Split Fragments Research Articles

Enhanced activity of split trehalase biosensors by interspecies domain combineering.

The split trehalase biosensor has potential as a versatile diagnostic technology. Split enzymes are engineered proteins, divided into inactive fragments, which can reassemble and regain activity when brought together by an analyte. The split TreA biosensor requires no sample processing and produces stable signals (in the form of glucose). Split trehalase reagents can function in blood, but periplasmic trehalase of E.coli requires blood acidification for maximal activity. The objective of this study was to obtain split trehalase with near physiological pH optimum. For this purpose, periplasmic trehalases of Cellvibrio spp. with higher activity at neutral pH, were split in analogy with the E. coli TreA into hood and catalytic domains. However, these split trehalases displayed self-complementation due to spontaneous reassembly. In contrast, when catalytic domains of Cellvibrio trehalases were combined with E.coli hood domains, these hybrids displayed conditional complementation capacity when split trehalase fragments fused to immunoglobulin-binding protein G (STIGA) were used to quantify immunoglobulin concentrations. Other hybrid combinations of Cellvibrio spp. had increased activity compared to the cognate pairs, albeit with strong self-complementation. A mutagenesis analysis of residues responsible for self-complementation led to uncoupling of self-complementation from allostery. The Michaelis-Menten kinetics of Cellvibrio enzymes and fragment pairs confirmed improved activity of a mutated hybrid pair of Cellvibrio hood and catalytic domains at physiological pH. In conclusion, the improvements in pH optimum and activity, demonstrated with STIGA, can now be leveraged to enhance other variations of the split trehalase biosensor platform, broadening its utility for testing clinical samples.

A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System

AbstractIn this study, a versatile method is developed to create an antibody/split‐enzyme complex for use in electrochemical immunosensors. SpyCatchers are genetically fused at each terminus of flavin adenine dinucleotide‐dependent glucose dehydrogenase (FAD‐GDH) and a protease recognition sequence is inserted into a loop region to prepare a soluble protein and the split GDH. SpyTag‐fused single‐chain variable fragments (scFvs) are employed, which leads to the simple preparation of antibody‐split enzyme complexes (split AECs). The addition of pentameric C‐reactive protein (CRP), as a representative multimeric protein, restored the GDH activity of the split AECs, as the two split AEC fragments formed active GDH when in close proximity. CRP in human serum is electrochemically detected within 5 min without any washing steps with a clinically relevant CRP detection range (0.03–1 mg dL−1). The detection system is expanded to detect hemoglobin, SARS‐CoV‐2 spike protein, and inactivated SARS‐CoV‐2 by exchanging the scFvs. These results show that the convenient preparation method of the split GDH and the split AEC by the insertion of the protease recognition sequence and the use of SpyCatcher/SpyTag can realize a rapid, homogeneous, and universal electrochemical detection system that can be integrated into a commercially available electrochemical glucose sensor.

Extracellular bimolecular fluorescence complementation for investigating membrane protein dimerization: a proof of concept using class B GPCRs.

Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins, however fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization.

Distraction of longitudinally split fragments using the Ilizarov method: a series of clinical cases of treating partial bone defects

Background. The Ilizarov method is a recognized technique for treating severe skeletal injuries, allowing for a comprehensive restoration of both bone and soft tissue components. Despite the fact that bone lengthening and transport are widely known techniques, distraction of a longitudinally split fragment is still used extremely rarely. The aim of the study is to describe a series of clinical cases involving patients operated on using this technique. Methods. We present a series of observations of five patients who underwent distraction of a longitudinally split fragment using the Ilizarov method between January 2006 and December 2022. Clinical information was obtained from case histories, all surgical interventions were documented. Postoperative examination was performed using radiography. Results. A case series demonstrates the successful application of this technique for reconstruction of partial bone defects resulting from trauma or osteomyelitis. The study included five patients (4 men and 1 woman) who underwent surgery 4.8-34.0 months after trauma for a partial defect of the proximal tibia ranging from 4 to 8 cm in length. Distraction was performed in different directions along the sagittal and longitudinal axes. The time of external fixation ranged from 3.5 to 4.8 months, the external fixation index ranged from 0.49 to 1.22. The ASAMI (Association for the Study and Application of the Methods of Ilizarov) functional score at the follow-up examination was excellent in all five patients. The ASAMI bone tissue assessment showed excellent results in all patients, except for one patient with residual equinus (good result). No other complications were reported. Conclusions. The Ilizarov method provides a minimally invasive and comprehensive approach to the elimination of partial bone defects, affecting simultaneously the skeletal and soft tissue components. Due to the longitudinal splitting during fragment transport and distraction osteogenesis, this method promotes bone and tissue regeneration and helps to avoid a volumetric bone defect and more complex segmental bone transport. Moreover, the role of transverse transport of the tibial cortex increases in the treatment of peripheral arterial diseases.

