Engineering functional and anthropomorphic models for surgical training in interventional radiology: A state-of-the-art review.

Training medical students in surgical procedures and evaluating their performance are both necessary steps to ensure the safety and efficacy of surgeries. Traditionally, trainees practiced on live patients, cadavers or animals under the supervision of skilled physicians, but realistic anatomical phantom models have provided a low-cost alternative because of the advance of material technology that mimics multi-layer tissue structures. This setup provides safer and more efficient training. Many research prototypes of phantom models allow rapid in-house prototyping for specific geometries and tissue properties. The gel-based method and 3D printing-based method are two major methods for developing phantom prototypes. This study excluded virtual reality based technologies and focused on physical phantoms, total 189 works published between 2015 and 2020 on anatomical phantom prototypes made for interventional radiology were reviewed in terms of their functions and applications. The phantom prototypes were first categorized based on fabrication methods and then subcategorized based on the organ or body part they simulated; the paper is organized accordingly. Engineering specifications and applications were analyzed and summarized for each study. Finally, current challenges in the development of phantom models and directions for future work were discussed.

Applications of Robotics, Artificial Intelligence, and Digital Technologies During COVID-19: A Review.

Many countries have enacted a quick response to the unexpected coronavirus disease 2019 (COVID-19) pandemic by using existing technologies. For example, robotics, artificial intelligence, and digital technology have been deployed in hospitals and public areas for maintaining social distancing, reducing person-to-person contact, enabling rapid diagnosis, tracking virus spread, and providing sanitation. In this study, 163 news articles and scientific reports on COVID-19-related technology adoption were screened, shortlisted, categorized by application scenario, and reviewed for functionality. Technologies related to robots, artificial intelligence, and digital technology were selected from the pool of candidates, yielding a total of 50 applications for review. Each case was analyzed for its engineering characteristics and potential impact on the COVID-19 pandemic. Finally, challenges and future directions regarding the response to this pandemic and future pandemics were summarized and discussed.

Quantification of polyclonal free light chains in clinical samples using a single turbidimetric immunoassay.

BACKGROUND: Elevated polyclonal serum free light chain (FLC) levels have been associated with increased mortality and disease activity in many conditions. Currently, polyclonal FLC quantification requires summation of individual FLCκ and FLCλ assays. Here we present a single assay for combined FLC (cFLC, Combylite) which reduces assay time and eliminates potential imprecision errors incurred by summating FLC assays (ΣFLC). METHODS: Sheep FLCκ- and FLCλ-specific antibodies were conjugated to latex microparticles to quantify FLCκ and FLCλ in a single assay. Combylite results were compared to ΣFLC (Freelite) in 132 healthy controls and 1127 patient samples. The utility of cFLC for predicting all-cause mortality in a haematological referral population was evaluated. RESULTS: cFLC and ΣFLC results were highly concordant (Passing-Bablok equation y=0.98x-1.59 mg/L, R²=0.96). Combylite assay imprecision was low at concentrations around the upper normal range [coefficient of variation (CV) 5.5%, 54 mg/L] and the upper limit of the measuring range (CV 5.5%, 170 mg/L). cFLC levels were significantly raised in disease states compared with healthy controls. Additionally, cFLC >65 mg/L was associated with shorter overall survival in a haematological referral population (hazard ratio=4.5, p<0.001). CONCLUSIONS: cFLC values obtained using Combylite were comparable to ΣFLC results over a wide concentration range, were elevated in diseases characterised by B cell activation and were associated with increased mortality in a haematological referral population. These observations indicate the Combylite assay has value for investigating the role of B cell activation in disparate disease groups and could be considered as a surrogate indication of B cell function.

Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting.

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.

Storage and handling guidelines for custom peptides.

