SAT synthesizes O-acetylserine from substrates l-serine and acetyl coenzyme A and is a key enzyme for regulating cellular cysteine levels by feedback inhibition of l-cysteine, and its involvement in the cysteine synthase complex. We have performed extensive structural and kinetic characterization of the SAT enzyme from the antibiotic-resistant pathogen Neisseria gonorrhoeae. Using X-ray crystallography, we have solved the structures of NgSAT with the non-natural ligand, l-malate  to 2.01 Å and with the natural substrate l-serine (2.80 Å) bound. Both structures are hexamers, with each monomer displaying the characteristic left-handed parallel β-helix domain of the acyltransferase superfamily of enzymes. Each structure displays both extended and closed conformations of the C-terminal tail. l-malate bound in the active site results in an interesting mix of open and closed active site conformations, exhibiting a structural change mimicking the conformation of cysteine (inhibitor) bound structures from other organisms. Kinetic characterization shows competitive inhibition of l-cysteine with substrates l-serine and acetyl coenzyme A. The SAT reaction represents a key point for the regulation of cysteine biosynthesis and controlling cellular sulfur due to feedback inhibition by l-cysteine and formation of the cysteine synthase complex. Data presented here provide the structural and mechanistic basis for inhibitor design and given this enzyme is not present in humans could be explored to combat the rise of extensively antimicrobial resistant N. gonorrhoeae.Serine acetyltransferase (SAT) catalyzes the first step in the two-step pathway to synthesize  Neisseria gonorrhoeae is an obligate human pathogen that causes the sexually transmitted infection gonorrhoea. With purported references to gonorrhoea in early texts including the bible -[GAED]-X2-[STAV]-X, that gives rise to the unique left-handed β-helix (LβH), characteristic of this family -X) which results in the formation of the LβH domain. There is a single deviation from the LβH domain structure between β-strands 7 and 8, which creates an extended loop (residues 191–199 in this structure) which is significant as this forms one half of the active site  from Soybean (Glycine max) [l-serine in the NgSAT + l-Ser structure.Comparing the two NgSAT structures, the active site residues, Asp96, Asp161, Arg196 and His197 , interacl-malate . The mosd His197 , whereasd His197 . This diine max) , with thl-malate when the tail is positioned near the active site . For the remaining single monomer in each trimer, the tail loops upward against the side of the monomer and tucks under the active site . There aive site . In the l-malate . The ‘cll-malate . Glutamiructures  that havl-malate was present in the crystallization condition and its occupation of the active site is expected to be based on charge in a similar manner to the inhibitor l-cysteine. Comparing the ‘closed’ active site of NgSAT with the homologous E. coli SAT (1T3D) (containing cysteine in the active site) we observe a similar arrangement of active site residues and the C-terminal tail position. Cysteine acts as a negative feedback regulator of SAT activity and it could be deduced that it does this by ‘closing’ the active site. Comparing the ‘open’ active site of NgSAT with the H. influenzae homologue (1SST) (containing CoA in the active site) we observe that the C-terminal tail is also orientated away from the active site and in addition the extended loop in 1SST is disrupted whereas in our structure is not. The substrate CoA is a large molecule and by occupying the active site the C-terminal tail cannot fold into this space. The structure presented here is unique in that we see both ‘open’ and ‘closed’ conformations in a monomer which gives insight into substrate binding.The tucked tail conformation of the NgSAT monomer is identical with homologous SAT structures that have the product cysteine bound  and analysis of these structures also reveals that there would be no access to the pocket of the active site . Due to an artifact of crystallographic packing, we have surprisingly been able to examine two active site states regardless of having a true substrate molecule bound. l-cysteine is well-documented across both bacterial and plant species [l-cysteine inhibition of NgSAT, relative to the substrates l-serine and acetyl-CoA. NgSAT activity was inhibited by increasing concentrations of l-cysteine with an IC50 of 6.4 ± 0.1 µM and a hill slope of −1.86 ± 0.15 (l-serine concentrations (KM(serine) from 1.16 mM with no l-cysteine bound to 4.83 mM in the presence of 8 µM l-cysteine, while the kcat did not change. This demonstrates that l-cysteine is a competitive inhibitor relative to l-serine. Competitive inhibition is supported by structural evidence from E. coli and E. histolytica where cysteine has been shown to bind directly to the l-serine active site, preventing l-serine from binding [h value (a measure of cooperativity) decreases steadily from 1 (non-cooperative binding) to 0.299 (negatively cooperative binding) with increasing l-cysteine concentrations (l-cysteine is present. This is consistent with the hill slope (−1.86) of the l-cysteine dose response curve (l-cysteine increases the affinity of other active sites in the multimer for l-cysteine (positive cooperativity) which reduces the affinity for l-serine  to 10.8 µM (hill coefficient 2.5 ± 0.1), consistent with a competitive inhibition mechanism [l-cysteine binding. Competitive inhibition is supported by the observation that in cysteine-bound SAT structures from other bacteria, the flexible C-terminal tail of SAT is folded against the active site [bacteria ,27. To itrations  in the petyl-CoA , demonstechanism  and posiive site ,27, as al-cysteine binding to NgSAT in solution. Crystallization attempts of NgSAT with l-cysteine were unsuccessful due to degradation of crystals upon addition of l-cysteine. The scattering data were consistent with a globular protein (r)) analysis support a hexamer of NgSAT monomers. The calculated molecular mass for the l-cysteine bound and unbound structures was the same (193.87 kDa). The molecular mass of the monomer including the His-tag is 31.6 kDa, therefore, 193.87 kDa divided by the molecular mass of the monomer (31.6 kDa) is 6.1 monomers per structure, consistent with the hexameric crystal structure and SEC presented above. The SAXS profiles were fit with the calculated theoretical scattering from our NgSAT crystal structure. The theoretical scattering profile shows a poor fit with the scattering data collected for unbound (χ2 = 5.31) and l-cysteine bound NgSAT scattering data (χ2 = 10.84)  analysis  was perf protein , and the= 10.84) , which il-cysteine shows clear differences  and P(r) analysis (Rg = 40.8 ± 0.14 Å for l-cysteine bound compared with Rg = 43.17 ± 0.14 Å for unbound). The scattering data, including the pairwise distribution analysis and Rg values from the Guinier analysis are consistent with SAXS data from SAT from E. coli [Comparison of the SAXS profiles in the presence and absence of ferences , consist E. coli .l-cysteine although the cysteine-free curve is slightly flattened suggesting slightly increased flexibility of the protein. Decreased flexibility and an increased compactness of the cysteine bound enzyme is consistent with previously reported SAT structures crystallized with the cysteine inhibitor [The bell-shaped curve observed in the Kratky plot  shows thnhibitor ,23,27. TO-acetylserine from l-serine and acetyl-CoA, and the second step catalyzed by O-acetylserine sulfhydrylase (OASS) that uses the substrates sulfide or thiosulfate to synthesize l-cysteine from O-acetylserine. The SAT and OASS-A enzymes form the cysteine synthase complex with the C-terminal tail of SAT binding into and inhibiting the active site of OASS-A. SAT is feedback inhibited by the end product of the pathway l-cysteine. Bacterial members of the SAT family are generally hexamers  and areN. gonorrhoeae (NgSAT). SEC and small angle X-ray scattering (SAXS) analysis confirm that NgSAT is a hexamer in solution as well as in the crystal structure, with no other oligomeric states detected. We have characterized the kinetics of NgSAT, and hypothesize a sequential mechanism, although the order of substrate binding is unknown. We suggest substrate inhibition at concentrations of acetyl-CoA ≥ 1 mM but due to limitations in experimental set-up were not able to confirm substrate inhibition. This is in contrast with other SAT enzymes from other bacteria including E. coli and H. influenzae that display substrate inhibition by l-serine [l-cysteine and the product CoA indicating an interconnectedness between active sites in the NgSAT hexamer. NgSAT is feedback inhibited by the end product of the pathway l-cysteine, and using SAXS we show small conformational changes in the cysteine bound enzyme. These changes suggest a less flexible, slightly more compact enzyme when bound to cysteine. This is consistent with the ‘closed’ active sites we see in the NgSAT crystal structure and crystal structures of other SAT enzymes bound to l-cysteine [Here we have presented structural and kinetic data for SAT from l-serine ,23. NgSAl-serine ,30. Coopcysteine ,23,27, wThe structure of NgSAT reveals that this enzyme is clearly part of the SAT family due to its hexameric nature and LβH. As with other related structures, NgSAT is a dimer of trimers, with each monomer comprising an N-terminal α-helical domain and a C-terminal LβH domain. The majority of SAT three-dimensional structures are incomplete and have up to 37 amino acids missing from the C-terminal end of the enzyme. We note that the NgSAT structure has the least number of missing residues from the distal C-terminal end compared with related structures, which has allowed us to investigate the topology of the active site with some confidence.l-malate is present in the active site of NgSAT due to its presence in the crystallography reagent. It occupies the same area and interacts with the active site residues that the natural substrate l-serine would, as seen in our NgSAT + Ser structure, with the exception of catalytic His162.A crystallographic artifact has led to two different C-terminal tail conformations in the NgSAT structure. Where