Patient-specific, deliverable, and self-expandable surgical guide development and evaluation using 4D printing for laparoscopic partial nephrectomy.

Accurate lesion diagnosis through computed tomography (CT) and advances in laparoscopic or robotic surgeries have increased partial nephrectomy survival rates. However, accurately marking the kidney resection area through the laparoscope is a prevalent challenge. Therefore, we fabricated and evaluated a 4D-printed kidney surgical guide (4DP-KSG) for laparoscopic partial nephrectomies based on CT images. The kidney phantom and 4DP-KSG were designed based on CT images from a renal cell carcinoma patient. 4DP-KSG were fabricated using shape-memory polymers. 4DP-KSG was compressed to a 10 mm thickness and restored to simulate laparoscopic port passage. The Bland-Altman evaluation assessed 4DP-KSG shape and marking accuracies before compression and after restoration with three operators. The kidney phantom's shape accuracy was 0.436 ± 0.333 mm, and the 4DP-KSG's shape accuracy was 0.818 ± 0.564 mm before compression and 0.389 ± 0.243 mm after restoration, with no significant differences. The 4DP-KSG marking accuracy was 0.952 ± 0.682 mm before compression and 0.793 ± 0.677 mm after restoration, with no statistical differences between operators (p = 0.899 and 0.992). In conclusion, our 4DP-KSG can be used for laparoscopic partial nephrectomies, providing precise and quantitative kidney tumor marking between operators before compression and after restoration.

Conformational dynamics of adenylate kinase in crystals.

Adenylate kinase is a ubiquitous enzyme in living systems and undergoes dramatic conformational changes during its catalytic cycle. For these reasons, it is widely studied by genetic, biochemical, and biophysical methods, both experimental and theoretical. We have determined the basic crystal structures of three differently liganded states of adenylate kinase from Methanotorrus igneus, a hyperthermophilic organism whose adenylate kinase is a homotrimeric oligomer. The multiple copies of each protomer in the asymmetric unit of the crystal provide a unique opportunity to study the variation in the structure and were further analyzed using advanced crystallographic refinement methods and analysis tools to reveal conformational heterogeneity and, thus, implied dynamic behaviors in the catalytic cycle.

Evaluation of the accuracy of an augmented reality-based tumor-targeting guide for breast-conserving surgery.

BACKGROUND AND OBJECTIVES: Although magnetic resonance imaging (MRI) is commonly used for breast tumor detection, significant challenges remain in determining and presenting the three-dimensional (3D) morphology of tumors to guide breast-conserving surgery. To address this challenge, we have developed the augmented reality-breast surgery guide (AR-BSG) and compared its performance with that of a traditional 3D-printed breast surgical guide (3DP-BSG). METHODS: Based on the MRI results of a breast cancer patient, a breast phantom made of skin, body, and tumor was fabricated through 3D printing and silicone-casting. AR-BSG and 3DP-BSG were executed using surgical plans based on the breast phantom's computed tomography scan images. Three operators independently inserted a catheter into the phantom using each guide. Their targeting accuracy was then evaluated using Bland-Altman analysis with limits of agreement (LoA). Differences between the users of each guide were evaluated using the intraclass correlation coefficient (ICC). RESULTS: The entry and end point errors associated with AR-BSG were -0.34±0.68 mm (LoA: -1.71-1.01 mm) and 0.81±1.88 mm (LoA: -4.60-3.00 mm), respectively, whereas 3DP-BSG was associated with entry and end point errors of -0.28±0.70 mm (LoA: -1.69-1.11 mm) and -0.62±1.24 mm (LoA: -3.00-1.80 mm), respectively. The AR-BSG's entry and end point ICC values were 0.99 and 0.97, respectively, whereas 3DP-BSG was associated with entry and end point ICC values of 0.99 and 0.99, respectively. CONCLUSIONS: AR-BSG can consistently and accurately localize tumor margins for surgeons without inferior guiding accuracy AR-BSG can consistently and accurately localize tumor margins for surgeons without inferior guiding accuracy compared to 3DP-BSG. Additionally, when compared with 3DP-BSG, AR-BSG can offer better spatial perception and visualization, lower costs, and a shorter setup time.

Development of a patient-specific chest computed tomography imaging phantom with realistic lung lesions using silicone casting and three-dimensional printing.

