates CHOP to induce lipoapoptosis66, thus inhibiting adipogenesis, probably through specific target genes67. Despite these studies, the role of EndoU proteins in lipid metabolism has remained largely unknown.Previous studies have implicated the EndoU protein family in lipid metabolism by affecting mRNA levels. The nematode homolog ENDU-2 protects germline immortality via downregulation of genes involved in lipid metabolism. However, in contrast to our findings, the lipid content is increased in 52. We found that a number of lipolytic genes are negatively regulated by Arlr via its binding to target mRNAs and that the EndoU domain is required for Arlr activity. Strikingly, knockdown of four lipolytic genes, Lsd-1, CG5162, yip2, and regucalcin, rescues the LD defects in arlr mutants  are regulated by Arlr . However, overexpressing full-length arlr or human ENDOU show normal LDs  is considered to be associated with lifespan extension76. Upon DR, flies shift their metabolism toward increasing fatty-acid synthesis and breakdown, and disruption of lipid synthesis or oxidation inhibits lifespan extension upon DR, indicating that lipid homeostasis is essential for lifespan extension42. In arlr mutants, lipid consumption is severely accelerated, indicating a disruption of lipid homeostasis, thus lifespan is reduced under standard food and starvation conditions  of deletion mutants (Bloomington Drosophila Stock Center/BDSC 78782). For targeted knockdown, overexpression and rescue experiments, the fat body-specific Gal4 drivers ppl-Gal4 (BDSC 58768) and LPP-Gal455 were used. UAS-GFP  driven by ppl-Gal4 was used as control. arlr–RNAi61997 (BDSC 61997) and arlr–RNAi14874 (Vienna Drosophila Resource Center/VDRC v14874) were used to knockdown arlr. dendoU–RNAi (VDRC v9916) was used to knockdown dendoU. Lsd-1–RNAi, regucalcin–RNAi, yip2–RNAi, and CG5162–RNAi  were used for the genetic interaction tests with arlr mutants. UAS-KDEL-mCherry, used to label the ER, was a gift from Yixian Cui .Flies were reared at 25 °C with a 12:12 h light/dark cycle on standard food unless otherwise mentioned. Ingredients of various foods are included in Supplementary Table arlr deletion mutants and UAS transgenic flies were established at the Drosophila resources and technology platform, Core Technology Facility of Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. sgRNAs targeting arlr for CRISPR/Cas9-midiated targeted mutagenesis were designed with the tools available at https://zlab.bio/guide-design-resources. Two sgRNAs (5’-ggacgcagtgatggccacgc-3’ and 5’-gccagtgaatactgtgtttg-3’) were injected into P{nos-Cas9.R}attP2 embryos. Putative transformants were crossed to the balancer strain  for 7 days. Then, DNA was extracted for PCR detection. Primers flanking the predicted lesions were designed for PCR. Two individual mutants were identified, arlr262  and arlr364 .The UAS-arlr-HA (encoding the full length 592 a.a.), UAS-arlrΔSP-HA , and UAS-arlrN-HA (encoding a truncated protein of 1–392 a.a.) were cloned by PCR. Primers are listed in Supplementary Table UAS-ENDOU-HA and UAS-dendoU-HA, the coding sequences of human ENDOU and Drosophila dendoU were de novo synthesized by Tsingke Biotechnology . Sequences were inserted into the pUASTattB vector through XbaI and EcoRI sites. All plasmids were injected into M{vas-int.Dm}ZH-2A w*; P{CaryP}attP2 (BDSC #68A4) embryos. Potential transformants (red eye) were crossed with the balancer strain  for 7 days. Subsequently, red-eye flies were picked out for DNA extraction and PCR detection.UAS transgenic lines, including arlr-GFP transgenic strain was established by Qidong Fungene Biotechnology . sgRNAs (5’-cctcattggcagcgcctatccgg-3’ and 5’-agcgcctatccggagatttgagg-3’) were designed by Chopchop (http://chopchop.cbu.uib.no/). Cas9 mRNA was transcribed using the template of a linearized plasmid containing the Cas9 cDNA (Addgene plasmid 42251). Homology 5’ and 3’ arms were amplified from the genomic DNA of arlr and linked to the backbone of the donor vector pBluescirpt SK vector (pBS) which we named “pBS-Arlr-arm”. pBS-Arlr-arm was linearized by PCR and linked to the GFP-loxp-3p3-RFP-loxp cassette (loxp-3p3-RFP-loxp was a marker with red eyes) to form the final donor construct “pBS-Arlr-GFP-LRL”. A formula of 30 μL containing 7.5 μg sgRNA, 15 μg Cas9 mRNA, and 9 μg donor plasmid in DEPC water was injected into w1118 embryos. P0 progenies were crossed with flies carrying the FM7a balancer. F1 flies were screened for RFP expression in the eyes. PCR was performed to validate the F1 flies. F2 flies from F1-positive tubes were crossed with Cre stocks  to remove the RFP marker between the two loxP sites. Flies without RFP were balanced with FM7a and kept for further study. All primers for PCR detection are listed in Supplementary Table The Fat bodies were dissected in ice-cold PBS and fixed in 4% paraformaldehyde at room temperature with gentle shaking for 15 min and washed in PBS for 15 min. Subsequently, the tissues were incubated with BODIPY493/503  or Nile Red  for 30 min at room temperature with gentle shaking in the dark and then washed in PBS for 30 min. Samples were mounted in Vectashield containing DAPI . For rabbit anti-GFP  and HA-Tag (C29F4) rabbit mAb  staining, tissues were incubated with the primary antibody overnight at 4 °C, washed in PBS for 30 min, followed by the secondary antibody goat anti-rabbit Alexa 647  or goat anti-rabbit Alexa 488  for 1 h, washed in PBS for 30 min and finally mounted in Vectashield containing DAPI. Confocal images were acquired using Zeiss LSM 800 confocal microscope with a 63X oil objective. Sequential scanning was performed every 0.2 μm for the reconstruction of the x-z and y-z sections. For immunostaining and the following experiments, including dietary tests, triglyceride, glucose and trehalose measurements, and lifespan, virgin females and males were measured separately.g for 10 min. Ten microliters of supernatant was used to measure the TAG level by Serum Triglyceride Determination Kits . Protein amounts were measured using Bradford Reagent . TAG level was normalized to the protein amount.For triglyceride measurements, ten adults were homogenized in 500 μL PBS containing 0.2% Triton X, heated at 70 °C for 5 min, and centrifuged at 18,400 × g for 10 min. The supernatant was used to measure the glucose level using Glucose Detection Kits . For trehalose measurements, ten adult flies were weighed and homogenized in 200 μL extracting solution. After standing at room temperature for 45 min, extractions were centrifuged at room temperature at 8000 × g for 10 min. The supernatant was used to measure the trehalose level using Trehalose Detection Kits . The glucose and trehalose levels were normalized to the weight. Each genotype was measured three times.For glucose measurements, ten adult flies were homogenized in 200 μL distilled water after being weighed and centrifuged at room temperature at 8000 × After eclosion, flies were reared in 4–8 vials  at 25 °C and transferred into new vials every three days. The number of survival individuals was recorded at each transfer. The survival rate was calculated by the percentage of total surviving flies. Surviving curves were generated using GraphPad Prism 9.5. Lifespan test was repeatable for each genotype and one of the two repeats was shown in the figures.arlr262 mutant flies including equal number of both males and females  were extracted from the abdomen of 5-week adults discarding the intestines and ovaries using TRIzolTM Reagent . Transcript libraries were constructed via Illumina novaseq 6000 (Illumina). Each treatment had 3 biological replicates. DESeq was used to analyze the differentially expressed genes (DEGs). Fold change ≥2 and false discover rate (FDR) < 0.01 were considered as significantly changed expression.Total RNA of control and arlr-GFP 5-week-old adult flies including equal number of males and females were dissected in ice-cold PBS, intestines and ovaries were discarded, and samples were collected in 1 mL ice-cold PBS. Tissues were centrifuged at 2000 × g for 2 min at 4 °C. Sediments containing RNAs and Arlr-GFP proteins were cross-linked by 180 μL 37% formaldehyde at room temperature and gently shaken for 15 min. To stop cross-linking, 20 μL glycine with a final concentration of 1.25 M was mixed using a vortex for 5 min. After centrifugation at 2000 × g for 2 min at 4 °C, the supernatant was discarded. Sediments were washed in 1 mL buffer A1  before centrifugation at 2000 × g for 2 min at 4 °C