other structures have missing residues, leading to an exposed active site cavity, NgSAT exhibits either a free C-terminal tail swept outwards from the enzyme or a constrained C-terminal tail tucked up into the active site. The outward C-terminal tail results in an ‘open’ active site, in contrast with the tucked tail which results in a ‘closed’ active site. The ligand The tucked C-terminal tail ‘closed’ conformation reveals interesting interactions with both the solvent and other residues. A key glutamine residue (Gln261) in the tail, interacts with two additional water molecules in the active site (assisted by a nearby Gly188) that were otherwise uninvolved in the ‘open’ conformation. The interaction of Gln261 in the C-terminal tail brings a serine residue near the active site into close-range to allow it to form polar contacts with an aspartate on the C-terminal tail, which in turn forms a polar contact with a lysine from a β-strand in the LβH. Analysis of the residues involved in these polar contacts to tuck the C-terminal tail into the active site reveals strict conservation between species. Overall, there is a chain reaction of polar contacts from the active site, downwards through the tail, securing it and closing the active site.N. gonorrhoeae lacks the ability to utilize sulfate as an inorganic sulfur resource, but its requirement for sulfur can be fulfilled by thiosulfate [N. gonorrhoeae lacks the variant of the OASS enzyme that utilizes thiosulfate (OASS-B/CysM), having just a single OASS enzyme with homology to OASS-A/CysK (the variant that utilizes sulfide not thiosulfate) [N. gonorrhoeae interacts with NgSAT to form the cysteine synthase complex although it is likely, as the C-terminal tail of NgSAT contains the conserved terminal isoleucine critical for binding of the tail into the active site of OASS [In bacteria the primary pathways of inorganic sulfur assimilation converge at cysteine biosynthesis and osulfate . Curioussulfate) . It rema of OASS ,14,58.N. gonorrhoeae and due to the unique nature of sulfur acquisition for cysteine biosynthesis in N. gonorrhoeae could represent a novel drug target for treating extensively antimicrobial resistant gonorrhoea.Our NgSAT structures presented here, alongside kinetic and inhibition data, gives unique insight into the structure, function and inhibition of SAT from UniProt Accession ID for NGFG_01496 (NgSAT): Q5F6X0 (Q5F6X0_NEIG1)


Methods: All patients who underwent robotic-assisted implantation of pedicle screws in the thoracolumbar spine were included in the study. Our workflow, consisting of preoperative imagining, screw planning, intraoperative imaging with automatic registration, fusion of the preoperative and intraoperative imaging with a review of the preplanned screw trajectories, robotic-assisted insertion of K-wires, followed by a fluoroscopy-assisted insertion of pedicle screws and control iCT scan, is described. Results: A total of 12 patients  underwent 13 surgeries using the Cirq® Robotic Alignment Module for thoracolumbar pedicle screw implantation. Spondylodiscitis, metastases, osteoporotic fracture, and spinal canal stenosis were detected. A total of 70 screws were implanted. The mean time per screw was 08:27 ± 06:54 min. The mean time per screw for the first 7 surgeries (first 36 screws) was 16:03 ± 09:32 min and for the latter 6 surgeries (34 screws) the mean time per screw was 04:35 ± 02:11 min (p < 0.05). Mean entry point deviation was 1.9 ± 1.23 mm, mean deviation from the tip of the screw was 2.61 ± 1.6 mm and mean angular deviation was 3.5° ± 2°. For screw-placement accuracy we used the CT-based Gertzbein and Robbins System (GRS). Of the total screws, 65 screws were GRS A screws (92.85%), one screw was a GRS B screw, and two further screws were grade C. Two screws were D screws (2.85%) and underwent intraoperative revision. There were no perioperative deficits. Conclusion: Brainlab’s Cirq® Robotic Alignment surgeon-controlled robotic arm is a safe and beneficial method for accurate thoracolumbar pedicle screw placement with high accuracy.Background: Robot-guided spine surgery is based on a preoperatively planned trajectory that is reproduced in the operating room by the robotic device. This study presents our initial experience with thoracolumbar pedicle screw placement using Brainlab’s Cirq There were approximately 3.6 million spine surgery cases in the United States between 2001 and 2010, with increasing prevalence each year  and reduWith the development of medical imaging technology and computer technology, both image navigation and robot technology have been gradually applied to pedicle screw placement ,8,9. It Guidance to the surgeon in robot-guided spine surgery is based on a preoperatively planned trajectory that is reproduced in the operating room by the robotic device. In one variant, this device is mounted to the patient’s spine for drilling holes in preparation for the placement of pedicle screws. The bone-mounting feature allows patient breathing and motion without altering the position of the robotic unit relative to the spine, which maintains the system’s accuracy ,12.® surgeon-controlled robotic arm . The robotic arm is mounted onto a standard radiolucent OR table, and the surgeon positions the arm, which, once roughly aligned above to entry point of the planned trajectory, automatically adapts to the planned trajectory, and provides a channel for drilling with a drill guide and the implantation of K-wires, followed by pedicle-screw placement. This is currently the only robotic system that provides automatic adaptation to the preplanned trajectory. To our knowledge, this is the first case series to evaluate the use of CIRQ® Robotic Alignment  for pedicle screw placement.Robot-assisted spine surgery, which can provide an efficient and accurate mode of hardware placement, is relatively new and still seldom used by spine surgeons . As withInformed consent was obtained from all individual participants included in this observational study. We obtained ethics approval for the prospective archiving of clinical and technical data, applying intraoperative imaging and navigation (study no. 99/18).All surgeries were performed by a team of two surgeons with three (M.P.) and five (B.S.) years of experience in complex spinal surgery. All patients who underwent robotic-assisted implantation of pedicle screws in the thoracolumbar spine were included in the study. A total of 70 pedicle screws were evaluated. Our workflow included the following: preoperative imagining, screw planning, intraoperative imaging with automatic registration, fusion of the preoperative and intraoperative imaging with a review of the preplanned screw trajectories, robotic-assisted insertion of K-wires followed by fluoroscopy-assisted insertion of pedicle screws and a control iCT scan. Prior to surgery, all patients received a 3D CT of the region of the spine that was planned for instrumentation. This dataset was exported to the navigation software . Using the crew planning application, the radius, length and trajectory of the pedicle screws were planned prior to surgery . The sof® 7500, Trumpf Medical Ditzinger, Germany) for spinal applications, connected to the CT scanner . Surgery was performed in the scanning position with anesthesia cables and lines routed through the gantry. Cirq® was attached and draped on the left side of the operating table on a metal holder. , was performed. Immediately after scanning, imaging data was automatically transferred to the navigation system  without user interaction for automatic patient registration with the acquired image data . To display preplanned screw trajectories in the recent imaging data, the co-registration of preoperative data, defining a region of interest with pre-planned screws, was conducted. Elements Spine Curvature Correction co-registers scans of the patient to compensate for inevitably varied spine positions during different imaging sessions. The software brings together scans from preoperative CT with iCT to update them for surgery. Additionally, if needed, preoperative magnetic resonance imaging (MRI) of the spine can be fused with preoperative and intraoperative imaging. Preoperative image data from CT and MRI are fused non-linearly, by applying the spine curvature element in a rigid and elastic fashion  .Figure . Rigid fRegistration accuracy was evaluated by placing the pointer tip on anatomical landmarks such as the spinous process, vertebra lamina, or the vertebral body surface, or on artificial landmarks, such as skin fiducials or mini-screws attached to the spinous process . A regisAn intraoperative CT was used for the robot-guided implantation of K-wires into the pedicles following the insertion of pedicle screws under fluoroscopy control. ® Robotic Alignment Module consists of an interchangeable head, which enables alignment with the preplanned trajectory and a kinematic unit for fine-tuning adjustment, based on pre-planned trajectories, during computer-assisted robotic surgery. A tracking array was attached to the kinematic unit for real-time tracking of the instruments and constant position feedback Surgery time was defined as the period between the first incision and closure.(b)The time taken for positioning and robot installation was defined as the time necessary to position the patient and completely install the robotic arm.(c)The robotic time, which was recorded by the robotic system, was the total time in which the robotic arm was in use. This period covers the point of initiation of the first entry point search to the implantation of all K-wires.(d)Time per screw—since we did not measure time needed for the implantation of each screw separately, time per screw was calculated per case by dividing the robotic time with the number of implanted screws. This provides a measurement of the time needed for robotic-assisted implantation of K-wires. Since K-wire implantation is the essential robotic-supported element of pedicle screw implantation, we labeled this time as time per screw.