The validation of the accuracy of the quantification software in computed tomography (CT) images is very challenging. Therefore, we proposed a CT imaging phantom that accurately represents patient-specific anatomical structures and randomly integrates various lesions including disease-like patterns and lesions of various shapes and sizes using silicone casting and three-dimensional (3D) printing. Six nodules of various shapes and sizes were randomly added to the patient's modeled lungs to evaluate the accuracy of the quantification software. By using silicone materials, CT intensities suitable for the lesions and lung parenchyma were realized, and their Hounsfield unit (HU) values were evaluated on a CT scan of the phantom. As a result, based on the CT scan of the imaging phantom model, the measured HU values for the normal lung parenchyma, each nodule, fibrosis, and emphysematous lesions were within the target value. The measurement error between the stereolithography model and 3D-printing phantoms was 0.2 ± 0.18 mm. In conclusion, the use of 3D printing and silicone casting allowed the application and evaluation of the proposed CT imaging phantom for the validation of the accuracy of the quantification software in CT images, which could be applied to CT-based quantification and development of imaging biomarkers.

An interactive and realistic phantom for cricothyroidotomy simulation of a patient with obesity through a reusable design using 3D-printing and Arduino.

BACKGROUND AND OBJECTIVES: Proper airway management during emergencies can prevent serious complications. However, cricothyroidotomy is challenging in patients with obesity. Since this technique is not performed frequently but at a critical time, the opportunity for trainees is rare. Simulators for these procedures are also lacking. Therefore, we proposed a realistic and interactive cricothyroidotomy simulator. METHODS: All anatomical structures were modeled based on computed tomography images of a patient with obesity. To mimic the feeling of incision during cricothyroidotomy, the incision site was modeled to distinguish between the skin and fat. To reinforce the educational purpose, capacitive touch sensors were attached to the artery, vein, and thyroid to generate audio feedback. The tensile strength of the silicone-cast skin was measured to verify the similarity of the mechanical properties between humans and our model. The fabrication and assembly accuracies of the phantom between the Standard Tessellation Language and the fabricated model were evaluated. Audio feedback through sensing the anatomy parts and utilization was evaluated. RESULTS: The body, skull, clavicle, artery, vein, and thyroid were fabricated using fused deposition modeling (FDM) with polylactic acid. A skin mold was fabricated using FDM with thermoplastic polyurethane. A fat mold was fabricated using stereolithography apparatus (SLA) with a clear resin. The airway and tongue were fabricated using SLA with an elastic resin. The tensile strength of the skin using silicone with and without polyester mesh was 2.63 ± 0.68 and 2.46 ± 0.21 MPa. The measurement errors for fabricating and assembling parts of the phantom between the STL and the fabricated models were -0.08 ± 0.19 mm and 0.13 ± 0.64 mm. The measurement errors internal anatomy embodied surfaces in fat part were 0.41 ± 0.89 mm. Audio feedback was generated 100% in all the areas tested. The realism, understanding of clinical skills, and intention to retrain were 7.1, 8.8, and 8.3 average points. CONCLUSIONS: Our simulator can provide a realistic simulation experience for trainees through a realistic feeling of incision and audio feedback, which can be used for actual clinical education.

Evaluation of formaldehyde, particulate matters 2.5 and 10 emitted to a 3D printing workspace based on ventilation.

Recently, the development of 3D printing (3DP) technology and its application in various fields have improved our quality of life. However, hazardous materials that affect the human body, such as formaldehyde and particulate matter (PM), are emitted into the air during 3DP. This study measured the formaldehyde, PM10, and PM2.5 emitted by 3DP with the ventilation operation using six materials in material extrusion (ME) and vat photopolymerization (VP) and compared them between the 3DP workspace and the control setting with test-retest validation by two researchers. The experiments were divided into four stages based on the 3DP and ventilation operation. A linear mixed model was used to analyze the mean differences and tendencies between the 3DP workspace and the control setting. The change as ventilation was switched from off to on was evaluated by calculating the area. The differences and tendencies were shown in the statistically significant differences from a post-hoc test (α = 0.0125) except for some cases. There was a significant difference in formaldehyde depending on the ventilation operation; however, only a minor difference in PM10, and PM2.5 was confirmed. The amount of formaldehyde exceeding the standard was measured in all materials during 3DP without ventilation. Therefore, it is recommended to operate ventilation systems.

Rehearsal simulation to determine the size of device for left atrial appendage occlusion using patient-specific 3D-printed phantoms.

Left atrial appendage (LAA) occlusion (LAAO) is used to close the finger-like extension from the left atrium with occlusion devices to block the source of thrombosis. However, selection of the devices size is not easy due to various anatomical changes. The purpose of this study is patient-specific, computed tomography angiography (CTA)-based, three-dimensionally (3D) printed LAAO phantoms were applied pre-procedure to determine the size. Ten patients were enrolled prospectively in March 2019 and December 2020. The cardiac structure appearing in CTA was first segmented, and the left atrium and related structures in the LAAO procedure were modeled. The phantoms were fabricated using two methods of fused deposition modeling (FDM) and stereolithography (SLA) 3D printers with thermoplastic polyurethane (TPU) and flexible resin materials and evaluated by comparing their physical and material properties. The 3D-printed phantoms were directly used to confirm the shape of LAA, and to predict the device size for LAAO. In summary, the shore A hardness of TPU of FDM was about 80-85 shore A, and that of flexible resin of SLA was about 50-70 shore A. The measurement error between the STL model and 3D printing phantoms were 0.45 ± 0.37 mm (Bland-Altman, limits of agreement from - 1.8 to 1.6 mm). At the rehearsal, the estimations of device sizes were the exact same with those in the actual procedures of all 10 patients. In conclusion, simulation with a 3D-printed left atrium phantom could be used to predict the LAAO insertion device size accurately before the procedure.