and 1 mL lysis buffer 1  before centrifugation at 2000 × g for 2 min at 4 °C. Tissues were resuspended in 300 μL 1% SDS lysis buffer 2 (0.5% N-lauroylasarcosine in fresh lysis buffer 1) and placed on ice for 1 h. Lysates were sonicated for 7.5 min on ice  to shear the chromatin into ~0.5 kb fragments. Sonicated lysates were centrifuged at 12,000 × g for 2 min at 4 °C, then the supernatant was retained in a new enzyme-free tubes.Abdomens from 20 g for 30 min at 4 °C. The precipitate was washed with pre-cold 75% ethanol twice before being dissolved in 20 μL enzyme-free water. The Ribo-off rRNA Depletion Kit  was used before RIP-seq to exclude the interference of ribosomal RNA. RIP-seq was performed by Novogene  using an Illumina Novaseq 6000 platform. There were 2 biological replicates for the input and IP groups. For genomic loci analysis, the bam raw document was analyzed using the Integrative Genomics Viewer (IGV) software . Potential motifs in the target sequences were predicted using Homer software .Fifty μL of the above solution was stored at −80 °C as an input control for later use. The remaining 250 μL solution was added with 2 μg anti-GFP antibody  as the IP group, remaining at 4 °C for 4 h with gentle shake. The solution was pre-washed using 30 μL protein A beads  at 4 °C overnight, and then washed by lysis buffer wash  for 4 times, 5 min each, followed by TE  for 2 times. The IP sample was incubated in 100 μL elution buffer 1  at 130 rpm for 15 min at 65 °C. The incubation of supernatant was then repeated in 150 μL elution buffer 2 . Next, both input and IP group samples were treated with 20 μg protease K at 130 rpm for 4 h at 65 °C for the reversal of cross-linking. Solutions with 1/10 volume of 3 M NaAC , 2.5 volume of pre-cold ethanol, 1 μL glycogen and 3 μL RNase inhibitor were used at −80 °C overnight for RNA purification before centrifugation at 12,000 × actin control. Other data were normalized to the internal control Rpl32. Primers are listed in Supplementary Table Following extraction of total RNAs, cDNA was synthesized using PrimeScriptTM Reagent Kit  and qRT-PCR was performed using PerfectStartTM Green qRT-PCR SuperMix (+Dye II)  using the QuantStudio 6 Flex platform. To prevent cross-linking and verify that the target genes directly bind to Arlr, we performed RIP–qRT-PCR analysis using a reagent kit without formaldehyde . These qRT-PCR data were normalized to the Lsd-1, regucalcin, yip2, and CG5162 were cloned into the pmirGLO dual-luciferase miRNA target expression vector . The full-length cDNA of arlr was cloned into a pcDNA3.1 vector . HEK293T cells  were cultured in DMEM medium  with 10% fetal bovine serum  and 1% antibiotic , and maintained in incubators with 5% CO2 at 37 °C. Cells were seeded in a 24-well plate. Transfection was performed at 70–90% confluence with Lipo8000 transfection reagent . Luciferase assays were performed 2 days after transfection using Dual-Lumi II luciferase reporter gene assay kit . Results were tested by SpectraMax i3x .Full-length cDNAs of Whole adults were homogenized in ice-cold RIPA buffer  supplemented with 100 μM protease inhibitors PMSF . Total protein concentrations were measured using the BCA assay Kit . Protein samples were diluted to equal concentrations using lysis buffer and loaded into SDS-PAGE gels for Western blot analysis. The primary antibodies were HA-Tag (C29F4) rabbit mAb  and mouse monoclonal anti-α-tubulin . The secondary antibodies were goat anti-rabbit IgG H&L (HRP)  and goat anti-mouse IgG (H + L), HRP .t test, whereas multiple comparisons between genotypes were determined by one-way ANOVA with Tukey’s or Mixed-effects analysis Dunnett’s multiple comparison test.Images were processed with Photoshop 2021. To quantify LD size, Image J V1.53 was used to measure the area of LDs on the same focal plane around the nuclei. For each genotype, a total of three adipocytes were measured in each fly sample and six flies were used. Lifespan data were subjected to survival analysis using a log-rank Mantel-Cox test and presented as survival curves. Statistical charts were produced by GraphPad Prism 9.5. Significance between two genotypes was determined by two-tailed Student’s Further information on research design is available in the Supplementary informationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting SummarySource Data


Graft fixation during cruciate ligament reconstruction using interference screws is a common and frequently used surgical technique. These interference screws are usually made of metal or bioabsorbable materials. This paper describes the development of an allograft interference screw from cortical human bone. During the design of the screw, particular attention was paid to the choice of the screw drive and the screw shape, as well as the thread shape. Based on these parameters, a prototype was designed and manufactured. Subsequently, the first biomechanical tests using a bovine model were performed. The test procedure comprised a torsion test to determine the ultimate failure torque of the screw and the insertion torque during graft fixation, as well as a pull-out test to asses the ultimate failure load of the graft fixation. The results of the biomechanical analysis showed that the mean value of the ultimate failure torque was 2633  The rupture of the anterior cruciate ligament (ACL), one of the major knee-stabilizing ligaments that connects the tibia to the femur, is one of the most common knee injuries in individuals who engage in physical activities and is the result of rapid deceleration or hyperextension ,2,3,4,5.Typically, the ACL reconstruction involves the following steps: removal of the ruptured ACL, graft harvesting (if an autograft is used), graft preparation, drilling of tibial and femoral tunnels in which the graft is placed similar to the original ACL, and graft fixation . For theIn this work, the focus is on the interference screw as an implant for graft fixation within the ACL reconstruction. These interference screws can either be made of metal or bioabsorbable materials ,20,21,22® . These allograft bone screws are used for fixation in fracture, osteotomy, and arthrodesis and therefore make additional application of non-human materials dispensable. Furthermore, these allogenic screws exhibit superior osteoconductive properties, which means they are integrated in the recipient bone by the continuous bone remodeling process, and therefore are completely converted into autologous bone [An alternative to metal or bioabsorbable screws are screws made of allograft human cortical bone, like the Shark Screwous bone ,27,28,29Therefore, the aim of this work was the development and the initial biomechanical analysis of an interference screw for ACL reconstruction made of allograft human cortical bone. For this purpose, a first prototype of the allograft interference screw was designed and manufactured. On the one hand, the focus was particularly on the design of the screw drive, since not every screw drive that is used in metal or bioabsorbable screws can be manufactured in human cortical bone  and, on At this point, it is worth mentioning a possible main advantage in terms of secondary stability of the allograft screws. Within a short time, rehydration of the freeze-dried screw leads to significant swelling. The screw diameter is thus slightly increased, which leads to an increase in compression on the graft and, thus, possibly to an increase in the stability of the fixation. In addition, the stability of the fixation should be further enhanced over time by the onset of the bone remodeling process ,27,28 anFor an interference screw, there are various requirements that must be met by the screw drive. The screw drive must be stable enough to withstand the torque that occurs when fixing the graft. Furthermore, the screw drive must allow the screw to be fully countersunk, which means that the diameter of the insertion tool must be smaller than the outer diameter of the screw. At the same time, however, the screw drive has to be as simple as possible to manufacture.