(e)Pedicle size—measurement of the pedicle size in the axial, coronal and sagittal plane, on the registration iCT scan, with a line perpendicular to the preplanned screw(f)entire intrapedicular position without a breach of the pedicle cortexexceeding the pedicle cortex < 2 mmexceeding the pedicle cortex 2–4 mmexceeding the pedicle cortex 4–6 mmexceeding the pedicle cortex > 6 mm or reaches outside of the pedicle.Pedicle screw accuracy—measured according to the CT-based Gertzbein and Robbins System (GRS). The grading system reflects the deviation of the screw from the “ideal” intrapedicular trajectory. The transpedicular screw position was graded from A to E, based on the extent to which the screw breaches the cortex of the pedicle 1–3:The following parameters for the assessment of the initial experience were used for evaluation:Grade A and B can be considered as satisfactory operation results. In grade C to E, neurological symptoms may occur and therefore, can be evaluated as an unsatisfactory surgical result . (g)Deviation of preplanned trajectory from actual pedicle screw position—offset of the screw compared to preplanned trajectory : mean deviation in entry point, the average deviation from the tip of the screw, and angular deviation. Deviations were determined using an image-overlay analysis to compare preoperative CT imaging with preplanned screw trajectories to a true screw position on intraoperative, control CT imaging . The meap value of <0.05 was considered to be statistically significant. For variables such as deviation of the entry point, screw tip and angular deviation, the mean was calculated with standard deviation (SD). A t test was used to measure statistically significant difference between the means. For calculating the differences between standard deviations, Leven’s Test for Equality of variances was performed prior to the t test. In cases where a statistically significant difference between the SDs was found, a t test was not performed. For comparison, we decided to compare roughly the first half of all implanted screws with the second half. In the first 7 surgeries there were 36 and in the following 6 surgeries a total of 34 implanted screws. An independent sample t test was used for a comparison of different mean values between the first 7 and the other 6 surgeries, i.e., between the first 36 and the remaining 34 screws. The analyses were performed using SPSS statistical software, version 20 . A In total, 12 patients  underwent 13 surgeries using the CIRQ Robotic Alignment Module for thoracolumbar pedicle screw implantation. One patient with spondylodiscitis underwent two surgeries for spinal fusion in the thoracic and lumbar spine (patient number one). Indications included spondylodiscitis (4 patients), metastases (3 patients), osteoporotic fracture (2 patients), and spinal canal stenosis (3 patients). A total of 70 screws were implanted. Six surgeries were performed using an open technique and the other 7 were performed using a minimally invasive percutaneous technique. The mean height of all patients was 171.4 ± 13.9 cm (range 145–189), with mean weight of 84.6 ± 23.2 kg (range 59–130). All patients underwent a registration CT scan for automatic navigation registration as well as at least one control CT scan for implant position control. The general characteristics of the patients are summarized in Patient 1. was admitted with severe spondylodiscitis of the cervical, thoracic, and lumbar spine. He initially underwent a stabilization on the cervicothoracic spine and decompression with empyema evacuation. Following this surgery and initial recovery, seethe was determined for stabilization in the thoracic and lumbar spine. The first surgery was performed using the open technique; the reference array was attached at the spinous process of Th8 for Th9/10 stabilization. Following a registration iCT scan, four K-wires were implanted along the preplanned screw trajectories in Th9 and Th10, followed by implantation of the pedicle screws under fluoroscopy control. Control iCT was performed and indicated the correct position of the implanted screws. In the second surgery, also using an open technique, the reference array was attached to the Th12 spinous process for LW1/2 stabilization; this was followed by XLIF-Surgery L1/2 in the right lateral decubitus position. Patient 2. underwent surgery Th11-L2 for spondylodiscitis. An open technique was used, and the reference array was attached at the spinous process of Th11. Following registration iCT, four screws were implanted using CIRQ Robotic Alignment, after control iCT, which confirmed the correct screw position. Patient 3. presented with spondylodiscitis L3/4 following dorsal surgery with interspinous spacer implantation. In the open technique, after registration iCT, robotic-guided screw implantation in L3 and L4 was performed, followed by control iCT with the correct placement of the screws. A registration array was attached at the L2 spinous process. The registration accuracy could have been monitored well using the interspinous spacer. Following this, a laminectomy and removal of the spacer was performed.Patient 4. underwent stabilization for breast-cancer metastases through a compression of the spinal cord in Th 12. An open technique was used because a laminectomy and a partial resection of the tumor in Th12 were performed following screw implantation. Since a longer construct was planned, the reference array was initially attached to Th8. After registration iCT, robotic-guided stabilization was performed for Th9-10, and a control iCT scan was performed. This scan demonstrated a GRS grade of D for the screw in Th10 on the right side, with the correct placement of all other screws. A registration accuracy check confirmed the high accuracy of the scan. The proposed mechanism leading to the misplacement of the screw is a medial breach of the corticalis of the pedicle, due to tumorous damage of the bony substance (skiving) since the entry point was correct. The screw was repositioned using robotic alignment. A reference array was then attached at Th12 to use the following scan at the same time as control iCT for the repositioned screw and as registration iCT for the lumbar screws. In the iCT scan, the correct position of the repositioned screw was observed (GRS A screw). Robotic-guided stabilization L1/2 was performed; a control iCT scan revealed a grade D screw in L2 on the left side, with the correct placement of all other screws (grade A). An accuracy check demonstrated suboptimal accuracy, possibly due to the movement of the reference array attached to the spinous process of Th12, which was infiltrated by the tumor and therefore unstable. We experienced a further technical issue at this point with the CIRQ Robotic Alignment, so the L2 screw on the left side was repositioned using iCT-based navigation, following another iCT scan with the reference array repositioned on the L1 spinous process. Following screw revision, the newly placed screw was of a GRS grade of A .Patient 5. underwent stabilization using the MIS technique for breast cancer metastasis of the L1 with Th12-L2; a reference array was attached to Th12 spinous process via small separate midline incision. Following robotic-guided screw implantation, a transpedicular biopsy of L1 from the left side using iCT-based navigation was performed.Patient 6. underwent MIS stabilization for multiple metastases on the thoracolumbar spine with instability. The reference array was attached at Th10 with a small separate incision; registration iCT and robotic-guided stabilization of Th11-Th12 was performed. The registration array was re-attached to L1, and iCT confirmed the correct placement of all screws, which was then used for the automatic registration of L2 and L3 screws. Following implantation of the screws in L1/2, a further iCT scan was performed, which showed GRS grade A screws in L1 left and right and L2 right with GRS Grade C L2 screw on the left side, with a lateral breach of less than 4 mm. This screw was not repositioned. Patient 7. underwent Th11-L2 stabilization through the MIS technique using robotic guidance; all screws were placed correctly. The patient also underwent corpectomy of L2 due to massive destruction caused by infection and implantation of the expandable cage, using the lateral approach.Patient 8. underwent a stabilization using the open technique for spinal canal stenosis with instability. The reference array was attached at L2; control iCT confirmed the correct position of all screws. Further decompression with TLIF-Cage implantation was performed in L3/4 and L4/5.Patient 9. presented with an L2 fracture including further severely osteoporotic changes in L4, leading to MIS-pedicle screw implantation in L1-3-5. The reference array was attached via separate incision on Th12. Following robotic-guided screw implantation, two control iCT scans were performed, since the first scan did not encompass the L5 level. All screws were placed correctly.Patient 10. underwent stabilization in MIS technique L5/S1, following spinal canal stenosis and instability after two surgeries for a herniated disc at L5/S1 on the left side. The reference array was attached via a separate small midline incision on L4. After control iCT confirmed the correct position of all screws, the reference array was removed, and the incision was extended so that the subperiostal preparation of L5/S1 level on both sides was performed. Decompression of L5/S1 with tranforaminal interbody lumbar fusion (TLIF)-Cage Implantation was subsequently performed.Patient 11. underwent thoracolumbar stabilization of Th10-L2 due to a previous Th12 fracture, and experienced a further fracture of L4 with adjacent segment disease in L2/3. In open technique, the reference array was attached at L1 and registration iCT was performed. Using robotic guidance, L3, L5 and S1 levels received screws, which all showed correct position. A decompression of L3-S1 was performed due to spinal canal stenosis. Loosened L2 screws were removed, and the newly implanted screws connected the Th10-L1 screws with one rod.Patient 12. experienced a Th12 burst fracture with maximal spinal canal stenosis. Stabilization using the MIS technique was performed for Th10/11 on L1/2; the reference array was attached via a separate incision on the spinous process of Th12. A control iCT scan confirmed a GBS Grade of A for the position of all screws. In a secondary surgery, corpectomy of Th12 was performed via the lateral approach, with