Dismantling and Rebuilding the Trisulfide Cofactor Demonstrates Its Essential Role in Human Sulfide Quinone Oxidoreductase.

Sulfide quinone oxidoreductase (SQOR) catalyzes the first step in sulfide clearance, coupling H2S oxidation to coenzyme Q reduction. Recent structures of human SQOR revealed a sulfur atom bridging the SQOR active site cysteines in a trisulfide configuration. Here, we assessed the importance of this cofactor using kinetic, crystallographic, and computational modeling approaches. Cyanolysis of SQOR proceeds via formation of an intense charge transfer complex that subsequently decays to eliminate thiocyanate. We captured a disulfanyl-methanimido thioate intermediate in the SQOR crystal structure, revealing how cyanolysis leads to reversible loss of SQOR activity that is restored in the presence of sulfide. Computational modeling and MD simulations revealed an ∼105-fold rate enhancement for nucleophilic addition of sulfide into the trisulfide versus a disulfide cofactor. The cysteine trisulfide in SQOR is thus critical for activity and provides a significant catalytic advantage over a cysteine disulfide.

A Catalytic Trisulfide in Human Sulfide Quinone Oxidoreductase Catalyzes Coenzyme A Persulfide Synthesis and Inhibits Butyrate Oxidation.

Mitochondrial sulfide quinone oxidoreductase (SQR) catalyzes the oxidation of H2S to glutathione persulfide with concomitant reduction of CoQ10. We report herein that the promiscuous activity of human SQR supported the conversion of CoA to CoA-SSH (CoA-persulfide), a potent inhibitor of butyryl-CoA dehydrogenase, and revealed a molecular link between sulfide and butyrate metabolism, which are known to interact. Three different CoQ1-bound crystal structures furnished insights into how diverse substrates access human SQR, and provided snapshots of the reaction coordinate. Unexpectedly, the active site cysteines in SQR are configured in a bridging trisulfide at the start and end of the catalytic cycle, and the presence of sulfane sulfur was confirmed biochemically. Importantly, our study leads to a mechanistic proposal for human SQR in which sulfide addition to the trisulfide cofactor eliminates 201Cys-SSH, forming an intense charge-transfer complex with flavin adenine dinucleotide, and 379Cys-SSH, which transfers sulfur to an external acceptor.

Structural and mutational analyses of psychrophilic and mesophilic adenylate kinases highlight the role of hydrophobic interactions in protein thermal stability.

Protein thermal stability is an important field since thermally stable proteins are desirable in many academic and industrial settings. Information on protein thermal stabilization can be obtained by comparing homologous proteins from organisms living at distinct temperatures. Here, we report structural and mutational analyses of adenylate kinases (AKs) from psychrophilic Bacillus globisporus (AKp) and mesophilic Bacillus subtilis (AKm). Sequence and structural comparison showed suboptimal hydrophobic packing around Thr26 in the CORE domain of AKp, which was replaced with an Ile residue in AKm. Mutations that improved hydrophobicity of the Thr residue increased the thermal stability of the psychrophilic AKp, and the largest stabilization was observed for a Thr-to-Ile substitution. Furthermore, a reverse Ile-to-Thr mutation in the mesophilic AKm significantly decreased thermal stability. We determined the crystal structures of mutant AKs to confirm the impact of the residue substitutions on the overall stability. Taken together, our results provide a structural basis for the stability difference between psychrophilic and mesophilic AK homologues and highlight the role of hydrophobic interactions in protein thermal stability.

Structural analyses of adenylate kinases from Antarctic and tropical fishes for understanding cold adaptation of enzymes.

Psychrophiles are extremophilic organisms capable of thriving in cold environments. Proteins from these cold-adapted organisms can remain physiologically functional at low temperatures, but are structurally unstable even at moderate temperatures. Here, we report the crystal structure of adenylate kinase (AK) from the Antarctic fish Notothenia coriiceps, and identify the structural basis of cold adaptation by comparison with homologues from tropical fishes including Danio rerio. The structure of N. coriiceps AK (AKNc) revealed suboptimal hydrophobic packing around three Val residues in its central CORE domain, which are replaced with Ile residues in D. rerio AK (AKDr). The Val-to-Ile mutations that improve hydrophobic CORE packing in AKNc increased stability at high temperatures but decreased activity at low temperatures, suggesting that the suboptimal hydrophobic CORE packing is important for cold adaptation. Such linkage between stability and activity was also observed in AKDr. Ile-to-Val mutations that destabilized the tropical AK resulted in increased activity at low temperatures. Our results provide the structural basis of cold adaptation of a psychrophilic enzyme from a multicellular, eukaryotic organism, and highlight the similarities and differences in the structural adjustment of vertebrate and bacterial psychrophilic AKs during cold adaptation.