®, where the wrench size is larger than the outer diameter of the screw, would be extremely suitable in terms of stability, but has the disadvantage that complete countersinking of the screws is not possible. A simple slot drive does not provide sufficient strength, and is also susceptible to tilting of the insertion tool, which could lead to unintentional damage to the implant. The internal hexagon socket would be ideally suited for this application on the one hand, and the internal hexalobular socket on the other. However, since these complex screw drives are very difficult to fabricate in cortical bone, as fabrication requires fragile micro-mills to achieve the small radii, they are of limited use for this application.Based on the above requirements, some conventional screw drives can already be excluded. A standard external hexagon head, which is also used in the Shark Screw®; second, the so-called three-bore drive, which was recently described in a prior publication [Following from the above considerations, three possible screw drives were shortlisted for further investigations. First, the so-called claw clutch, which is already used with the Shark Screwlication ; and thiIn order to make an estimation of whether the screw drives are able to withstand the torques occurring during graft fixation, a finite element analysis (FEA) using the software Fusion 360 , similarly to a previous publication , was per problem . For theThe FEA was designed as a static stress analysis with linear isotropic material properties in order to reduce the complexity of the problem and thus also processing power. Since the idea is to find the torque that the screw can just withstand , we remain in the linear range of the stress–strain diagram. This approach is therefore a good approximation ,32. The Structural constraints were applied to both the screw and the insertion tool  from which the allograft screws are made ,35, the e screws  and can e common ,38,39. TBased on these considerations, three different versions ((a), (b), and (c)) of prototypes of the allograft interference screw shown in Bovine tendons with a total length of about 14  to 9 mm a. Prior The prepared grafts were fixed at the stitched end in bovine cancellous bone blocks, which were stored at − 18 ntersunk b,c. Duri−1 until failure of the graft fixation. At the same time, the tensile force acting on the graft was measured, and the maximum value at fixation failure was recorded. The tensile force was applied in the direction of the axis of the bore tunnel, so that the worst-case scenario was simulated.In order to measure the ultimate failure load of the graft fixation, a motorized force test bench PCE-MTS 500  with an S-type load cell PCE-PFG 2K  was used. The cancellous bovine bone block in which the graft was fixed with the interference screw was clamped into a vice connected to the moving table of the force test bench. The graft was attached to the stationary upper clamp, connected to the load cell, of the force test bench using the loop resulting from the single tendon fold. For this purpose, the screw of the upper clamp was threaded through the loop of the graft. The screw was not tightened, which prevented the graft from jamming and possibly being damaged. After the specimen was installed properly d, the moThe ultimate failure torque was measured, as described in a previous publication , by clamt-tests were performed to examine significant differences in results between 7 Independent-samples In total, The mean value for the maximum insertion torque of the 7 mm graft group was In this work, an allograft interference screw manufactured from cortical human bone was developed and biomechanically analyzed. During development, special attention was paid to the design of the screw drive, the screw thread, and the screw dimensions and shape. Several biomechanical tests were performed, including measurements of the insertion torque during graft fixation, ultimate failure load of the graft fixation, and ultimate failure torque of the screw.The observed mean insertion torque of the interference screw during graft fixation was pecimens . This wapecimens .Compared to the ultimate failure torque of bioabsorbable interference screws, the allograft interference screw developed here is in the mid-range at .75 N mm . This spThe FEA in The measured mean ultimate failure load of the graft fixation with the allograft interference screw was mensions . Therefoscrew in , and thescrew in . Neverthscrew in . These dWeiss et al. performed a biomechanical analysis of ACL graft fixation in porcine and bovine models, respectively, using, among other methods, metallic interference screws with comparable dimensions (9×30 mm) . In the ained by  and by tained by . The resectively . Moreoveively [N . From thCompared to the previous study analyzing the biomechanical stability of the allograft interference screw in human proximal tibia specimens , the ult setting . At this setting .Of course, the selected transplant itself can also influence the strength of the fixation. Different folding and suturing techniques may well have an influence on the ultimate failure load of the graft fixation and the insertion torque of the screw . For theFurthermore, in agreement with previous publications , a signiThe sample size of the 9 Looking at early functional rehabilitation programs after ACL injuries, in which loads of approximately 450 reported , the priperature , rehydraperature ,27,28, aHere, it is especially important and interesting to analyze the surrounding tissue of the graft fixation in more detail. In a study, Tecklenburg et al. were able to show the differences in the healing process of ACL reconstruction between bioabsorbable and allograft interference screws over a period of two years . For theIn addition to ACL reconstruction, another interesting field of application for the allograft interference screw is ACL revision. As ACL reconstruction is a very successful operation, revision rates range from 3% to 25% . The risThis work is intended to serve as an initial starting point for more detailed future investigations, and to provide a first glimpse of the performance of an allograft interference screw for ACL reconstruction. The biomechanical tests of the allograft interference screw developed in this work showed good results in the first step, compared to conventional interference screws. In terms of ultimate failure torque, the allograft screw is in the mid-range among bioabsorbable screws. However, the primary stability of the graft fixation with the allograft interference screw (235 diameter ). The in


To develop a bidirectional slide guide to assist screw placement in the axial lamina and to preliminarily discuss the accuracy and feasibility of auxiliary screw placement.CT data from 40 randomly selected patients were imported into the software for modelling, and cross-pinning was used to simulate pinning. According to the different crossing methods of the upper and lower laminar screws, they are divided into two groups. In the software, the position of the needlepoint of each screw is accurately measured, and the needle point is kept unchanged to simulate the movable range of the screw tail under the condition that the body does not penetrate the cortical bone. The data were compared by grouping and gender. Finally, the guide was designed by combining the screw exit point and fine adjustment angle data of all patients with the centripetal principle of the slide rail.p > 0.05).The needle exit data L1/L2/L3/L4 were 6.44 ± 0.52 mm, 7.05 ± 0.48 mm, 3.55 ± 0.75 mm and 5.09 ± 0.74 mm, respectively, and the fine adjustment angle of the slide rail was 10.51° ± 0.87°. There was no significant difference between the two groups or between men and women (In this experiment, using the data obtained from the simulation of screw insertion, a two-way slide guide was designed to assist the insertion of axial laminar screws. The guide locks the screw outlet point to position and guides the screw inlet point, which improves the accuracy and safety of screw placement. In the clinic, spinal instability often occurs due to trauma, metastatic tumours, inheritance, infection, and other reasons. Atlantoaxial instability, which is located at the stress concentration of the head and neck, is the most common in the cervical vertebra, and this kind of instability often requires internal fixation surgery. The atlantoaxial position is deep, and there are important nerves and blood vessels nearby, which increases the risk and difficulty of internal fixation. At present, the main methods of posterior atlantoaxial fixation include articular process fixation, lateral mass fixation, and transpedicular internal fixation , 2. AlthIn this experiment, 40 patients with 3D CT data were randomly selected, including 19 female patients and 21 male patients, with an average height of 172.9 ± 9.5 cm and an average age of 51.1 ± 12.8 