Efficient EndoNeRF reconstruction and its application for data-driven surgical simulation.

PURPOSE: The healthcare industry has a growing need for realistic modeling and efficient simulation of surgical scenes. With effective models of deformable surgical scenes, clinicians are able to conduct surgical planning and surgery training on scenarios close to real-world cases. However, a significant challenge in achieving such a goal is the scarcity of high-quality soft tissue models with accurate shapes and textures. To address this gap, we present a data-driven framework that leverages emerging neural radiance field technology to enable high-quality surgical reconstruction and explore its application for surgical simulations. METHOD: We first focus on developing a fast NeRF-based surgical scene 3D reconstruction approach that achieves state-of-the-art performance. This method can significantly outperform traditional 3D reconstruction methods, which have failed to capture large deformations and produce fine-grained shapes and textures. We then propose an automated creation pipeline of interactive surgical simulation environments through a closed mesh extraction algorithm. RESULTS: Our experiments have validated the superior performance and efficiency of our proposed approach in surgical scene 3D reconstruction. We further utilize our reconstructed soft tissues to conduct FEM and MPM simulations, showcasing the practical application of our method in data-driven surgical simulations. CONCLUSION: We have proposed a novel NeRF-based reconstruction framework with an emphasis on simulation purposes. Our reconstruction framework facilitates the efficient creation of high-quality surgical soft tissue 3D models. With multiple soft tissue simulations demonstrated, we show that our work has the potential to benefit downstream clinical tasks, such as surgical education.

Efficient Deformable Tissue Reconstruction via Orthogonal Neural Plane.

Intraoperative imaging techniques for reconstructing deformable tissues in vivo are pivotal for advanced surgical systems. Existing methods either compromise on rendering quality or are excessively computationally intensive, often demanding dozens of hours to perform, which significantly hinders their practical application. In this paper, we introduce Fast Orthogonal Plane (Forplane), a novel, efficient framework based on neural radiance fields (NeRF) for the reconstruction of deformable tissues. We conceptualize surgical procedures as 4D volumes, and break them down into static and dynamic fields comprised of orthogonal neural planes. This factorization discretizes the four-dimensional space, leading to a decreased memory usage and faster optimization. A spatiotemporal importance sampling scheme is introduced to improve performance in regions with tool occlusion as well as large motions and accelerate training. An efficient ray marching method is applied to skip sampling among empty regions, significantly improving inference speed. Forplane accommodates both binocular and monocular endoscopy videos, demonstrating its extensive applicability and flexibility. Our experiments, carried out on two in vivo datasets, the EndoNeRF and Hamlyn datasets, demonstrate the effectiveness of our framework. In all cases, Forplane substantially accelerates both the optimization process (by over 100 times) and the inference process (by over 15 times) while maintaining or even improving the quality across a variety of non-rigid deformations. This significant performance improvement promises to be a valuable asset for future intraoperative surgical applications. The code of our project is now available at https://github.com/Loping151/ForPlane.

Human cytochrome p450c17: single step purification and phosphorylation of serine 258 by protein kinase a.

Cytochrome P450c17 (P450c17) is the single microsomal enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities. The ratio of lyase to hydroxylase activity of human P450c17 determines whether steroidogenesis leads to the synthesis of cortisol or sex steroids. This ratio is regulated posttranslationally by factors that influence the efficiency of electron transfer from P450 oxidoreductase to P450c17. One factor favoring more efficient electron transfer and 17,20 lyase activity is cAMP-dependent serine/threonine phosphorylation of P450c17. Identifying the responsible kinase(s) and the P450c17 residues that undergo phosphorylation has been challenging, partly because of difficulties in preparing biochemically useful amounts of pure, catalytically active P450c17. We describe a modified strategy for preparing P450c17 in which the traditional carboxy-terminal 4xHis tag is replaced by 3xGly6xHis. This construct permits more rotational freedom of the protein when bound to the nickel affinity column, reducing steric associations between the protein and the column, and permitting a single-step chromatographic purification to apparent homogeneity. Using this vector, we explored P450c17 phosphorylation by mutagenesis of Ser and/or Thr residues to Asp or Glu to mimic the approximate size and charge of phospho-Ser or phospho-Thr. This strategy did not identify Ser and/or Thr site(s) that increase the ratio of lyase to hydroxylase activity, suggesting that the regulatory phosphorylation strategy of human P450c17 is very complicated. Although previous work has excluded protein kinase A (PKA) as the responsible kinase, the cAMP-inducible nature of the phosphorylation-associated increase in lyase activity suggests that PKA may play a role, possibly as a priming kinase. Using our novel vector and a series of mutations, we identified the P450c17 site phosphorylated by PKA as Ser258.