Structure and Stability of the Dimeric Triosephosphate Isomerase from the Thermophilic Archaeon Thermoplasma acidophilum.

Thermoplasma acidophilum is a thermophilic archaeon that uses both non-phosphorylative Entner-Doudoroff (ED) pathway and Embden-Meyerhof-Parnas (EMP) pathway for glucose degradation. While triosephosphate isomerase (TPI), a well-known glycolytic enzyme, is not involved in the ED pathway in T. acidophilum, it has been considered to play an important role in the EMP pathway. Here, we report crystal structures of apo- and glycerol-3-phosphate-bound TPI from T. acidophilum (TaTPI). TaTPI adopts the canonical TIM-barrel fold with eight α-helices and parallel eight ß-strands. Although TaTPI shares ~30% sequence identity to other TPIs from thermophilic species that adopt tetrameric conformation for enzymatic activity in their harsh physiological environments, TaTPI exists as a dimer in solution. We confirmed the dimeric conformation of TaTPI by analytical ultracentrifugation and size-exclusion chromatography. Helix 5 as well as helix 4 of thermostable tetrameric TPIs have been known to play crucial roles in oligomerization, forming a hydrophobic interface. However, TaTPI contains unique charged-amino acid residues in the helix 5 and adopts dimer conformation. TaTPI exhibits the apparent Td value of 74.6°C and maintains its overall structure with some changes in the secondary structure contents at extremely acidic conditions (pH 1-2). Based on our structural and biophysical analyses of TaTPI, more compact structure of the protomer with reduced length of loops and certain patches on the surface could account for the robust nature of Thermoplasma acidophilum TPI.

Effectiveness and limitations of local structural entropy optimization in the thermal stabilization of mesophilic and thermophilic adenylate kinases.

Local structural entropy (LSE) is a descriptor for the extent of conformational heterogeneity in short protein sequences that is computed from structural information derived from the Protein Data Bank. Reducing the LSE of a protein sequence by introducing amino acid mutations can result in fewer conformational states and thus a more stable structure, indicating that LSE optimization can be used as a protein stabilization method. Here, we describe a series of LSE optimization experiments designed to stabilize mesophilic and thermophilic adenylate kinases (AKs) and report crystal structures of LSE-optimized AK variants. In the mesophilic AK, thermal stabilization by LSE reduction was effective but limited. Structural analyses of the LSE-optimized mesophilic AK variants revealed a strong correlation between LSE and the apolar buried surface area. Additional mutations designed to introduce noncovalent interactions between distant regions of the polypeptide resulted in further stabilization. Unexpectedly, optimizing the LSE of the thermophilic AK resulted in a decrease in thermal stability. This destabilization was reduced when charged residues were excluded from the possible substitutions during LSE optimization. These observations suggest that stabilization by LSE reduction may result from the optimization of local hydrophobic contacts. The limitations of this process are likely due to ignorance of other interactions that bridge distant regions in a given amino acid sequence. Our results illustrate the effectiveness and limitations of LSE optimization as a protein stabilization strategy and highlight the importance and complementarity of local conformational stability and global interactions in protein thermal stability.

An integrated approach for thermal stabilization of a mesophilic adenylate kinase.

Thermally stable proteins are desirable for research and industrial purposes, but redesigning proteins for higher thermal stability can be challenging. A number of different techniques have been used to improve the thermal stability of proteins, but the extents of stability enhancement were sometimes unpredictable and not significant. Here, we systematically tested the effects of multiple stabilization techniques including a bioinformatic method and structure-guided mutagenesis on a single protein, thereby providing an integrated approach to protein thermal stabilization. Using a mesophilic adenylate kinase (AK) as a model, we identified stabilizing mutations based on various stabilization techniques, and generated a series of AK variants by introducing mutations both individually and collectively. The redesigned proteins displayed a range of increased thermal stabilities, the most stable of which was comparable to a naturally evolved thermophilic homologue with more than a 25° increase in its thermal denaturation midpoint. We also solved crystal structures of three representative variants including the most stable variant, to confirm the structural basis for their increased stabilities. These results provide a unique opportunity for systematically analyzing the effectiveness and additivity of various stabilization mechanisms, and they represent a useful approach for improving protein stability by integrating the reduction of local structural entropy and the optimization of global noncovalent interactions such as hydrophobic contact and ion pairs.