years. Patients with axial lamina defects and hypoplasia were excluded.The cervical spine CT data of 40 patients were imported into mimics21.0  software for 3D reconstruction. The reconstructed 3D model of the axis was imported into solidworks2021  for simulated screws insertion and distance and angle measurements. The screw insertion point and angle were selected by the standard screw insertion method to simulate screw insertion. (1) According to the up-and-down position of the laminar screw, it can be divided into upper laminar screws and lower laminar screws. (2) The upper lamina screw entry point is approximately 5 mm from the midline of the spinous process, and the lower laminar screw entry point is approximately 9 mm from the midline of the spinous process on the other side, all of which pass through the intersection of the lateral outer edge of the opposite lamina and articular process. Forty patients were randomly divided into two groups according to different directions of screw placement, with 20 patients in each group. In the first group, the superior laminar screw entered the left lamina, and the lateral edge of the right lamina passed out. The lower lamina screw passes through the right lamina and passes through the left lamina. The direction of screw placement in the second group was opposite to that in the first group. (3) The distance between the upper vertebral screw and the lateral edge of the vertebral plate is L1, the distance between the upper vertebral screw and the upper edge is L2, the distance between the lower vertebral screw and the lateral edge of the vertebral plate is L3, and the distance between the lower edge is L4  and printed.During the operation, the posterior arch of the atlas and the vertebral lamina of the axis are fully exposed, and the structure of the posterior part of the segment to be operated on is exposed. Then, the screw outlet socket of the guide is fixed at the intersection of the posterior edge of the vertebral lamina of the axis and articular process. The position of the guide sleeve of the slide rail is adjusted to find the ideal screw entry point. Then, the three-stage guide sleeve is locked at the screw entry point. After the position of the fixing frame is determined, Kirschner wires are inserted for fixation Fig. . Then, tAccording to statistics, the positions L1/L2/L3/L4 of the total sample size are 6.44 ± 0.52 mm, 7.05 ± 0.48 mm, 3.55 ± 0.75 mm, and 5.09 ± 0.74 mm, respectively, and the fine adjustment angle of the slide rail is 10.51° ± 0.87°. In this experiment, based on the data obtained by measuring the total sample size and the centripetal principle of the arc slide rail, a two-way slide guide was designed to assist the insertion of the axial laminar screw. The guide device comprises a fixing frame main body, a three-stage sleeve, an arc-shaped centripetal slide rail, a slide rail connecting ring, and other parts. The upper end of the fixing frame body is a spinous process locking sleeve, which can penetrate Kirschner wire to fix the guide on the spinous process, and the middle part is an arc-shaped centripetal slide rail. The guide sleeve can be fixed inside the slide rail through the slide rail connecting ring to ensure the centripetal nature of the sleeve. At the same time, the guide sleeve has a three-level structure, which can complete all screw setting operations. To avoid the grinding and collision of cross-screws, the optimal angle of cross-screws is simulated in 3D. The main body of the guide is made of metal, which reduces deformation during operation and achieves better stability.The atlanto-axis belongs to the structure of the upper cervical vertebra, and its area is deep. There are important nerves and blood vessels near the axis, so it is difficult to operate, and there is high risk of injury during the surgery. Currently, the following methods are commonly used for fixation of the axis screw, including lateral mass screws, C1/2 joint screws, C2/3 joint screws, and transpedicular fixation techniques. However, all of the above techniques have the risk of vertebral artery injury in clinical application. In 2004, Wright  proposedSince C2 vertebral plate screw fixation technology was reported, it has been recognized by clinicians because of its simple operation and high safety. However, in clinical practice, most surgical operators completely rely on anatomical markers of the lamina to locate, and the accuracy of screw placement will be affected by factors such as operation experience, fluoroscopy effect, and intraoperative posture of surgical operators. At present, it has been reported that the direction deviation of screws during surgery breaks through the abdominal wall of the vertebral plate and enters the spinal canal, resulting in high spinal cord injury . In addiThree-dimensional data simulation and synthesis technology are applied to orthopaedics. By extracting the three-dimensional CT data of the cervical vertebra and establishing a personalized auxiliary guide device, the accuracy of axial laminar screw placement can be improved to some extent. The personalized guide device is made by reconstructing the preoperative cervical spine image data to obtain a three-dimensional model. The manufacture of a personalized guide device can reconstruct the preoperative cervical spine image data of the patient to obtain a three-dimensional model. On the model, the structural data of the guide device are obtained by simulating the ideal screw track, and finally, it is achieved by processing and manufacturing special technologies such as three-dimensional printing. Three-dimensional printing process technology makes the three-dimensional model by spraying technology, which saves laser equipment in the process and can obtain faster printing speed and higher three-dimensional reduction degree. This process improves the printing efficiency and accuracy, reduces the printing cost, and controls the density of the printing model. Lu et al.  made a cAt present, the existing axial guide uses RP (rapid prototyping) technology to make the physical model –19, but In the design of this guide, many factors, such as axial lamina deformity and fracture, are considered. Forty cases of 3D modelling samples are compared according to different screw placement methods and genders, and the position of screw exit points is measured. According to the obtained data, a slide guide with a wider application range is designed. Only the screw outlet interface contacts the outer edge of the vertebral plate, and it can guide accurately even if soft tissue remains or the vertebral plate structure is damaged. The guide is a titanium frame structure with high hardness and low deformation rate, which greatly improves structural stability. Two-way slide guidance does not require repeated fluoroscopy in navigation operations, which can greatly reduce the operation time and radiation dose. Because it can be reused, it is superior to the custom guide in cost. The guide can lock the screw entry point and the screw exit point of the lamina screw at the same time and then fix the guide on the spinous process by using two cross-fixing frames, which can lock the safe screw path and stabilize the guide, thus improving the double cortex rate of screw placement and ensuring the accuracy of screw placement. The entire operation can be completed visually, which has great clinical prospects. There are also shortcomings in this study, such as the limitation of sample size and lack of data measurement of bidirectional sliding guides in actual screw placement.In summary, this guide is universal, and it has many advantages in the process of axis laminar screw placement, such as a stable main structure, firm multipoint fixation, accurate guidance, simple operation, and simultaneous placement of bilateral laminar screws. It greatly reduces the operation time, improves the operation efficiency, and provides an economical and reliable auxiliary method for the placement of axial laminar screws.According to the centripetal principle, locking the screw exit point and positioning and guiding the screw entry point can prevent the screw tip from going into the spinal canal due to the deviation in the insertion process. Two screw placement methods and data comparisons of different sexes ensure the universal applicability of this guide. With the help of spinous locking sleeves and titanium frames, the stability of screw placement is high, which improves the accuracy and safety of screw placement.