Protein-Controlled Split DNAzyme to Enhance Catalytic Activity: Design and Performance.

In this study, we utilized proteins to control the assembly of split DNAzyme to establish protein-controlled split DNAzymes (Pc SD), with the aim of enhancing its catalytic activity. To achieve this, simultaneous recognition of protein by affinity ligands at both ends of split DNAzyme fragments induced an increased local concentration of each split fragment, leading to reassembly of the split catalytic core with a rigid conformation and higher affinity to its cofactor. As a result, under protein control, Pc SD exhibits unexpected cleavage efficiency compared to free split DNAzyme. To further explore the catalytic features, we then systematically positioned split sites within the catalytic core of three popular DNAzyme-based Pc SDs, thus revealing the important nucleic acids that influence Pc SDs activity. Based on the excellent analytical performance of Pc SD for streptavidin (with a LOD of 0.1 pM in buffer),we equipped Pc SD with antibodies as rapid diagnostic tools for inpatient care (AFP as biomarker) with a minimized workflow (with a LOD of 2 pM in 5% human serum). The results of this study offer fundamental insights into external factors for boosting DNAzyme catalysis and will be promising for applications that utilize split DNAzymes.

Development of Luminescent Biosensors for Calprotectin.

Calprotectin, a metal ion-binding protein complex, plays a crucial role in the innate immune system and has gained prominence as a biomarker for various intestinal and systemic inflammatory and infectious diseases, including inflammatory bowel disease (IBD) and tuberculosis (TB). Current clinical testing methods rely on enzyme-linked immunosorbent assays (ELISAs), limiting accessibility and convenience. In this study, we introduce the Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement. FESCA utilizes two new fragment antigen binding proteins (Fabs), CP16 and CP17, that bind to different epitopes of the calprotectin complex. These Fabs are fused with split NanoLuc luciferase fragments, enabling the reconstitution of active luciferase upon binding to calprotectin either in solution or in varied immobilized assay formats. FESCA's output luminescence can be measured with standard laboratory equipment as well as consumer-grade cell phone cameras. FESCA can detect physiologically relevant calprotectin levels across various sample types, including serum, plasma, and whole blood. Notably, FESCA can detect abnormally elevated native calprotectin from TB patients. In summary, FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples, with the potential to improve the diagnosis and monitoring of inflammatory diseases, especially in at-home or point-of-care settings.

DFT investigation of [formula omitted]-split interstitial paramagnetic centers in diamond

The density functional theory (DFT) method, in combination with the cluster approach, has been utilized to investigate a series of paramagnetic interstitial defects in diamond with an electron spin of S = 1. A good correlation has been achieved between simulated and experimental zero-field splitting D-tensor eigenvalues and eigenvectors for the paramagnetic centers R2, R1, and O3, identified previously as 100-split interstitial, di-100-split interstitial, and planar three-100-split interstitial, respectively. Based on the calculations, two new configuration models have been proposed for the low-symmetry centers R13 and R3, as configurations of non-planar three- and five-100-split interstitial, respectively. The formation energies for the series of defects under study have been evaluated, suggesting that the aggregation of split interstitial fragments is energetically favored.

Exon-junction complex association with stalled ribosomes and slow translation-independent disassembly

Exon junction complexes are deposited at exon-exon junctions during splicing. They are primarily known to activate non-sense mediated degradation of transcripts harbouring premature stop codons before the last intron. According to a popular model, exon-junction complexes accompany mRNAs to the cytoplasm where the first translating ribosome pushes them out. However, they are also removed by uncharacterized, translation-independent mechanisms. Little is known about kinetic and transcript specificity of these processes. Here we tag core subunits of exon-junction complexes with complementary split nanoluciferase fragments to obtain sensitive and quantitative assays for complex formation. Unexpectedly, exon-junction complexes form large stable mRNPs containing stalled ribosomes. Complex assembly and disassembly rates are determined after an arrest in transcription and/or translation. 85% of newly deposited exon-junction complexes are disassembled by a translation-dependent mechanism. However as this process is much faster than the translation-independent one, only 30% of the exon-junction complexes present in cells at steady state require translation for disassembly. Deep RNA sequencing shows a bias of exon-junction complex bound transcripts towards microtubule and centrosome coding ones and demonstrate that the lifetimes of exon-junction complexes are transcript-specific. This study provides a dynamic vision of exon-junction complexes and uncovers their unexpected stable association with ribosomes.