implantation of the expandable cage. Patients number 9. and 11. had paraparesis which did not improve following surgery. Patient number 12. had a severe ataxia which slightly improved following surgery. All other patients did not have neurological deficits prior to surgery and underwent thoracolumbar stabilization due to intractable pain and instability in the spine due to infection or tumor. Complications occurred intraoperatively in patient number 4., where two screws were intraoperatively revised. The patient did not experience any neurological deficits following surgery. Patients number 6. and 8 experienced wound-healing deficits due to seroma, which required additional surgery. Patient number 7. developed wound healing problems on the lateral, XLIF wound, and needed a revision surgery. Patient number 11. developed a CSF leak, which required revision surgery. The mean surgery time was 03:37:14 ± 01:30:29 h.p < 0.05).The mean positioning and robot installation time was 53:37 ± 36:46 min. The mean time for the first 7 surgeries was 64:43 ± 45:9 min and for the latter 6 surgeries the mean time was 40:6 ± 12:9 min. This difference was not significant . The mean robotic time for open surgery was 52:01 ± 41:30 min and for MIS surgery 42:56 ± 30:36 min; this difference was not significant.The mean robotic time was 45:40 ± 34:29 min. The mean robotic time for the first 7 surgeries (36 screws) was 76:18 ± 35:10 min and for the latter 6 surgeries (34 screws) 25:14 ± 10:45 min; this difference was considered to be significant .The mean time per screw was 08:27 ± 06:54 min. The mean time per screw for the first 7 surgeries (first 36 screws) was 16:03 ± 09:32 min. The mean time per screw for the latter 6 surgeries (34 screws) was 04:35 ± 02:11 min. This difference was significant  and patients number 3,4,5,6, 10 and 12 by surgeon M.P. . Patient number 9. underwent surgery by both surgeons. The mean surgery time for surgeon B.S. was 03:06:50 ± 01:22:38 h, and the mean surgery time for surgeon M.P. was 04:20:10 ± 01:21:34 h. Excluding patient 9., the mean time per screw for surgeon B.S. was 11:35 ± 09:57 min and the mean time per screw for surgeon M.P. was 10:56 ± 08:44 min. Patient number 4, who underwent revision for two screws, was operated on by a surgeon with less experience (M.P.).Patients underwent one registration iCT and one control iCT scan. Patient number 9. underwent 3 iCT scans and patient number 4. underwent 4 iCT scans.® Robotic Alignment, with an average of 5.38 screws placed for the patients and a mean of 2.46 ± 1.21 levels fused. A total of 44 screws were implanted in the lumbar and 22 screws in the thoracic spine; there were four screws implanted in the sacrum (S1). The most common screw dimensions were 6.5 × 50 mm (26 screws) and 6.5 × 45 mm (24 screws), followed by 6.5 × 40 mm (8 screws), 6.5 × 60 mm (4 screws), 5.5 × 50 mm (4 screws), 6.5 × 55 mm (2 screws) and 7.5 × 40 mm (2 screws). There were no perioperative neurological deficits.A total of 70 pedicle screws were implanted using CirqThe mean axial pedicle size measured on the registration scan was 11 ± 4 mm, mean coronal 12.1 ± 2.9 and mean sagittal 12.1 ± 2.9. In the thoracic spine, mean axial, coronal and sagittal size were 8.5 ± 1.9, 11.9 ± 1.37) and 10.2 ± 1.33 mm, respectively. In the lumbosacral spine, mean axial, coronal and sagittal size were 12.25 ± 4, 12.3 ± 2.2 and 13 ± 3 mm, respectively. Mean entry point deviation ranged from 0.5 to 5 mm and was 1.9 ± 1.23 mm for all screws . The mean deviation from the tip of the screw ranged from 0 to 7 mm and was 2.61 ± 1.6 mm for all screws . Angular deviation ranged from 1° to 9.30° and the mean was 3.5° ± 2° for all screws . For screw placement accuracy, we used the CT-based Gertzbein and Robbins System (GRS). Out of 70 screws, 68 screws were placed in a way that intraoperatively revision was not required. Of these screws, 65 screws were GRS A screws (92.85%), one screw was GRS B screw with a lateral breach of less than 2 mm (1.4%) and the remaining two screws were of C grade, with a lateral breach of less than 4 mm (2.85%). Two screws were D screws (2.85%), with a medial breach of more than 6 mm. These screws underwent intraoperative revision. One screw was revised using the robotic arm and one further screw was revised using iCT-based navigation with robot abandonment. This was also the only case of abandonment of the robot (Patient Number 4.). Following revision, both mispositioned screws were found to have a GRS grade of A. For the 70 screws placed, a rate of non-revised screws of 97.1% was recorded (GRS grade A/B/C screws), with 68 correctly positioned screws without need for revision. There were two mispositioned screws in one patient, which required revision surgery, both with a medial breach, one of which was repositioned using the robotic arm, and the other was repositioned using iCT-based navigation. ® Brain Lab Munich, Germany have been published. The first known study provided a 2D-surgical video, depicting the surgical workflow with the O-arm and instrument holder module of the robot [So far, only a few studies examining the novel robotic system Cirqhe robot . There a® Instrument Holder, the first in the cervical spine to achieve minimally invasive C1-C2 stabilization for an odontoid fracture [® arm are intended to be more ergonomic and less disruptive to operative workflow relative to larger robotic units [®’s potential cross-compatibility with implants from multiple manufacturers [Two further studies include the application of the Cirqfracture , and thefracture . In theifracture  althoughfracture . The litfracture . The ligic units . Furtheracturers .MAZOR Spine Assist was the first robotic system to be approved for use in spine surgeries in the USA in 2004 . Prior w®; Globus Medical, Inc. Audubon, PA, USA [®, used on a small number of patients, 97.1% of screws did not require intraoperative revision. This rate is slightly lower than the reported accuracy of existing robotic systems. However, this initial study provides guidance on operative workflow and aimed to identify and prevent possible events that could lead to less accuracy in pedicle screw placement.Most spinal surgery robots are shared-control robots that simultaneously provide both the surgeon and robot the ability to control instruments and motions . There a PA, USA ,31,32, R PA, USA , Orthbot PA, USA  and TiRo PA, USA . Unfortu PA, USA . Most st PA, USA ,38,39. T PA, USA .The ROSA PA, USA ,41. In oCahil et al. described the operative workflow of Mazor Spine Assist . Most co® is the Mazor’s second-generation spine robot, replacing the SpineAssist in 2011, with upgraded image recognition algorithms and the ability for the surgeon to flatten the bone around screw entry points before drilling for prevention of skidding for the guiding cannula on a sloped anatomy [® is the most recent release by Mazor, and includes an integrated linear optic camera that allows the robot to perform a volumetric assessment of the work environment in order to self-detect its location and provide collision avoidance intraoperatively, allowing each vertebral body to be registered independently and hence has its own accuracy [®. One prospective study which compared Mazor-X assisted pedicle-screw implantation to computerized-navigation based implantation of the pedicle screw has demonstrated that robotic technology exposed patients to a reduced fluoroscopy time, decreased time-per-screw placement and shorter hospital stay than 3D-CT navigation [The Renaissance anatomy ,48. Mazoaccuracy . The autvigation . The Excelsius GPS System features real-time intraoperative imaging, automatic compensation for patient movement, and direct screw insertion through a rigid external arm–obviating the need for K-wires or clamps, with feedback provided instantly via the robot’s monitor if the drill skives or the reference frame moves . Farber ® SPINE was approved by the FDA in 2016. Similar to Mazor X, the free-standing ROSA utilizes a robotic arm and navigation camera—each mounted to their own floor-fixable mobile bases—in order to optimize and guide pedicle entry points and trajectories [® , Curexo®  and Fusion Robotic System®. ROSAectories . Lonjon ectories . Operatiectories . Current®) [® Spine) [®) [The mean surgery time in our cohort was 03:37:14 ± 01:30:29 h and differed only slightly between open and MIS surgery. A long operative time is attributed to the decompression of the spinal canal stenosis, discectomy, cage implantation, tumor resection or biopsy, which are unrelated to screw implantation. This is consistent with previously published results on the initiation of a robotic system for which the operative time was reported to be 224 min (193 to 307 min) in a study of Bydon et al. (Spine Assist TM®) . In thei®) . A signi® Spine) . A prolo® Spine) ,55. Kantine) [®) .