The p53 family member TAp63α plays an important role in maintaining the genetic integrity in oocytes. DNA damage, in particular DNA double strand breaks, lead to the transformation of the inhibited, only dimeric conformation into the active tetrameric one that results in the initiation of an apoptotic program. Activation requires phosphorylation by the kinase CK1 which phosphorylates TAp63α at four positions. The third phosphorylation event is the decisive step that transforms TAp63α into the active state. This third phosphorylation, however, is ~ 20 times slower than the first two phosphorylation events. This difference in the phosphorylation kinetics constitutes a safety mechanism that allows oocytes with a low degree of DNA damage to survive. So far these kinetic investigations of the phosphorylation steps have been performed with the isolated CK1 kinase domain. However, all CK1 enzymes contain C-terminal extensions that become auto-phosphorylated and inhibit the activity of the kinase. Here we have investigated the effect of auto-phosphorylation of the C-terminus in the kinase CK1δ and show that it slows down phosphorylation of the first two sites in TAp63α but basically inhibits the phosphorylation of the third site. We have identified up to ten auto-phosphorylation sites in the CK1δ C-terminal domain and show that all of them interact with the kinase domain in a “fuzzy” way in which not a single site is particularly important. Through mutation analysis we further show that hydrophobic amino acids following the phosphorylation site are important for a substrate to be able to successfully compete with the auto-inhibitory effect of the C-terminal domain. This auto-phosphorylation adds a new layer to the regulation of apoptosis in oocytes. One of the proteins that is involved in eliminating oocytes with DNA double strand breaks is p635, a member of the p53 family of transcription factors6. In mammals, oocytes spend most of their time in a quiescent state before meiosis I is completed and therefore contain a fourfold set of the genetic information. While this situation allows for efficient DNA repair via the process of homologous recombination8 the high DNA content makes oocytes also vulnerable to DNA damage. The challenge during evolution was to develop a surveillance system that effectively detects and eliminates oocytes with severe DNA damage to protect the genetic integrity but that is triggered only above a certain threshold level of damage. While many processes in oocytes are downregulated, DNA can get damaged, for example, by the remaining metabolism creating reactive molecules or by background ionizing radiation. A too sensitive trigger level for oocyte cell death would therefore endanger the survival of the species by eliminating oocytes before reproduction occurs. Several components contribute to the tight regulation of the apoptotic program in oocytes. First, TAp63α, the central transcription factor that upregulates the apoptotic program in oocytes4, adopts in resting primary oocytes an inactive state by forming a closed, only dimeric conformation9. This conformation is stabilized by interaction between the C-terminal transactivation inhibitory domain (TID)10 and residues located between the N-terminal transactivation and the DNA binding domains  and CHk2 (mainly through double strand breaks) which leads to the phosphorylation of S58218, only requiring a priming site on the substrate, for example provided by the phosphorylation of another kinase20 . In case of TAp63α, CHk2 acts as such a priming kinase. However, a situation in which CK1 only amplifies a CHk1/CHk2 signal would be highly dangerous for the long term survival of oocytes as the reproductive potential would be determined only by the activity of CHk1/CHk2. By measuring the phosphorylation kinetics of the four CK1 phosphorylation steps we could show that they follow different kinetic regimes25. While the first two phosphorylation events are very fast, the third one is ~ 20-fold slower. Interestingly, it is this third phosphorylation event that is necessary to produce the open state25 . This slow kinetics of the decisive step serves as a safety measure. Activated, tetrameric TAp63α gets degraded fast, thus when the activation is slow because of a low level of DNA damage, the oocyte survives as activated TAp63α does not accumulate. Only when the damage level is high a corresponding fast activation would outcompete degradation of activated TAp63α, leading to cell death25. The trigger level for the activation of TAp63α, however, is species dependent. In humans the LD50 dose required for elimination of 50% of the ovarian reserve is between 2 and 20 Gy26, depending on the age of the patient, while in mice the LD50 value is 0.15 Gy. In guinea pigs, an LD50 value of 4 Gy was found and primordial oocytes of Rhesus monkeys are eliminated only after exposure to 70 –120 Gy27. Despite evolutionary divergence in radiation-sensitivity, TAp63α is highly conserved in mammals with only 9 amino acids difference between mouse and humans17, suggesting that additional safety mechanisms with different threshold levels exist in individual species. One such mechanism might be regulation through the C-terminal tail of CK1 kinases which have been shown to get auto-phosphorylated and inhibit the kinase activity32. Here we investigated the effect of auto-phosphorylation in the C-terminus of CK1δ on the phosphorylation kinetics of p63and show that auto-phosphorylation can almost completely inhibit the third, decisive phosphorylation step. Interaction between the kinase domain and the auto-phosphorylated C-terminus does not require a particular site but all phosphorylated serines seem to contribute. Important for a substrate to be able to compete with the inhibitory C-terminal domain of the kinase are hydrophobic residues following the phosphorylation site and that align with a nonpolar binding site on the kinase. We have chosen to investigate CK1δ as the best characterized kinase for TAp63α for which also co-crystal structures exist25. CK1ε and CK1α can phosphorylate TAp63α as well. As auto-inhibitory effects from the C-termini of these kinases have been reported32, similar effects as reported here can be expected for their phosphorylation kinetics of TAp63α. To better distinguish amino acids in the kinase from residues in the p63 phosphorylation activation domain (PAD) peptide we will use the three letter nomenclature for the kinase and single letters for the p63 peptide.CK1 kinases belong to a group of enzymes that do not require phosphorylation in the activation loop for high activity and are therefore sometimes described as constitutively active19. For human CK1δ and its yeast homologues Hhp1 and Hhp2 it was found that phosphorylation of Thr220 located at the N-terminus of helix αG close to the substrate binding cleft results in changes in the phosphorylation kinetics33. While for most substrates the kinetics was slower in the Thr220 phosphorylated state, phosphorylation of S585 and S588 of TAp63α was surprisingly accelerated. We wanted to investigate the influence of further auto-phosphorylation events that mainly occur in the C-terminal domain of CK1δ to evaluate their potential influence on the activation of TAp63α in oocytes. We, therefore, mixed a p63 peptide containing all CK1 phosphorylation sites (G569 to R598) and primed on S582 with activated MK2 kinase17 with either the CK1δ isolated kinase domain or the full length kinase in a 1:2000 ratio (100 µM peptide concentration) and measured the phosphorylation kinetics by mass spectrometry. Here and in all following experiments we used a T586A mutant of the p63 PAD peptide. In our initial characterization of the phosphorylation kinetics we had seen that phosphorylation of T586 (which is not part of the CK1 consensus sequence) also occurs during the reaction at a slow rate. Mutating it does not change the kinetics of the four main phosphorylation sites but simplifies the analysis25. Figure 25.Phosphorylation of the kinase itself either in the kinase domain or in other domains can have a strong influence on the enzymatic activity29.Phosphorylation of the first two sites, S585 and S588, is very fast with the isolated kinase domain. To investigate if the only partial inhibition of their phosphorylation with the full length kinase is due to this very fast kinetics which occurs in parallel with the auto-phosphorylation of the C-terminus, we pre-incubated the full length CK1δ kinase with ATP for four hours before adding the p63 peptide. The phosphorylation kinetics of all four sites became only slightly slower, suggesting that the auto-phosphorylation is fast and no pre-incubation is necessary for an inhibitory effect in vitro Fig. , consist32. To confirm that these sites play a role in inhibition of TAp63α activation in our in vitro assay, we mapped phosphorylation sites using mass spectrometry. These experiments resulted in the identification of full length kinase with a maximum of ten phosphorylated residues located at Thr323, Ser331, Thr344, Ser361, Ser382, Ser384, Thr397, Ser406, Ser407 and Ser411 following incubation with ATP for four hours  resulted in only a few very minor chemical shift perturbations, suggesting that the interaction between the C-terminus and the kinase domain is very weak . To ensure that the observed chemical shift differences are not due to slight pH changes during the titration which would influence the protonation state and thus also the chemical shift, we repeated the titration experiment