Identification of N-terminal lobe motifs that determine the kinase activity of the catalytic domains and regulatory strategies of Src and Csk protein tyrosine kinases.

Csk and Src protein tyrosine kinases are structurally homologous but use opposite regulatory strategies. The isolated catalytic domain of Csk is intrinsically inactive and is activated by interactions with the regulatory Src homology 3 (SH3) and SH2 domains, while the isolated catalytic domain of Src is intrinsically active and is suppressed by interactions with the regulatory SH3 and SH2 domains. The structural basis for why one isolated catalytic domain is intrinsically active while the other is inactive is not clear. In this study, we identified structural elements in the N-terminal lobe of the catalytic domain that render the Src catalytic domain active. These structural elements include the alpha-helix C region, a beta turn between the beta4 and beta5 strands, and an Arg residue at the beginning of the catalytic domain. These three motifs interact with one another to activate the Src catalytic domain, but the equivalent motifs in Csk directly interact with the regulatory domains that are important for Csk activation. The Src motifs can be grafted to the Csk catalytic domain to obtain an active Csk catalytic domain. These results, together with available Src and Csk tertiary structures, reveal an important structural switch that determines the kinase activity of a catalytic domain and dictates the regulatory strategy of a kinase.

Subdomain switching reveals regions that harbor substrate specificity and regulatory properties of protein tyrosine kinases.

Csk and Src are two protein tyrosine kinases that share a similar overall multidomain structural organization and a high degree of sequence homology but have different substrate specificities and regulatory properties. In this study, we generated chimeric kinases of Csk and Src by switching the C-terminal lobes of their catalytic domains, and we characterized their substrate specificity and regulatory properties. First, both Csk and Src phosphorylate Src as a common substrate, but on different Tyr residues. The C-terminal lobes of the kinase catalytic domain determined the site of phosphorylation on Src. Furthermore, toward several physiological substrates of Src, the substrate specificity was also determined by the C-terminal lobe of the catalytic domain regardless of the regulatory domains and the N-terminal lobe of the catalytic domain. Second, Csk and Src represent two general regulatory strategies for protein tyrosine kinases. Csk catalytic domain is inactive and is positively regulated by the regulatory domains, while Src catalytic domain is active and suppressed by its interactions with the regulatory domains. The regulatory properties of the chimeric kinases were more complicated. The regulatory domains and the N-lobe did not fully determine the response to a regulatory ligand, suggesting that the C-lobe also contributes to such responses. On the other hand, the intrinsic kinase activity of the catalytic domain correlates with the identity of the N-lobe. These results demonstrate that the chimeric strategy is useful for detailed dissection of the mechanistic basis of substrate specificity and regulation of protein tyrosine kinases.

Synthesis and evaluation of 3-phenylpyrazolo[3,4-d]pyrimidine-peptide conjugates as Src kinase inhibitors.