®) [The mean robotic time was 45:40 ± 34:29 min. For the first 7 surgeries (36 screws) it was 76:18 ± 35:10 min and for the latter 6 surgeries (34 screws) it was 25:14 ± 10:45 min, with significant differences. Although measured on a small sample size, this improvement shows a certain learning curve. Unfortunately, we did not measure the pedicle screw insertion time for each screw given the workflow of implantation of the screw, where first a set of K-wires was implanted followed by serial implantation of screws under fluoroscopy. This is the reason why time per screw was measured as a division of robotic time through the number of screws for each case. Using a similar method of screw insertion time measurement , screw placement times for the latter 6 surgeries (25:14 ± 10:45 min) were found to be similar to a study by Feng et al.  and the ®) [The mean time per screw was 08:27 ± 06:54 min, and for the first 7 surgeries (first 36 screws) it was 16:03 ± 09:32 min, and for the latter 6 surgeries (34 screws) was 04:35 ± 02:11 min. This difference was significant. In a study that compared robot-guided and 3D-CT navigation with O-Arm, the mean time-per-screw placement was found to be 3.7 min for the robotic group and 6.8 min for the 3D-CT navigation group with fluoroscopy time, time-per-screw placement and length of stay significantly lower in the robotic group . Robotic®) . In our The mean positioning and robot installation time was 53:37 ± 36:46 min. The mean time for the first 7 surgeries was 64:43 ± 45:9 min and for the latter 6 surgeries 40:6 ± 12:9 min, with one case of robot abandonment. Robot abort time has been reported in up to 9.7% of cases  with a 1®) [®). Furthermore, an average 2 ± 1 mm error at the midpoint of the screw, which corresponded to the location of the pedicle, was observed [The mean entry point deviation ranged from 0.5 to 5 mm and was 1.9 mm SD ± 1.23), the average offset from the tip of the screw ranged from 0 to 7 mm and was 2.61 mm (SD ± 1.6), and angular deviation ranged from 1° to 9.30° and the mean was 3.5° (SD ± 2°). The mean entry point deviation is consistent with the findings of van Dijk et al., who, in a study on 112 robot-guided spine surgery patients, reported a mean deviation of entry point of 2.0 ± 1.2 mm, compared to preoperative plan   with an observed . One of observed .For screw placement accuracy, we used the CT-based Gertzbein and Robbins System (GRS). Out of 70 screws, 68 screws were placed so that intraoperative revision was not needed. Of these, 65 screws were GRS A screws (92.85%), one was a GRS B screw with a lateral breach of less than 2 mm (1.4%) and two further screws were grade C, with a lateral breach of less than 4 mm (2.85%). Van Dijk et al. found no correlation between GRS grading and screw offset from planned trajectory, meaning that a screw that deviates from the planned trajectory does not constitute inaccurate screw placement . Jiang eAccording to the Gertzbein-Robbins grading system, Jiang et al. reported that, in their series, 184 screws (72%) were classified as grade A and 70 screws (28%) as grade B . The higIntraoperative complications pertaining to robotic guidance usually occur due to skiving of the screw and low entry points . A meta-Prolonged surgery time, especially in the first cases following the initiation of the robotic system in the department, could lead to a higher infection rate compared to fluoroscopy guided or navigation-guided pedicle screw implantation. However, Menger et al. conducted a large, retrospective study of 557 patients and found robotic surgery to be cost-effective, resulting in reduced length of stay, fewer revision surgeries, lower infection rates, and shorter operative time . They deThere were several studies that compared robotic guided with fluoroscopy guided implantation of pedicle screws. In their retrospective review on 70 patients with metastatic spine disease and a total of 406 screws, Solomiichuk et al. described a misplacement rate of 15.6% in the robotic and 16.4% in the fluoroscopy group, with no difference in surgical site infection, duration of surgery and radiation time . InteresIt has been argued that the accuracy of screw placement with the use of navigation is high enough, so that use of a robotic arm is not necessary. However, the possibility of screw planning prior to surgery can significantly decrease surgical time, maximize the fidelity of screw placement and mitigate the human error that is ever present in repetitive manual tasks . A recenAs of yet, only one single-surgeon study has been conducted, which evaluated differences between O-arm navigation and Mazor robotic-guide navigation (46 vs. 39 cases), revealing that screw placement was significantly more accurate and precise with robotic assistance when considering Gertzbein-Robbins A placement whereas mean operative times, estimated blood loss, wound revision rates and clinically acceptable instrumentation placement did not demonstrate significant differences between the 2 groups . LaudatoNo clear economic benefit of robotic systems has been shown. Key elements that impact economic feasibility include the efficient use of surgical time and a decrease in revision and complication rates . The hig®) [The learning curve is largely dependent on experience and the dedication of the operator . For com®) . In addi®) . This in®) . The lea®) . This su®) .® Robotic Module. This data provides valuable initial benchmarks for comparison and lessons for wider scale implementation [There are several limitations of our study. First, this is a retrospective review of prospectively archived data. Patients comprise a heterogenous group for various indications using open surgery and the minimally invasive technique. We did not have a control group but compared our data to the data from the literature. A comparison of the initial experience of our study, using robotic-guided spine surgery with several hundreds of cases, a free-hand/fluoroscopy guided technique and CT-based navigation, performed by the two surgeons, did not seem practical. We selected relatively easy cases for the robotic-guided implantation of pedicle screws to first gain experience with a novel robotic system, with the intention to use the robotic arm for more complex cases in the future. In future studies, we plan to use the robotic arm for complex cases, including scoliosis surgery and revision. Furthermore, after our initial experience and set up of the operative workflow, further studies that compare pedicle screw implantation in more complex cases using iCT-based navigation vs. robotic-guided pedicle screw implantation are needed, to validate the value of the new operative method compared to the surgical standard. We did not measure the pedicle screw time but the total time for which the robotic arm was in use, divided by the number of implanted screws. Furthermore, an assessment of the accuracy of the pedicle screw implantation was performed in only a 2-dimensional fashion. However, this initial experience described the workflow and gave rise to relevant observations on the operative setting. Prospective studies that compare CT-navigation-based pedicle screw placement and the placement of pedicle screws using a robotic arm are needed for the evaluation of the efficacy of the Cirqentation .® Robotic Alignment surgeon-controlled robotic arm is a safe and beneficial method for accurate thoracolumbar pedicle screw placement with high accuracy. The high accuracy observed in this study is consistent with the data from the literature. A learning curve was observed through the reduction of the robotic time and time per screw compared to total surgery time. Prospective studies are needed for a better evaluation of the novel robotic system. An optimization of the workflow should be considered for a reduction in the surgery time. The Brainlab’s Cirq


In Helmholtz’s illusion, a square with horizontal stripes appears taller than an identical square with vertical stripes. This effect has also been observed in experiments with human stimuli, where a human figure wearing a dress with horizontal stripes appears thinner than a drawing clad in vertical stripes. These findings do not agree with the common belief that clothes with horizontal stripes make someone appear wider, neither do they disentangle whether the horizontal or vertical stripes account for the thinning effect. In the present study, we focused on the effect of horizontal stripes in clothes comparing horizontal stripes against no-stripes , using photos of a real-life female model, and controlling for the average luminance of the stripes (Experiment 2). Results showed that horizontal stripes and lower luminance have—independently—a small-to-moderate thinning effect on the perceived size of the body, and the effect is larger when the two variables are combined. In Experiment 3, we further show that the thinning effect due to the luminance of the dress is enhanced when the general background gets darker. Helmholtz illusion. Do clothes with horizontal stripes make us look fatter? The common belief is that they do. Interestingly, not only horizontal stripes are frequently quite fashionable, but empirical evidence also supports the opposite: horizontal stripes seem to have a thinning effect on the perceived body width. The first to report a thinning effect of horizontal stripes was A number of studies have investigated whether the Helmholtz illusion generalizes to clothes. For instance, In a more recent study, However, not all research findings on Helmholtz illusion are unequivocal. For example, Imai  and to non-stripes (Cohen's d = 0.61), corroborating the common belief that horizontal stripes make someone look wider. The study by Swami and Harris added ecological validity to the effect, since it resembled the judgment process followed in natural surroundings . However, the experimental method lacked a clear focus on the perceptual nature of the task and rather introduced confounding variables, since participants were asked to assess from memory a stimulus they were not instructed to pay attention to. It is well known that people are poor at perceiving unattended stimuli in their environment  and non-stripes was tested by The comparison between horizontally and non-striped stimuli was tested in a laboratory setting by To test whether this also applies to clothes, and since no previous study has tested whether a human figure wearing a garment with horizontal stripes results in a thinning effect compared to an object without stripes , we compared the perceived width of a female model wearing a dress with horizontal stripes against a similar stimulus without stripes. Following As compared to previous experiments, the present study differentiates in two ways. First, the stimuli used in previous studies were either line drawings of human figures . Although the PSE in previous studies may reflect a perceptual effect, in some cases it might reflect a response bias, since participants may consistently prefer choosing one stimulus over another , and another one wearing the same dress without stripes . The width of one model (the standard) was fixed, whereas the width of the other (the variable) was randomly determined. An example display is illustrated in SD = 3.8 years, ranging from 18 to 35 years) took part in Experiment 1, which lasted approximately 10 min. All participants were naïve as to the purpose of the experiment and were paid €4 or received course credits for their participation. All participants provided written consent. The experiment was approved by the local ethics committee of the Vrije Universiteit Amsterdam and conducted in accordance with the Declaration of Helsinki.Forty-three participants . 