at pH 7.5 . As we cannot exclude that Ser370 also gets auto-phosphorylated we mutated it as well. Mutating the last four phosphorylation sites  to alanine resulted in an increase in the kinase activity relative to the wild type sequence but it was still significantly slower than the kinetics with the isolated kinase domain , protein kinase B (PKB), protein kinase Cα (PKCα), CDC-like kinase 2 (CLK2), and checkpoint kinase 1 (Chk1) in vitro and in vivo and its phosphorylation was shown to have a strong inhibitory effectain Fig. . Interesain Fig. . Mutatioain Fig. .Figure 325. The residues C-terminally following each phosphorylation site interact with a small non-polar patch (centered around Leu173), helping to position the side chain of the serine or threonine residues in the active site. For S585, A586 (or the hydrophobic part of T586 in the wild type sequence) and the side chain of V587 provide this effect. For S588 the side chain of V589 acts as the hydrophobic anchor point. With S591 in the active site, however, S592 has to occupy this position which is less favorable than interaction of this site with the hydrophobic side chains following S585 and S588. The second important interaction is that of the side chain of E593 with a basic cluster in the CK1δ kinase domain . This cluster has also been implicated in substrate selectivity for the different phosphorylation sites (degron and FASP peptides) in Per240. The interaction of E593 with the basic cluster occurs when S588 is in the active site and also helps to anchor the peptide in the optimal position for S588 phosphorylation. Moving S591 into the active site requires that this interaction is broken. Mutating these two sites (S592V and E593G) indeed significantly accelerated the phosphorylation of S591, without much effect on the other sites25. The strongest effect was seen by mutating S592 to the hydrophobic residue valine, underlining the importance for a hydrophobic residue C-terminal to the phosphorylation site. To further investigate the importance of this previous result we mutated the two residues V587 and V589 to asparagine . These mutations indeed resulted in a slower phosphorylation kinetics of S585 and a strong inhibition of the phosphorylation of S588 with the full length CK1δ  are strong binding sites for CK1δ that can effectively compete with the phosphorylated C-terminal domain while S591 is a weak site. In a previous investigation we had shown that S592 and E593 are two key residues that determine the slow kinetics of the third siteK1δ Fig. . These d23. The relevant peptide stretch  contains five consecutive phosphorylation sites with a perfect CK1 spacing (+ 3). We wanted to investigate the influence of phosphorylation in the CK1δ C-terminus on the phosphorylation kinetics of PER2 as a member of this second class of targets (un-primed) that does not show such a big gap in the kinetics between different phosphorylation sites as TAp63α. To counterbalance the low solubility of this peptide which initially prevented the use of NMR spectroscopy to follow the phosphorylation kinetics we fused the small protein immunoglobulin G binding domain 1 of Streptococcal protein G (GB1) to its N- as well as its C-terminus. GB1 is often used as a solubility enhancer42 and this construct was soluble enough for our kinetic studies. After assignment of the GB1-PER2 peptide-GB1 construct using standard NMR experiments , if certain sequence requirements are fulfilled. One such example is the PER2 protein which plays an essential role in the regulation of the circadian clockwer Fig. . A simil662 Fig.  as well 662 Fig.  show a s662 Fig. .Figure 545. Their predisposition for pre-phosphorylated sites enables them to add many phosphate groups in a row, thus amplifying the original phosphorylation signal. The requirement for such an amplification process is that phosphorylatable residues—serines or threonines—are located within a certain spacing. In case of CK1 kinases, this spacing is 3 residues in the direction of the C-terminus and in the case of the CK2 and GSK kinases three and four residues in the direction of the N-terminus, respectively46. The model that these kinases are not regulated at all has, however, changed with the characterization of the auto-phosphorylation as well as phosphorylation events by other kinases in the long, unstructured C-terminus32 as well as the observation of auto-phosphorylation within the kinase domain33. Inhibitory effects of C-terminal phosphorylations have been found for all members of the CK1 kinases, including CK1α47, CK1γ1-348, CK1δ32 and CK1ε30. In addition to auto-inhibition, phosphorylation of the C-terminal tail of CK1δ was reported for the kinases PKA, Akt, CLK2, PKC and CHK138. The distribution of the serine and threonine residues in the C-terminal domain do not follow a classical + 3 CK1 pattern. Still, previous data and the results reported here show that auto-phosphorylation of the C-terminus occurs fast and efficient. These data also suggest that all residues in the C-terminus of CK1δ that become phosphorylated contribute to auto-inhibitory effects. Our list of identified phosphorylated residues might not be complete and alternative methods to mass spectrometry might be necessary to obtain a complete overview of modified serines and threonines. For the interpretation of the data as a fuzzy interaction of the C-terminus with the kinase domain a completeness of the list is, however, not necessary.Members of the CK1, CK2 and GSK kinase families are not activated by phosphorylation in the activation loop and are often described as constitutively activehttps://doi.org/10.1101/2023.04.24.538174). However, this study found a predominant inhibitory effect for phosphorylation sites in the last 16 amino acids of the C-terminus. Interestingly, two splice forms of the CK1δ kinase differ by exactly these 16 amino acids19, suggesting that regulation of the activity of CK1δ could also be achieved by differential splicing.A very recent study of the auto-inhibitory effect of the C-terminus of CK1δ comes to similar conclusions , we have observed that the already phosphorylated sequence of the PAD peptide forms an α-helical structure and interacts via their phosphate groups with the basic pocket centered at Arg178 and Lys22425. The second basic pocket, located at Arg127, Lys154 and Lys171, interacts with the carboxyl group of E593 which contributes to the slow kinetics of the third phosphorylation site. Similar interactions of phosphorylated residues with the basic pockets were found in a recent crystal structure of the phosphorylated PER peptide in complex with the CK1δ kinase domain43.The general mechanism of the autoinhibition of the kinase activity by the C-terminal domain does not seem to be that a specific phosphorylated sequence interacts strongly with the kinase domain, thereby blocking its enzymatic activity, but that of a fuzzy relatively weak interaction of many phosphorylated sites competing with substrates for access to the active site, similar to the interaction seen between the multiple-phosphorylated disordered protein Sic1 and Cdc425. The peptide positioning for the phosphorylation of S591 thus constitutes a weak substrate with which the phosphorylated C-terminus of the kinase can compete effectively. The result is that the activation of TAp63α is further slowed down. Activated TAp63α gets degraded fast which is most likely a safety mechanism to avoid that spontaneous activation, low levels of kinase activity or low levels of DNA damage result in elimination of the oocyte.For the activation of p63 the results shown here suggest that phosphorylation of the C-terminal tail of CK1 proteins by other kinases or by auto-phosphorylation has small effects on the kinetics of the first two sites. In contrast, the third site, which is the decisive one that leads to the opening of the closed dimeric state and the formation of the active tetramer is severely impacted. The first two sites are very good substrates for the CK1δ kinase. In contrast, our previous structure determination as well as Molecular Dynamics simulations have revealed that binding of the PAD peptide in a substrate mode with S591 in the active site is energetically less favorable as it would require interaction of a serine reside (S592) with the small hydrophobic patch of the kinase around Leu173 as well as breaking of the interaction between the side chain of E593 with the basic pocket of Arg127, Lys154, Lys17132. Treatment with calyculin A, a general phosphatase inhibitor, resulted in phosphorylation of both kinases. These results suggested that CK1δ is constantly de-phosphorylated by a phosphatase in the cellular environment to keep it active. If the C-terminal domain gets constantly de-phosphorylated as these data indicate, what role could it play in regulating the activity of the kinase in oocytes? In the case of CK1δ it was shown that S370 can get phosphorylated by the stress kinase CHK1. This kinase becomes activated upon DNA damage, in particular DNA single strand breaks. Phosphorylation of CK1δ by CHk1 could lead to partial inhibition of CK1 while at the same time phosphorylating TAp63α on S582, the priming site. As our results have shown that C-terminally phosphorylated CK1δ is still able to phosphorylate S585 and S588 but not S591 such a scenario would result in the built-up of S585 and S588 phosphorylated but still dimeric TAp63α. After de-phosphorylation of CK1δ, this pool of pre-activated TAp63α could then be effectively transformed into the active tetrameric