Development of a new caged intein for multi-input conditional translation of synthetic mRNA.

mRNA medicines can be used to express therapeutic proteins, but the production of such proteins in non-target cells has a risk of adverse effects. To accurately distinguish between therapeutic target and nontarget cells, it is desirable to utilize multiple proteins expressed in each cell as indicators. To achieve such multi-input translational regulation of mRNA medicines, in this study, we engineered Rhodothermus marinus (Rma) DnaB intein to develop "caged Rma DnaB intein" that enables conditional reconstitution of full-length translational regulator protein from split fragments. By combining the caged Rma DnaB intein, the split translational regulator protein, and target protein-binding domains, we succeeded in target protein-dependent translational repression of mRNA in human cells. In addition, the caged Rma intein showed orthogonality to the previously reported Nostoc punctiforme (Npu) DnaE-based caged intein. Finally, by combining these two orthogonal caged inteins, we developed an mRNA-based logic gate that regulates translation based on the expression of multiple intracellular proteins. This study provides important information to develop safer mRNA medicines.

A putative design for the electromagnetic activation of split proteins for molecular and cellular manipulation.

The ability to manipulate cellular function using an external stimulus is a powerful strategy for studying complex biological phenomena. One approach to modulate the function of the cellular environment is split proteins. In this method, a biologically active protein or an enzyme is fragmented so that it reassembles only upon a specific stimulus. Although many tools are available to induce these systems, nature has provided other mechanisms to expand the split protein toolbox. Here, we show a novel method for reconstituting split proteins using magnetic stimulation. We found that the electromagnetic perceptive gene (EPG) changes conformation due to magnetic field stimulation. By fusing split fragments of a certain protein to both termini of the EPG, the fragments can be reassembled into a functional protein under magnetic stimulation due to conformational change. We show this effect with three separate split proteins: NanoLuc, APEX2, and herpes simplex virus type-1 thymidine kinase. Our results show, for the first time, that reconstitution of split proteins can be achieved only with magnetic fields. We anticipate that this study will be a starting point for future magnetically inducible split protein designs for cellular perturbation and manipulation. With this technology, we can help expand the toolbox of the split protein platform and allow better elucidation of complex biological systems.

Splittable systems in biomedical applications.

Splittable systems have emerged as a powerful approach for the precise spatiotemporal control of biological processes. This concept relies on splitting a functional molecule into inactive fragments, which can be reassembled under specific conditions or stimuli to regain activity. Several binding pairs and orthogonal split fragments are introduced by fusing with other modalities to develop more complex and robust designs. One of the pillars of these splittable systems is modularity, which involves decoupling targeting, activation, and effector functions. Challenges, such as off-target effects and overactivation, can be addressed through precise control. This review provides an overview of the design principles, strategies, and applications of splittable systems across diverse fields including immunotherapy, gene editing, prodrug activation, biosensing, and synthetic biology.

Simulation-guided engineering of split GFPs with efficient β-strand photodissociation

Green fluorescent proteins (GFPs) are ubiquitous for protein tagging and live-cell imaging. Split-GFPs are widely used to study protein-protein interactions by fusing proteins of interest to split GFP fragments that create a fluorophore upon typically irreversible complementation. Thus, controlled dissociation of the fragments is desirable. Although we have found that split strands can be photodissociated, the quantum efficiency of light-induced photodissociation of split GFPs is low. Traditional protein engineering approaches to increase efficiency, including extensive mutagenesis and screening, have proved difficult to implement. To reduce the search space, key states in the dissociation process are modeled by combining classical and enhanced sampling molecular dynamics with QM/MM calculations, enabling the rational design and engineering of split GFPs with up to 20-fold faster photodissociation rates using non-intuitive amino acid changes. This demonstrates the feasibility of modeling complex molecular processes using state-of-the-art computational methods, and the potential of integrating computational methods to increase the success rate in protein engineering projects.

Split Luciferase-Fragment Reconstitution for Unveiling RNA Localization and Dynamics in Live Cells.