2; in all stimuli we measured the average luminance in the belly-waist area), or black (<0.5 cd/m2), whereas the striped dress was a combination of both colors . The height of both images was fixed , whereas the width of the bodies varied between 13.20° to 18.41° (width measured as the maximum distance between stimulus’ left-right hand), in steps of 0.087°. From the two images on the screen, the width of one body (the standard) was fixed (15.81°), whereas the initial width of the other body (the variable) was randomly determined (±1.73°) in order to avoid a hysteresis effect. The center of the images was located on the left and the right from the center of the screen (distance was 9.11°), and the x and y coordinates were jittered (±0.43°). The body wearing the striped dress was randomly presented on the left or the right side of the screen, with equal probabilities to appear on either side.On every trial, participants saw a female body wearing a dress with stripes, and another one wearing a dress without stripes on a general light gray background . The cue was either the word “stripes” when participants were asked to adjust the width of the body wearing the striped dress, and “green” or “black” when participants were instructed to adjust the width of the body wearing the non-striped dress. Subsequently, the two bodies were shown and participants were instructed to adjust the width of the variable body until they perceived both bodies to be equally wide . Participants pressed either the upper or lower arrow key to increase or decrease the body width, respectively, and the spacebar to submit their response once they were satisfied. There was no time limit in their responses. The dependent variable was the mean PSE. The independent variables were the type of stripes on the dress (stripes or non-stripes), and the color of the non-striped dress (black or green). Each condition was counter balanced and the order was randomly determined. Each combination was repeated 15 times, making a total of 60 trials. Participants received instructions on the screen prior to the experiment and practiced a couple of trials to familiarize themselves with the task.1For illustrative purposes, the distribution of responses per image is shown in t-tests were conducted, to avoid family-wise error rate, all p-values across the manuscript have been adjusted following the Bonferroni-Holm method ,2 and the mean PSE was significantly smaller than zero when the variable body was wearing a dress without stripes . When the striped dress was compared to the black dress, no significant difference was observed neither when the variable body was the striped  nor when it was the black dress .In the comparison between the striped and non-striped light-green dress, the mean PSE was significantly larger than zero when the variable body was wearing a dress with stripes  points toward the dress that is perceived as thinner. As revealed by a two-tailed t-test, the striped dress was perceived as thinner than the non-striped light-green dress . However, the body wearing stripes was not significantly different than the body wearing a black dress .Provided that we are not interested in whether participants were adjusting the body wearing stripes or the one without stripes, as this will simply result in opposing effects see , we deciThe results suggest that the striped dress was perceived as thinner than the non-striped dress. However, this effect was predominantly observed when the non-striped dress was light-green, but not when it was black. A likely explanation for this contrasting effect might be the luminance of the non-striped dresses. According to the irradiation illusion , bright The difference between the striped and non-striped green dress could also be attributed to luminance, since by removing the—black—stripes, the luminance of the non-striped dress increases. If the “horizontal” and “luminance” effects combine , this would explain why the striped dress looked thinner when compared to the non-striped light-green dress, but not when compared to the black dress, since the luminance effect is in the opposite direction of the stripes effect. To disentangle between the potential stripe and luminance effects, we conducted Experiment 2.The aim of Experiment 2 was twofold. First, we aimed to replicate the findings of Experiment 1. Second, we aimed to further explore the nature of the illusion by decomposing it into two separate effects: the potential effect of horizontal stripes controlling for any effect of luminance, and the potential effect of luminance controlling for the effect of horizontal stripes. The procedure was identical to Experiment 1, but we introduced two novel stimuli to control for the effect of stripes and luminance. We expected that a body clad in a striped dress would be perceived as thinner than a body clad in a non-striped dress when both stimuli have the same luminance (stripes effect). Furthermore, we expected that the luminance of a dress plays a crucial role in the perception of the body width, such that darker stimuli will be perceived as thinner, regardless of whether those dresses have stripes or not (luminance effect).SD = 3.93 years, ranging from 18 to 39 years) took park in Experiment 2. The experiment lasted approximately 40 min. All participants were naïve as to the purpose of the experiment and were paid €7 or received course credits for their participation.Forty-five participants , and the striped version of the same dark-green dress . The two new dresses were a non-striped dark-green dress with luminance 2 cd/mdark-green versus stripes light-green (the two dresses have the same luminance). Second, the luminance effect controlling for the effect of stripes was tested across two conditions: stripes light-green versus stripes dark-green and light-green versus dark-green dress. Third, to test whether the effects of stripes and luminance sum together we used five conditions: dark-green versus stripes dark-green, light-green versus stripes dark-green, black versus stripes light-green, light-green versus stripes light-green, and black versus stripes dark-green. As in Experiment 1, each dress served as the variable stimulus on 50% of the trials and as the standard on the remaining trials. The word cue was either “stripes dark-green” or “stripes light-green” when participants were asked to adjust the width of the body wearing the dress with stripes, and “dark-green,” “light-green,” or “black” when participants were asked to adjust the width of the body wearing the dress without stripes. In total, there were 240 trials . The order of the trials was randomized for all participants.The inclusion of the extra stimuli allowed to test the effect of one variable  when controlling for the effect of the other, but also to test the aggregating effect of both variables. More specifically, the experimental conditions allowed to test for three effects. First, the effect of horizontal stripes controlling for the effect of luminance was tested in one condition: t44 = 2.50, p = .049, d = 0.37, 1 − β = 0.69). Thus, when the effect of luminance was controlled for (the comparison included only stimuli of same luminance), striped stimuli were perceived as thinner compared to non-striped stimuli.The effect of stripes is shown in t-tests were conducted on the PSE. Stimuli with lower luminance were perceived as thinner, both for striped  and non-striped dresses . Thus, when the effect of stripes was controlled for, dresses with lower luminance were perceived as thinner.The effect of target luminance is shown in t44 = 3.64, p = .004, d = 0.54, 1 − β = 0.94), non-striped dark-green  and light-green . The dress with light-green stripes was perceived as thinner compared to non-striped light-green  and marginally significantly thinner when compared to black . These results replicate the findings of Experiment 1.The combined effect of luminance and stripes is shown in 2, meaning that in these two conditions the effect of luminance contributes very little to the overall effect, and the PSE difference is rather attributable to the thinning effect of stripes rather than that of luminance.Although our design did not allow for a direct test of the interaction between the two main factors , the data seem to indicate that when stripes and low luminance are taken together, they produce an even larger thinning effect. Bars 3 and 5  illustraAlthough the results seem to support the notion that both stripes and dress luminance have a thinning effect, one might argue that dress luminance is confounded with background luminance, since across all conditions two relatively dark stimuli (variable and standard) are placed on a relatively bright background . Consequently, the thinning effect could theoretically be the result of the higher contrast produced between dark dresses and the light-grey background, and not of the low luminance of the dress itself. To test whether figure-background contrast affected the perceived thinning effect, we conducted Experiment 3.The role of the contrast between the dress and its background in the thinning effect was not explicitly studied in Experiments 1 and 2. It could be argued that the higher the contrast, the stronger the thinning effect that was observed across conditions. However, this would be difficult to justify in the case of the striped dress condition. In the stripes effect , the twoIn Experiment 3, we kept the luminance of the dark and bright dresses constant and manipulated the background luminance to test whether it affected size perception. We decided to replicate the findings of SD = 4.59 years, ranging from 18 to 35 years) took park in Experiment 3. The experiment lasted approximately 15 min. All participants were naïve as to the purpose of the experiment and received course credits for their participation. One participant was excluded from analyses, as he or she selected the first random variable dress (without any adjustment of the stimulus) as being equal to the standard dress on 35% of the trials (compared to the group mean: 1.9%).Twenty participants  and the light-green (39 cd/m2) dress (both without stripes). Second, we manipulated the background luminance, creating dark- (1.5 cd/m2), average- (20.5 cd/m2) and bright-grey  backgrounds. In