state. Building-in such a delay could be an additional safety step for the oocyte to avoid cell death in case the damage is not severe as phosphatases might not only be important for increasing the activity of CK1 kinases but also for removing phosphorylation sites on pre-activated TAp63α before phosphorylation of S59111. A regulatory effect of a phosphatase, protein phosphatase 5, has indeed been shown for the regulation of the kinase activity of CK1ε as part of the circadian clock49. The role of CK1 kinases in oocytes, however, is complex and requires further investigations. In addition to CK1δ, CK1ε and CK1α can also phosphorylate TAp63α, although inhibitor studies have suggested that CK1ε plays a less important role17. CK1 isoforms, in particular CK1α, are also involved in regulation of DSB repair in oocytes50 and effective DSB repair would prevent oocyte death. Thus the level of DNA damage that results in oocyte death that is encoded in the activation kinetics of TAp63α could be fine-tuned by several kinases (CHK1/CHK2 and different CK1 enzymes). The results reported here suggest that in addition to kinases, phosphatases might also represent important regulatory elements and could contribute to the different sensitivities of oocytes toward DNA damage observed in different species.The question remains how TAp63α can get effectively activated when auto-phosphorylation of the C-terminal domain of CK1δ effectively inhibits phosphorylation of S591. Previous investigations have shown that CK1ε and CK1δ in NIH 3T3 mouse fibroblasts are mostly unphosphorylatedRecombinant human CK1δ (gift from the laboratory of Stefan Knapp) was subcloned into the pMAL-c4x vector .MBPtag—TEVsite—Full length CK1δ—Strep-Tag). The protein, bearing a C-terminal Strep-tag, was co-expressed with lambda phosphatase in BL-21(DE3)-R3-Rosetta (SGC Oxford) in 2xYT medium. Protein expression was induced with 1 mM IPTG and expression was carried out for 18 h at 16 °C. Cells were harvested by centrifugation, resuspended in Buffer A  supplemented with RNAse (Sigma), DNAse (Sigma), protease inhibitor cocktail (homemade) and lysed by sonication. The lysate was cleared by centrifugation at 4 °C. The supernatant was passed through an equilibrated dextrose column, washed with 5 column volumes (CV) of buffer A and eluted with 20 mM Maltose in Buffer A. Afterwards, the MBP tag was cleaved using TEV protease at a 1:50 (w/w) ratio of TEV to protein over night at 4 °C. The protein was loaded onto a Strep column  and eluted using 2 mM desthiobiotin (Sigma) and finally purified by size exclusion chromatography, SEC  in kinase NMR buffer  for 4 h and quenching the reaction with 20 mM EDTA. The phosphorylated protein was purified using SEC  in kinase NMR buffer.8—TEVsite—hPER2—GB1 Strep-tag) in a pET16b expression vector and was isotopically labeled by expression in M9 minimal media for 18 h at 20 °C after induction with 0.8 mM IPTG at an OD of 0.6. After resuspension in Buffer A  and sonication the lysate was loaded on an immobilized metal affinity chromatography (IMAC) column , washed with 10 CV of Buffer A and eluted with 300 mM imidazole in Buffer A. Binding and elution from a Strep column eliminated truncated constructs and the protein was finally purified by SEC  in NMR Kinase buffer. Phosphorylation was done by incubating the protein at 298 K with 10 mM ATP (sigma) with a 1:100 ratio of kinase to protein for 24 h. The reaction was quenched by addition of 20 mM EDTA. The phosphorylated protein was purified by SEC  into kinase NMR buffer. The phosphorylation state was confirmed by Mass Spectrometry. The sample was used for the assignment of the peptide.The human PER2(645–676) peptide was expressed as a C670A mutant, fused at its C- as well as its N-terminus with the immunoglobulin G binding domain 1 of Streptococcal protein G  was isotopically labeled by expression in M9 medium for 16 h at 22 °C following induction with 500 µM IPTG at an OD of 0.6. The purification protocol followed the one established for the hPER2 peptide . The expression tag and the peptide were cleaved by incubation with 3C protease overnight at 4 °C along with dialysis to reduce the NaCl and imidazole concentrations to 50 mM. Cation exchange chromatography was performed to eliminate GFP and the protease. The protein was subjected to SEC in kinase NMR buffer . Phosphorylation was done incubating the protein at 298 K with 10 mM ATP (Sigma) with (1:100) ratio of kinase to protein for 48 h. Quenching of the reaction was done with EDTA. The phosphorylated protein was purified using SEC  in kinase NMR buffer. Furthermore, the phosphorylation state was confirmed by Mass Spectrometry.The C-terminal domain of CK1δ . Experiments were measured at a sample temperature of 298 K. Assignments of GB1-hPER2-GB1 and the C-terminal domain of CK1δ at different phosphorylation states were performed using constant-time HNCACB, HN(CO)CACB and HN(CA)CO experiments.Samples for NMR experiments were prepared in kinase NMR buffer , protease inhibitor (1×), phosphatase inhibitor (1×), ATP (10 mM), CK1 kinase (125 nM), buffer (to 200 µl).Phosphorylation kinetics of p63 were recorded using sample volumes of 180 µl placed in 3-mm capillaries and final peptide concentrations of 250 µM. The kinase concentration was 125 nM (1:2000). The phosphorylation kinetics of GB1-hPER2-GB1 were measured in standard NMR tubes of 5 mm with a volume of 600 µl using 150 µM of PER2 and 600 nM (1:250) or 1.5 µM (1:100) kinase. All NMR experiments with peptides were performed at least twice under identical conditions. The general sample composition of a kinetic sample was as follows:15N, 1H]-BEST-TROSY correlation spectra were recorded throughout the kinetic reaction with NMR instruments operating at 1H frequencies ranging from 600 to 950 MHz.A series of [17. Full length kinase and pre-phosphorylated full length kinase were passed through SEC column on the day of the experiment to avoid soluble aggregates. The reaction mixture contained 100 µM p63 PAD peptide, prephosphorylated at S582, 50 nM kinase, 10 mM ATP, 1× Protease inhibitor, 0.5 mM TCEP in NMR Buffer. Kinetics were measured at 298 K. Aliquots of these reactions were quenched by the addition of 0.1% TFA at certain time points.Kinetics were measured similar as reported before17.All the kinase was subjected to SEC prior to the kinetics on the same day at 50 nM concentration in the same buffer as described for the NMR experiments. The reaction mixture contained 100 µM p63 PAD peptide, prephosphorylated at S582, 50 nM kinase, 10 mM ATP, 1 × Protease inhibitor, 0.5 mM TCEP in NMR Buffer. Kinetics were measured at 298 K. Aliquots of these reactions were quenched by the addition of 0.1% TFA at certain time points. All the reactions were carried out in triplicates. The samples were measured on a LC-ESI-TOF (Agilent 6230B) Mass Spectrometry instrument. An external standard was used to correct for the deviating behavior of differentially phosphorylated peptidesThe purified CK1δ protein was reduced with 10 mM TCEP and alkylated with 40 mM chloroacetamide in 50 mM Tris–HCL and NaCl buffer. 6 µg of the protein were subjected to enzymatic digestion using Trypsin  at a weight-to-weight ratio of 1:100 (Trypsin:Protein). The digestion was carried out overnight at 37 °C in a final Urea concentration of 1 M. The peptides were cleaned up with Empore C18 stage tipping and dried right away for shooting.51. Simply, peptides were separated with Easy-nLC 1200 (Thermo Fisher Scientific) and kept at 50 °C using an integrated column oven. Individual peptides were eluted by a nonlinear gradient from 4 to 32% acetonitrile over 50 min, followed by a step-wise increase to 76% acetonitrile in 6 min, which was kept for another 9 min and sprayed into a QExactive HF massspectrometer (Thermo Fisher Scientific). Full-scan MS spectra (300–1650 m/z) were acquired with a resolution of 60,000 at m/z 200, maximum injection time of 20 ms and AGC target value of 3 × 106.Dried peptides of the sample were resuspended in 2% (v/v) acetonitrile/1% (v/v) formic acid solution and shot similar to earlier publication5. Ions were then fragmented using higher energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 27; and the dynamic exclusion was set to 20 s to minimize the acquisition of fragment spectra of already acquired precursors.The 10 most intense precursors were selected for fragmentation (Top 10) and isolated with a quadrupole isolation window of 1.4Th. MS2 scans were acquired in centroid mode with a resolution of 30,000 at m/z 200, a maximum injection time of 54 ms, AGC target value of 1 × 1052 partner repository with the dataset identifier PXD043233.Raw data was analyzed with Proteome Discoverer 2.4 (ThermoFisher Scientific). SequenceHT node was selected for database searches of MS2-spectra. Human trypsin digested proteome ) was used for protein identifications. Contaminants (MaxQuant “contamination.fasta”) were determined for quality control. Carbamidomethyl  at cysteine residues were set as fixed modifications. Phosphorylation (+ 79.966 Da) on Ser, Thr, and Tyr residues, methionine oxidation  and acetylation (+ 42.011) at the protein N-terminus were set for dynamic modifications. Precursor mass tolerance was set to 7 ppm and fragment mass tolerance was set to 0.5 Da. Default percolator settings in PD were used to filter perfect spectrum matches (PSMs). Reporter ion quantification was achieved with default settings in consensus workflow. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDESupplementary Figures.