3-Phenylpyrazolo[3,4-d]pyrimidine (PhPP) derivatives substituted with an alkyl or aryl carboxylic acid at the N1-endocyclic amine, such as PhPP-CH(2)COOH (IC(50)=250 microM), and peptides Ac-CIYKYY (IC(50)=400 microM) and Ac-YIYGSFK (IC(50)=570 microM) were weak inhibitors of polyE(4)Y phosphorylation by active c-Src. A series of PhPP-peptide conjugates were synthesized using PhPP as an ATP mimic and CIYKYY or YIYGSFK as a peptide substrate to improve the inhibitory potency against active c-Src kinase. PhPP derivatives were attached to the N terminus or the side chain of amino acids in the peptide template. Two N-terminal substituted conjugates, PhPP-CH(2)CO-CIYKYY (IC(50)=0.38 microM) and PhPP-CH(2)CO-YIYGSFK (IC(50)=2.7 microM), inhibited the polyE(4)Y phosphorylation by active c-Src significantly higher than that of the parent compounds. The conjugation of PhPP with the peptides produced a synergistic inhibition effect possibly through creation of favorable interactions between the conjugate and the kinase domain as shown by molecular modeling studies.

Design and evaluation of hydroxamate derivatives as metal-mediated inhibitors of a protein tyrosine kinase.

Protein tyrosine kinases use two Mg(2+) ions as cofactors in catalysis, one as the ATP-Mg complex (M1) and the other as an essential activator (M2). The M2-binding site has high affinity for transition metal cations such as cobalt and zinc. Taking advantage of this high affinity, we examined hydroxamates as metal-mediated inhibitors against C-terminal Src kinase (Csk), a protein tyrosine kinase. Of a small group of amino acid hydroxamates, tyrosine and phenylalanine hydroxamates inhibited Csk activity only in the presence of Co(2+). Four classes of phenylalanine and tyrosine hydroxamate derivatives were synthesized and evaluated as metal-mediated inhibitors of Csk, leading to improved inhibition and a better understanding of the structure-activity relationships. This study suggests that hydroxamates may serve as a general scaffold for developing metal-mediated inhibitors against protein tyrosine kinases. To the best of our knowledge, this is the first report of designing metal-mediated inhibitors against a protein tyrosine kinase by targeting a metal binding site.

Bacterial expression and characterization of catalytic loop mutants of SRC protein tyrosine kinase.

Protein tyrosine kinase Src is a key enzyme in mammalian signal transduction and an important target for anticancer drug discovery. Although recombinant expression in bacterial cells offers a convenient and rapid way for producing several other protein tyrosine kinases, active Src is difficult to produce in bacterial systems. However, a kinase-defective Src mutant (due to a single point mutation, Lys295Met) is expressed strongly in bacteria. We hypothesize that the difficulty with expressing active Src in bacteria is due to toxicity caused by Src kinase activity. To test this hypothesis, we generated a series of Src mutants by altering certain residues, especially His384, in the catalytic loop and examined their expression in the bacteria and their kinase activity. The results demonstrate that Src mutants with kinase activity above a certain threshold could not be purified from a bacterial expression system, while a variety of mutants with a kinase activity below this threshold could indeed be expressed and purified. These observations support the conclusion that Src activity is toxic to the bacteria, which prevents high-level expression of fully active Src. We further demonstrated that His384, a universally conserved residue among protein tyrosine kinases, is not essential for Src catalysis or its inactivation by C-terminal tail Tyr phosphorylation. Interestingly, His384 mutants undergo autophosphorylation on Tyr416 like wild-type Src but are not activated by autophosphorylation. The potential role of His384 in Src activation by autophosphorylation is discussed in the context of Src structure.

A new strategy to produce active human Src from bacteria for biochemical study of its regulation.

Enzymological studies of Src protein tyrosine kinase have been hindered by the lack of a suitable bacterial expression system. Poor expression of active Src appears to be due to toxicity associated with its kinase activity. To overcome this problem, we fused Src to a protein tyrosine phosphatase with an affinity tag and an appropriate thrombin cleavage site. Upon affinity purification of the fusion protein, Src was released by thrombin digestion and further purified by FPLC. This strategy has been used to produce several Src mutants that display catalytic and regulatory properties similar to those from eukaryotic expression systems. Characterization of the Src mutants confirmed that inactivation of Src by Csk through tail tyrosine phosphorylation required the Src SH3 domain.