total, there were 90 trials . The order of the trials was randomized for all participants.The experiment was identical to Experiment 2, with two exceptions. First, in contrast to Experiment 2, we used only the dark-green . The thinning effect was non-significant in the light background , but significant in the average  and dark  backgrounds. These results directly reject the hypothesis that the thinning effect obtained for darker dresses was due to their higher contrast with the light-grey background. If that was the case, we would expect the dark green dress in the first bar of F = 9.04, p = .001, β = 0.96), and post hoc pairwise comparisons revealed that the only significant difference was between the light and dark backgrounds .To test this effect, we run a repeated measures analysis of variance. Indeed, the main effect of background absolute luminance was significant (p = .049), the overall results were in the same direction and the magnitude of the effect was similar to Experiment 2 (PSE Experiment 2 = .05° vs. PSE Experiment 3 = .03°). In general, the results corroborated our previous finding that darker stimuli are perceived as thinner. On top of that, the contrast between the stimuli and the background luminance did not significantly affect the thinning effect of darker dresses. Instead, the absolute luminance of the background seemed to have contributed to the overall effect, since darker dresses were perceived even thinner in darker backgrounds.Although, for the light background condition we failed to replicate the thinning effect reported in Experiment 2  to be significantly thinner than an identical non-striped (light-green) version of the same dress, but not when compared to a non-striped black version. Since the thinning effect was significant only when the striped dress was compared to the light-green dress, we hypothesized that luminance might moderate the thinning effect. In Experiment 2, we dissociated the stripes and luminance effects by including two extra stimuli. In this way, it was possible to measure the effect of horizontal stripes and luminance on the perceived body width independently, as well as their aggregating effect. The results showed that, first, when the effect of luminance was controlled for, horizontal stripes had a thinning effect against non-striped stimuli. Second, when the effect of horizontal stripes was controlled for, stimuli with lower luminance had a thinning effect against stimuli with higher luminance, either when both stimuli were striped or non-striped. Third, when both stripes and luminance were taken together, the effect was even larger.Although it seems that the stripes effect holds when the luminance of the stimuli and the background are held constant, the luminance of the background—against which the stimuli were placed—might have affected the thinning effect of darker stimuli. This is because, in the luminance effect, the darker (vs. lighter) stimuli were further away from the luminance of the background and this luminance difference might have affected size perception. In Experiment 3, we manipulated the background luminance  to investigate whether the luminance effect of Experiment 2 was due to the increased contrast between the dark dresses and the light-grey background. The results showed that the thinning effect of dark dresses could not be attributed to stimuli-background contrast. Instead, we discovered that when the luminance of the general background is darker, the thinning effect becomes more pronounced.The thinning effect of low luminance is in line with the irradiation illusion , accordiSo far only one study has tested the perceptual effect of horizontal stripes against non-striped stimuli. Our results also extend and partially explain the Helmholtz illusion. In the original paradigm, a square with horizontal stripes is compared to a similar square with vertical stripes. In that context, the thinning effect of horizontal stripes cannot be attributed to luminance, since both stimuli have the same luminance regardless of stripe orientation. It can be attributed, however, to the thinning effect of horizontal stripes, as Experiment 2 showed. Whether vertical stripes also contribute to the overall effect or not, remains an open empirical question.The present results open two main venues for future research. The first is to further probe the boundary conditions that might account for the effect. In the present study we tested the effect of horizontal stripes, luminance, and contrast but the list of confounding variables may be longer. Some variables that might account for the effect include the orientation of the stripes , their spatial frequency (the thickness of the stripes), or color. Moreover, previous studies reported that the body size moderates the effect, with bigger body types decreasing  or even 3 This inconsistency was also described by The second line of research calls for an interdisciplinary approach between the fields of social cognition (participants’ previous beliefs) and psychophysics (participants’ perception). It is striking that, although the results of the present experiments support the thinning effect of horizontal stripes, this finding clashes with participants’ beliefs and leads to a paradox: during the behavioral task in the lab participants perceived horizontal stripes in clothes as thinner, but in self-reports they stated the opposite.The story of stripes in clothes is old and inconsistent. From a symbol of sorcery in medieval times to the sartorial choice of inmates in previous decades, stripes today are a symbol of style and fashion . Contrar


Models for ice sublimation from a freeze-drying vial rely on the assumption of a planar ice interface up to ~25% loss of ice mass  whereas single-vial determinations of the sublimation endpoint (by temperature sensors) are based on the point when the observed temperature reaches a plateau, which cannot differentiate between sublimation and desorption-drying. In this work, the real part capacitance of TVIS vial(s) containing frozen water (during sublimation drying) was measured at 100 kHz. This parameter  Freeze drying or lyophilization is a multi-stage down-stream processing method for preserving unstable liquid drug formulations  . The forSince the introduction of the US Food and Drugs Administration (FDA) Process Analytical Technology (PAT) initiative in 2003  and Nailthe vial . Recent the vial ,8,9.Here we highlight some of the assumptions in application of the heat and mass balance equation for the determination of (1)The ice interface is expected to be planar over the initial sublimation period, and into the steady state period, and therefore the area term used in the determination of (2)The vial is surrounded by its nearest neighbor in a hexagonal array and the relative contributions to (3)The above conditions continue to apply up to the first 25–30% loss of ice mass.It is important to recognize, whether it’s the determination of re vials . The perablished ,11,12.Drying rates of individual vials containing pure water/ice can be observed using a microbalance technique  and fromThermocouples could be used to define a period of temperature stability, from which a steady state may be inferred, but the additional heat contribution during drying and the impact on ice nucleation and growth during the freezing stage all contribute to difference in the way the TC-containing vials dry.Neutron imaging and in situ tomography are able to scan the partially dried matrix in a glass vial and visualize the sublimation kinetics and in doing so provide true topographic information on the changing structure of the ice front. However, they require a line of sight to the vial(s) under scrutiny, which then impacts the drying process and results in anomalous ice shapes developing during the process .It remains the case, that no technique has been established which can qualify the time period over which the sublimation interface remains unchanged, and steady state conditions apply, without perturbing the system that one is attempting to measure.®, Tempris GmbH, Holzkirchen, Germany) [It is well known that primary drying is the longest phase of a lyophilization cycle and manufacturers should focus on optimizing primary drying to make the whole process more efficient ,15. In tGermany) ,16. One Germany) .All these technologies involve the insertion of a probe into the product to measure its ‘point’ temperature during the lyo cycle, with the sublimation end point being assessed after the product temperature increases above that of the shelf temperature, as the process of sublimative cooling ends . HoweverThe issues with these product-invasive probes has stimulated the development of non-product invasive, individual-vial sensors such as (i) plasma-sputtered thermocouples deposited externally to the glass vial ; (ii) BrUltimately, any technique for the determination of sublimation end point which is based purely on changes in the thermal properties of the vial and contents will be compromised by the fact that the contribution to temperature changes from the desorption of water from the unfrozen fraction (leading to the attainment of thermodynamic equilibrium of water vapor distributed between the solid fraction and the gaseous phase) is conflated with that from the sublimation of ice. In other words, one process inevitably merges with the other.Another non-invasive technique for the determination of primary drying end point is through-vial impedance spectroscopy (TVIS). In its current manifestation, a pair of thin film or metal foil electrodes are attached on opposite faces of the glass wall and connected to a purpose built broad-band (10 to 1 MHz) electrical impedance spectrometer . The objIn this study, we present a universal parameter, The rationale for choosing pendence . Given tIn future articles we shall demonstrate that the TVIS derived primary drying endpoint can be clearly differentiated from thermal changes associated with water loss from the unfrozen fraction and hence provide a precise assessment of the true sublimation endpoint in the freeze-drying of solutions.The aim of this work is to develop an application for through vial spectroscopy (TVIS) for monitoring the changes in the shape of the ice mass during the process