The intracellular distribution and dynamics of RNAs play pivotal roles in various physiological phenomena. The ability to monitor the amount and localization of endogenous RNAs in living cells allows for elucidating the mechanisms of various intracellular events. Protein-based fluorescent RNA probes are now widely used to visualize and analyze RNAs in living cells. However, continuously monitoring the temporal changes in RNA localization and dynamics in living cells is challenging. In this study, we developed a bioluminescent probe for spatiotemporal monitoring of RNAs in living cells by using a split-luciferase reconstitution technique. The probe consists of split fragments of a bioluminescent protein, NanoLuc, connected with RNA-binding protein domains generated from a custom-made mutation of a PUM-HD. The probe showed rapid luminescence intensity changes in response to an increase or decrease in the amount of a target RNA in vitro. In live-cell imaging, temporal alteration of the intracellular distribution of endogenous β-actin mRNA was visualized in response to extracellular stimulation. Furthermore, the application of the probe to the visualization of the specific localization of β-actin mRNA in primary hippocampal neurons was conducted. These results demonstrate the capability of the bioluminescent RNA probe to monitor the changes in localization, dynamics, and the amount of target RNA in living cells.

SplitPlace: AI Augmented Splitting and Placement of Large-Scale Neural Networks in Mobile Edge Environments

In recent years, deep learning models have become ubiquitous in industry and academia alike. Deep neural networks can solve some of the most complex pattern-recognition problems today, but come with the price of massive compute and memory requirements. This makes the problem of deploying such large-scale neural networks challenging in resource-constrained mobile edge computing platforms. To solve this, a promising solution is to split resource-hungry neural networks into lightweight disjoint smaller components for pipelined distributed processing. At present, there are two main approaches to do this: semantic and layer-wise splitting. The former partitions a neural network into parallel disjoint models that produce a part of the result, whereas the latter partitions into sequential models that produce intermediate results. However, there is no intelligent algorithm that decides which splitting strategy to use and places such modular splits to edge nodes for optimal performance. To combat this, this work proposes a novel AI-driven online policy, SplitPlace, that uses Multi-Armed-Bandits to intelligently decide between layer and semantic splitting strategies based on the input task's service deadline demands. SplitPlace places such neural network split fragments on mobile edge devices using decision-aware reinforcement learning for efficient and scalable computing. Moreover, SplitPlace fine-tunes its placement engine to adapt to volatile environments. Our experiments on physical mobile-edge environments with real-world workloads show that SplitPlace can significantly improve the state-of-the-art in terms of average response time, deadline violation rate, inference accuracy, and total reward by up to 46, 69, 3 and 12 percent respectively.

Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold.

RNA processing and metabolism are subjected to precise regulation in the cell to ensure integrity and functions of RNA. Though targeted RNA engineering has become feasible with the discovery and engineering of the CRISPR-Cas13 system, simultaneous modulation of different RNA processing steps remains unavailable. In addition, off-target events resulting from effectors fused with dCas13 limit its application. Here we developed a novel platform, Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST), which can simultaneously execute multiple RNA modulation functions on different RNA targets. In CREST, RNA scaffolds are appended to the 3' end of Cas13 gRNA and their cognate RNA binding proteins are fused with enzymatic domains for manipulation. Taking RNA alternative splicing, A-to-G and C-to-U base editing as examples, we developed bifunctional and tri-functional CREST systems for simultaneously RNA manipulation. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and/or PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce nearly 99% of off-target events otherwise induced by a full-length effector. The flexibility of the CREST framework will enrich the transcriptome engineering toolbox for the study of RNA biology.

Trauma, memory and the impact of redecision therapy

It is concluded that redecision therapy is a form of exposure therapy. Whilst redecision therapy has much wider goals than exposure therapy, the Gouldings created a potent form of exposure therapy that forms part of the process of redecision. There is a very large body of research evidence verifying the efficacy of exposure therapy as a treatment for trauma and PTSD especially. This article shows how redecision therapy is an exposure therapy and then how exposure therapy can assist in reintegrating the split off fragments of the personality that are formed in the schizoid process.

A triple-cycle amplification biosensor for colorimetric detection of mutant PIK3CAE545K based on cascade-driven DNA walker and branched hybridization strand reaction

Circulating tumor DNA (ctDNA) is an ideal candidate for liquid biopsy biomarkers. Therefore, detecting a low abundance of ctDNA is essential for early cancer diagnosis. Here, we developed a novel triple circulation amplification system integrating entropy and enzyme cascade-driven three-dimensional (3D) DNA walker and branched hybridization strand reaction (B-HCR) for ultrasensitive detection of breast cancer-related ctDNA. In this study, the 3D DNA walker was constructed by inner track probes (NH) and complex S on a microsphere. Once the DNA walker was triggered by the target, the strand replacement reaction ran first and kept circulating to rapidly displace the DNA walker containing 8–17 DNAzyme. Secondly, the DNA walker could repeatedly cleave NH autonomously along the inner track, generating numerous initiators, and then promoting B-HCR to activate the third cycle. Subsequently, the split G-rich fragments were brought in close to form the G-quadruplex/hemin DNAzyme by adding hemin, with the addition of H2O2 and ABTS, the target could be observed. Benefiting from triplex cycles, the PIK3CAE545K mutation detection possesses a good linear range from 1-103 fM, and the limit of detection was 0.65 fM. Due to the low cost and high sensitivity, the proposed strategy has great potential in early diagnosis of breast cancer.