Botrytis cinerea and Monilinia spp., are fungal diseases responsible for significant losses during the storage of fruit and vegetables. Nowadays, the control of postharvest diseases is shifting towards more sustainable strategies, including the use of plant secondary metabolites. In this study, the antifungal activity of Origanum vulgare, Thymus vulgaris, Thymus serpyllum, Melaleuca alternifolia, Lavandula officinalis, Lavandula hybrida, Citrus bergamia, Rosmarinus officinalis, Cinnamomum zeylanicum essential oils (EOs) in vapor phase was tested in vitro against B. cinerea, Monilinia fructicola, Monilinia fructigena, and Monilinia laxa. For the experiments, a protocol using a volatile organic compounds (VOC) chamber was designed. Results indicate a dose-dependent inhibitory activity of all the tested EOs, with O. vulgare, T. vulgaris, and T. serpyllum being the most active ones, with minimum inhibitory concentrations (MIC) of 22.73, 45.45, and 22.73 µl/L, respectively, against B. cinerea and a range between 5.64 and 22.73 µl/L against the three Monilinia spp. Overall, B. cinerea presented lower sensitivity to vapor-phase EOs than any of the Monilinia strains, except for the C. zeylanicum EO, which consistently showed higher inhibition against B. cinerea. Among the three Monilinia spp., M. fructicola was the least sensitive, while M. fructigena was the most sensitive. The use of VOC chambers proved to be a reliable protocol for the assessment of antimicrobial activities of EOs. These results suggest that the VOC emitted by the tested EOs are effective towards important decay-causing fungi, and that they could be used for the control of gray mold and brown rot in in vivo trials.Gray mold and brown rot, caused respectively by   They prEO composition vary not only according to the plant species, but also to the variety, cultivar, plant part, physiological state, and extraction method . This ovin vitro methodologies derived from the protocol proposed by EO complexity leads to challenges in both the study and subsequent application of EO VOCs for microbial control. EO hydrophobicity and volatility compromise their effectivity in direct contact, affecting the availability and diffusion of the individual active compounds, and imposing the use of solvents or emulsifiers that may affect the resulting outcome . The tesNevertheless, biological interactions mediated by volatile organic compounds (VOCs) from plants, fungi, yeasts and bacteria have been explored for decades . AlthougBotrytis cinerea Pers. and Monilinia spp. can be counted among some of the most destructive and widespread postharvest fungal diseases  Honey, Monilinia fructigena (Pers.) Honey, and Monilinia laxa (Aderh. and Ruhland) Honey are among the most common postharvest fungal pathogens affecting stone and pome fruits, and are the causal agents of brown rot and blossom blight on fruit trees, resulting in important yield losses, postharvest fruit waste, and reduced shelf life  to develop an effective protocol for the evaluation of these interactions using VOC chambers; and (ii) to evaluate and compare the 22.1B. cinerea , and M. fructicola, M. fructigena, and M. laxa isolated from infected nectarines in Italy . These strains had been previously identified in our laboratory by morphological and molecular methods using multiplex PCR  composed of 25% M. alternifolia, 25% O. vulgare, 25% C. zeylanicum, and 25% T. vulgaris was also assayed. The EOs, including the mixture, were kindly provided by Flora srl  and GreenVet . The extraction method, plant material, and purity grade of the tested EOs (100% pure) were certified by the suppliers .Twelve commercial essential oils were tested individually to assess their vapor-phase antifungal activity against the referred fungal strains: uppliers Table 12.3Fungi were exposed to the volatiles of the listed EOs Table 1 From three up to eight doses were assessed depending on the specific EO, having been selected considering the bibliography and the sensitivity of each fungal strain in accordance with preliminary assays. Concentrations are expressed as microliters of EO per liter of air inside the VOC chamber (µL/L) by calculating the air volume of the headspace . Lowest concentrations were set up at 2.82 µL/L for all EOs, while the highest ones were determined by assessing the inhibitory activity of each individual treatment. The EO volumes were measured using a micropipette . The specific concentrations tested for each EO and fungal strain can be found in the results section alongside their corresponding percentages of inhibition . Inoculated plates without any EO were used as controls. Three replicates were performed per treatment in a single assay. PIs were determined using the following equation: PI = [(C - T)/C] x 100 . Where CB. cinerea; and 5 dpi for M. fructicola, M. fructigena, and M. laxa, when mycelial growth of one of the treatments reached the edge of the plate, and PIs were subsequently calculated in comparison to the growth of the untreated control. The Minimum Inhibitory Concentration (MIC) for each EO and fungal strain was determined as the lowest tested concentration that showed no mycelial growth inhibition at the last day of the experiment  for periment . A rank periment  was perfThe inoculation plugs from those treatments presenting complete inhibition were re-inoculated on fresh PDA to evaluate whether the activity of the volatile EOs was fungicidal or fungistatic , and eve2.4post hoc test (p ≤ 0.05). A non-parametric Kruskal–Wallis H-test was used, and means were separated with the Mann-Whitney U-test (p ≤ 0.05) when data were not normally distributed or homoscedastic. All statistical analyses were performed using IBM SPSS Statistics 26 .After testing the data normality and equality of variances with Kolmogorov-Smirnov´s test and Levene´s test, statistical analyses were carried out using one-way analysis of variance (ANOVA), and means were separated with Tukey’s 33.1B. cinerea in comparison to all three Monilinia spp.  exerted a considerable inhibition, mostly in the range of the two tested Thymus spp., with MICs: 45.45 μL/L against B. cinerea, 22.73 μL/L for M. fructicola and M. laxa, and 11.76 μL/L against M. fructigena. Some MIC differences were also observed between EOs derived from the same plant but from a different provider . MICs of all the tested EOs against the four fungal strains are presented in Results indicate a dose-dependent inhibitory activity of VOCs from all tested EOs against the fungal strains. Overall, they exerted lower antifungal effects against nia spp. Table 2.3.2The antifungal effects of all tested EOs were demonstrated to be dose dependent, with percentage of inhibition (PI) rising as concentrations increased 6.B. cinerea  was reported to be double that on M. laxa (13.3 μL/L) (M. laxa). These differences were even higher when testing the effects of its individual components, namely thymol and p-cymene. Conversely, in the present study, C. zeylanicum EO VOCs were the only ones that produced a consistently higher antifungal activity on B. cinerea than against any of the tested Monilinia spp. strains. These results could point to a different mode of action, thus further research would be of interest to understand the specific traits of this EO.This study indicates that EO VOCs show antimicrobial activity toward  cinerea . These a.3 μL/L) , which aO. vulgare, T. vulgaris, and T. serpyllum presented the highest antifungal activity of all the tested EOs. This is again in accordance with previous research that showed their strong inhibitory effects , similar to that of T. vulgaris, but stronger than that of M. alternifolia and C. zeylanicum. These results do not indicate a synergistic activity of the listed EOs. Previous studies have reported both synergistic and non-synergistic results concerning microbial inhibition from EO mixtures , while they reported a MIC of around 15.63 μg/L for carvacrol and thymol. We could hypothesized that these differences derive from the lower level of carvacrol in the O. vulgare EO used by these authors (15% in comparison to 69% and 79% in the present study). Several studies have also demonstrated the strong antifungal effect of T. vulgaris and C. zeylanicum EOs against B. cinerea . These same authors reported that EOs from Melaleuca spp., Lavandula spp., and R. officinalis were less effective against B. cinerea, as also found in our trials. For example, they reported a PI of 72.3%, very similar to the 78.4% we observed at 181.82 μL/L for R. officinalis EO. Other studies further support these findings, with L. hybrida showing a half maximal inhibitory concentration (IC50) around one order of magnitude higher that of T. vulgaris . In vivo assays indicate that T. vulgaris and C. zeylanicum EO VOCs can also protect peaches from brown rot caused by M. laxa via the induction of physiological and defensive responses in the fruit  show higher antifungal activities than the others. This is supported by previous reports, in which these individual compounds demonstrated strong antifungal activity .SÁ-G: Conceptualization, Validation, Data curation, Formal Analysis, Investigation, Methodology, Software, Visualization, Writing – original draft. MM: Formal Analysis, Investigation, Software, Validation, Writing – review & editing. GR: Validation, Writing – review & editing, Conceptualization, Funding acquisition, Project administration, Resources, Supervision.