Conformational basis for SH2-Tyr(P)527 binding in Src inactivation.

Src protein-tyrosine kinase contains a myristoylation motif, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a catalytic domain, and a C-terminal tail. The C-terminal tail contains a Tyr residue, Tyr527. Phosphorylation of Tyr527 triggers Src inactivation, caused by Tyr(P)527 binding to the SH2 domain. In this study, we demonstrated that a conformational contribution, not affinity, is the predominant force for the intramolecular SH2-Tyr(P)527 binding, and we characterized the structural basis for this conformational contribution. First, a phosphopeptide mimicking the C-terminal tail is an 80-fold weaker ligand than the optimal phosphopeptide, pYEEI, and similar to a phosphopeptide containing three Ala residues following Tyr(P) in binding to the Src SH2 domain. Second, the SH2-Tyr(P)527 binding is largely independent of the amino acid sequence surrounding Tyr(P)527, and only slightly decreased by an inactivating mutation in the SH2 domain. Furthermore, even the unphosphorylated C-terminal tail with the sequence of YEEI suppresses Src activity by binding to the SH2 domain. These experiments demonstrate that very weak affinity is sufficient for the SH2-Tyr(P)527 binding in Src inactivation. Third, the effective intramolecular SH2-Tyr(P)527 binding is attributed to a conformational contribution that requires residues Trp260 and Leu255. Although the SH3 domain is essential for Src inactivation by Tyr(P)527, it does not contribute to the SH2-Tyr(P)527 binding. These findings suggest a conformation-based Src inactivation model, which provides a unifying framework for understanding Src activation by a variety of mechanisms.

Synthesis and structure-activity relationships of linear and conformationally constrained peptide analogues of CIYKYY as Src tyrosine kinase inhibitors.

A series of peptide analogues of Ac-CIYKYY (1) were synthesized by functional group modifications in peptide side chains or by introducing conformational constraints, to improve the inhibitory potency against active Src kinase. Ac-CIYKF(4-NO2)Y (2, IC50 = 0.53 microM) and conformationally constrained peptide 31 (IC50 = 0.28 microM) exhibited 750- and 1400-fold higher inhibitory activities, respectively, versus that of 1 (IC50 = 400 microM). Compound 2 exhibited a partial competitive inhibition pattern against ATP.

Structural basis for domain-domain communication in a protein tyrosine kinase, the C-terminal Src kinase.

The catalytic activity of protein tyrosine kinases is commonly regulated by domain-domain interactions. The C-terminal Src kinase (Csk) contains a catalytic domain and the regulatory SH3 and SH2 domains. Both the presence of the regulatory domains and binding of specific phosphotyrosine-containing proteins to the SH2 domain activate Csk. The structural basis for both modes of activation is investigated here. First, the SH3-SH2 linker is crucial for Csk activation. Mutagenic and kinetic studies demonstrate that this activation is mediated by a cation-pi interaction between Arg68 and Trp188. Second, Ala scanning and kinetic analyses on residues in the SH2-catalytic domain interface identify three functionally distinct types of residues in mediating the communication between the SH2 and the catalytic domains. Type I residues are important in mediating a ligand-triggered activation of Csk because their mutation severely reduces Csk activation by the SH2 domain ligand. Type II residues are involved in suppressing Csk activity, and their mutation activates Csk, but makes Csk less sensitive to activation by the SH2 ligand. Both type I and type II residues are likely involved in mediating SH2 ligand-triggered activation of Csk. Type III residues are those located in the SH2 domain whose mutation severely decreases Csk catalytic activity without affecting the SH2 ligand-triggered activation. These residues likely mediate SH2 activation of Csk regardless of SH2-ligand interaction. These studies lead us to propose a domain-domain communication model that provides functional insights into the topology of Csk family of protein tyrosine kinases.