Protein Ligation-Assisted Reconstitution of Split HRP Fragments for Facile Production of HRP Fusion Proteins in E. coli.

Horseradish peroxidase (HRP) is a pivotal biocatalyst for biosensor development and fine chemical synthesis. HRP proteins are mostly extracted and purified from the roots of horseradish because the solubility and productivity of recombinant HRP in bacteria are significantly low. In this study, we investigate the reconstitution system of split HRP fragments to improve its soluble expression levels in E. coli allowing the cost-effective production of bioactive HRPs. To promote the effective association between two HRP fragments (HRPn and HRPc), we exploit SpyTag-SpyCatcher chemistry, a versatile protein coupling method with high affinity and selectivity. Each HRP fragment was genetically fused with SpyTag and SpyCatcher, respectively, exhibiting soluble expression in the E. coli cytoplasm. The engineered split HRPs were effectively and irreversibly reconstituted into a biologically active and stable assembly that can catalyze intrinsic enzymatic reactions. Compared to the chaperone co-expression system, our approach shows that the production yield of soluble HRP is comparable, but the purity of the final product is relatively high. Therefore, our results can be applied to the high-yield production of recombinant HRP variants and other difficult-to-express proteins in bacteria without complex downstream processes.

Assembly of split aptamers by dynamic pH-responsive covalent ligation.

Reversible pH-responsive N-methoxyoxazolidine formation is used to ligate split aptamer fragments. Two twice-split models and one thrice-split model of CBA (cocaine-binding aptamer) were examined. The aptamer assembly was dynamic, proportional to the substrate concentration and occurred without interfering background ligation.

Fracture Line Morphology of Greater Tuberosity Fragments of Neer Three- and Four-Part Proximal Humerus Fractures.

In complicated Neer three- and four-part proximal humerus fracture (PHF), greater tuberosity (GT) fragments are often comminuted, and the currently widely used locking plate may not fix GT fragments effectively. A further understanding of morphological characteristics of the GT fragments may help explore new fixation devices. This study aimed to determine the fracture line morphology of the GT fragment of Neer three- or four-part PHF and analyze the location relationship between the locking plate and the GT fragment. Seventy-one three-dimensional computed tomography scans of Neer three- and four-part PHF were retrospectively reviewed between January 2014 and June 2019. Fracture fragments were reconstructed and virtually reduced in the Mimics software, and fracture lines of GT fragments were depicted on a humerus template in the 3-matic software and then were superimposed altogether. The common sites of the GT fracture were identified, and the location relationship between the locking plate and GT fragments was analyzed in a computer-simulated scenario. The fracture line morphology of GT fragments was similar between Neer three- and four-part PHF. The overall morphology of GT fragments was in a fan shape, which could be summarized as anterior, superior, posterior, and middle lines. Of these, we identified 51 split and 29 avulsion type GT fragments based on the Mutch classification, and they could occur simultaneously in a PHF. The overall morphology of split type fragments was in a fan shape, and avulsion type fragments showed a quite distinguishable distribution pattern. A GT fragment could be classified as anterior-split, posterior-split, complete-split, anterior -avulsion, and posterior-avulsion type based on its morphology and location. The median percentage of fragment area covered by the plate was 32.3% in all of the fragments, and it was 69.4%, 23.0%, 37.2%, 21.8%, 0.0% in anterior-split, posterior-split, complete-split, anterior-avulsion, and posterior-avulsion type GT fragments. We defined the posterior-split, anterior-avulsion, and posterior-avulsion type GT fragments as the risky GT fragments, and they occurred in 43 (60.6%) Neer three- and four-part PHFs. The fracture line morphology of GT fragments of Neer three- and four-part PHF was in a fan shape. GT fragments could be classified based on their location and morphology. The extent of GT fragment coverage provided by the locking plate differed in various fragment types, and we identified the anterior-avulsion, posterior-avulsion, and posterior-split type fragments as the risky GT fragments with a high incidence rate in Neer three- and four-part PHFs.