of sublimation and for determining the endpoint of ice sublimation in TVIS vials at different locations of the shelf of a small-scale development dryer.For the TVIS measurement, a Schott 10R Type 1 glass tubing vial  was modified with foil electrodes of dimensions 10 mm (height) by 19 mm (width) cut from a copper adhesive tape (3M 1181 Tape Copper Foil with Conductive Adhesive) and attached to the surface of the glass wall, at a distance of 3 mm from the bottom of the vial a. This T(i)The complex capacitance spectra of the TVIS vial between 10 Hz and 1 MHz (with 10 data points per decade), (ii)The freeze-dryer process parameters of the shelf temperature and chamber pressure and the product temperature from the thermocouple in the unmodified glass vial(iii)Photographic images of the TVIS vial using a Canon DSLR cameraA batch of 160 × 10R vials were filled each with 3 g of double distilled water. Of the 160 vials, three were TVIS vials with copper electrodes (19 mm by 10 mm) positioned at 3 mm from the base of the vial and hence from the freeze-drying shelf were placed in the front row (edge), the ninth row  and the last row (edge) of the batch as shown in It should be pointed out that there are differences between the two lyo cycles. In the lyo cycle for the single vial, the shelf temperature is lowered to −40 °C and primary drying is carried out at −15 °C, whereas for the batch the shelf temperature is lowered to −45 °C and primary drying is carried out at −5 °C. The lower shelf temperature for the single/isolated vial ensures a slower drying rate and one that we hope might be comparable to the drying rate for the front vial in the batch experiment hence facilitating a more direct comparison of the drying profiles recorded by the TVIS system. The first is that the magnitude of the dielectric relaxation process  decreases during primary drying. This might be expected given that the process is due to the dielectric relaxation of ice and so as the ice volume decreases within the electrode region then one would expect to see a progressive decrease in the magnitude of this process. The second observation is that the relaxation process decreases to zero before all the ice has been removed from the vial. In other words, the absence of a measurable relaxation does not in itself indicate the sublimation end point. The third, and possibly unexpected observation, is that the magnitude of the real part capacitance  to C (3:30), the trajectory of and door  and fromand door . It is cDuring the period between point C (3:30) and point D (10:42), the capacitance profile changes course as the magnitude of ckground . This enIn the previous sections, we considered a single (isolated) TVIS vial to demonstrate the features of the high frequency capacitance timeline from the beginning of primary drying to the visual endpoint of this individual vial. In this section we consider a shelf full of vials  which inLet us first consider the response from the TC-containing vials. (i)The gradual detachment of the TC sensor from the ice mass, which might start as early as point X, which is followed with a notable inflection in the temperature profile at point Y, which appears to be associated with the full detachment of the sensing bead from the ice mass vii.(ii)A clear inflection at point D which corresponds to the visual endpoint ix(iii)The visual end point is followed by gradual increase in temperature until it reaches a plateau at point E, which means that the vial has come to a thermal equilibrium with its surroundings .The profiles for the back and the core vials share some of the features of the front vial, in that they appear to reach thermal equilibrium at point E at the same time as the front vial, however, the corresponding point D is less clearly defined.Next, we shall consider the timelines of the VIS vial . Plot  qualifies that the end of the steep rise in the high frequency capacitance at 12:56  to the thermal equilibration point E. The point E therefore corresponds to the thermal equilibration of the vial with its surroundings. This may also be taken as the sublimation end point of the batch, but only for the specific case of the sublimation from pure water. For the TVIS vials located at the back and core of the array, it is then assumed that the sublimation end point may be also taken from the point D, and occurs at times 16:22 and 16:48, respectively. Although there is no direct photographic evidence to support this assumption, the fact that the point D for the core vial takes place 20 min after the back vial is consistent with the relative locations across the shelf of the dryer. As was the case for the TC containing vials, the thermal equilibration point E is also quite similar for all three TVIS vials, again pointing to the significance of this event as the time when the temperature becomes quite uniform and constant across the vial array. In general, it can be concluded that the edge vials do not reach thermal equilibrium until the internal vials reach their own equilibrium temperature. In other words, after the visual endpoint is reached, the temperature sensed by the front TVIS vial continues to stay low, as long as the center vials are still drying. In this study, a unique time profile of the real part capacitance of the TVIS vial, at an excitation frequency of 100 kHz, has been identified which displays characteristics that reflect key features of the evolving shape of the ice mass during the primary drying phase. The first feature is the linear region in the trajectory of the Our next studies will explore whether the characteristic profile from the high frequency capacitance of the TVIS vial is universal for solutions, as well as for ice. The cake is likely to impact  the way the topography of the ice front develops, as so we might expect the same principal shape but with different durations for each phase; For example, the stabilization of the ice front might allow for a longer linear phase (from points A to B). The main difference, however, might be a result of the existence of the solids fraction that is left behind once the ice has been removed. It is known that this partially dry material liberates water from the desorption of that portion of water which did not crystalize as ice but instead hydrates the solutes within the solids fraction. The desorption of the adsorbed water requires energy and given that the vial heat transfer is poor then the energy is drawn to a large extent from the thermal mass of the vial and its contents. We have already seen, from this study, how a drift in the temperature of a vial can be observed after the sublimation end point is reached for pure water within that vial, as a consequence of the heat being removed by neighboring vials that are still in the active process of ice sublimation. We might also expect there to be a similar thermal effect from the desorption of water from the solids fraction within an individual vial and for this to contribute to an extension to the period D to E. The preliminary observations, that we have made to date, indicate similar behavior is indeed observed for simple sugar solutions. In subsequent studies, we shall be able to make some use of the photographic evidence from a front/edge vial; even though we might be able to see the shape of the ice front we can nevertheless detect the height of the ice cylinder in intimate contact with the inside perimeter of the vial; given that ice which intimate contact with the inside wall will take on a different appearance to that of the dried solids fraction and any ice fraction that has pulled away from the wall owing to preferential drying. We shall compare the photographic evidence for the rate of decrease in the height of the ice cylinder in contact with the side wall with the rate of decrease in the high frequency capacitance, and if both are linear with time then that will confirm that we can use the linear region (A to B) of the The next point to qualify is the dip at point C, where the The more challenging transition is to establish whether point D provides a universal indicator for the condition when all the ice has been removed from the frozen solution. Photographic images cannot see what is happening on the underside of the vial and so an alternative method is proposed, in which a series of TVIS vials is placed at various positions from the front (edge) to the core. The primary drying cycle is then interrupt once the In industrial practice, the preference is for the determination of the sublimation end point is the use of a comparative pressure measurement (CPM) using a Pirani gauge and the capacitance manometer, given that this approach determines the batch end point rather than a single vial. However, when drying frozen solutions (as opposed to simply ice) the CPM approach will also detect water vapor generated from the unfrozen fraction as well as that from sublimation of ice. It is a characteristic of the time profile of the pressure measured by the Pirani gauge that it attains a steady state value when the chamber is saturated with moisture vapors but then as the sublimation of ice comes to an end, across the population of vials in the batch, the measured pressure decreases in a sigmoidal way. However, during this sigmoidal descent of the measured pressure, the Pirani gauge senses water vapors desorbing from the solids fraction and only comes to a new steady state value once an equilibrium has been achieved between the residual water in the solids fraction and the water vapor in the chamber. The two processes overlap which then obscures the true endpoint of the sublimation phase. The potential for TVIS to be able to differentiate between the ice sublimation end point and the ongoing desorption of moisture from the solids fraction, will be based largely on whether the sublimation of ice from frozen solutions results in a distinct inflection in the high frequency capacitance profile, from the steep, almost linear rise which then transitions abruptly to a more gradual rise once the individual vial sublimation end point has been reached. If this turns out to be the case, then TVIS might in future be used to provide a clue as to which characteristic of the CPM time profile can be taken as the true sublimation end point of the batch.