Synthetic custom peptides offer a rational, scalable, and ever more affordable approach for exploring protein-protein interactions or even more complex phenomena, such as immune responses directed against specific epitopes. As the use of such peptides increases, it becomes increasingly important to consider all relevant factors when designing and using peptides. This unit aims to provide guidelines for the storage and handling of custom peptides, to enable the user to achieve optimal results when using peptides in their experiments. It also discusses the implications of the presence of certain amino acids in the peptide sequence and suggests design considerations to help achieve desired outcomes.

The structure of the transition state of the heterodimeric topoisomerase I of Leishmania donovani as a vanadate complex with nicked DNA.

Type IB topoisomerases are essential enzymes that are responsible for relaxing superhelical tension in DNA by forming a transient covalent nick in one strand of the DNA duplex. Topoisomerase I is a target for anti-cancer drugs such as camptothecin, and these drugs also target the topoisomerases I in pathogenic trypanosomes including Leishmania species and Trypanosoma brucei. Most eukaryotic enzymes, including human topoisomerase I, are monomeric. However, for Leishmania donovani, the DNA-binding activity and the majority of residues involved in catalysis are located in a large subunit, designated TOP1L, whereas the catalytic tyrosine residue responsible for covalent attachment to DNA is located in a smaller subunit, called TOP1S. Here, we present the 2.27A crystal structure of an active truncated L.donovani TOP1L/TOP1S heterodimer bound to nicked double-stranded DNA captured as a vanadate complex. The vanadate forms covalent linkages between the catalytic tyrosine residue of the small subunit and the nicked ends of the scissile DNA strand, mimicking the previously unseen transition state of the topoisomerase I catalytic cycle. This structure fills a critical gap in the existing ensemble of topoisomerase I structures and provides crucial insights into the catalytic mechanism.

Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines.

The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-His-Asp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly126, implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 A (where 1 A=0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity.

Eukaryotic arylamine N-acetyltransferase. Investigation of substrate specificity by high-throughput screening.

Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

Screening and characterizing human NAT2 variants.

Acetyl CoA:arylamine N-acetyltransferase (NAT; E.C. 2.3.1.5) enzymes play a key role in the metabolic activation of aromatic amine and nitroaromatic mutagens to electrophilic reactive intermediates. We have developed a system in which the activation of mutagens by recombinant human NAT2, expressed in Escherichia coli, can be detected by the appearance of Lac+ revertants. The mutagenesis assay is based on the reversion of an E. coli lacZ frameshift allele; the host strain for the assay is devoid of endogenous NAT activity and a plasmid vector is used for expression of human NAT2. A high-throughput version of the assay facilitates rapid screening of pools of NAT2 variants generated (for example) by random mutagenesis. Along with the methods for these assays, we present selected results of a screening effort in which mutations along the length of the NAT2 sequence have been examined. Homology modeling and simulated annealing have been used to analyze the potential effects of these mutations on structural integrity and substrate binding.

The structure of arylamine N-acetyltransferase from Mycobacterium smegmatis--an enzyme which inactivates the anti-tubercular drug, isoniazid.

Arylamine N-acetyltransferases which acetylate and inactivate isoniazid, an anti-tubercular drug, are found in mycobacteria including Mycobacterium smegmatis and Mycobacterium tuberculosis. We have solved the structure of arylamine N-acetyltransferase from M. smegmatis at a resolution of 1.7 A as a model for the highly homologous NAT from M. tuberculosis. The fold closely resembles that of NAT from Salmonella typhimurium, with a common catalytic triad and domain structure that is similar to certain cysteine proteases. The detailed geometry of the catalytic triad is typical of enzymes which use primary alcohols or thiols as activated nucleophiles. Thermal mobility and structural variations identify parts of NAT which might undergo conformational changes during catalysis. Sequence conservation among eubacterial NATs is restricted to structural residues of the protein core, as well as the active site and a hinge that connects the first two domains of the NAT structure. The structure of M. smegmatis NAT provides a template for modelling the structure of the M. tuberculosis enzyme and for structure-based ligand design as an approach to designing anti-TB drugs.

The pharmacogenetics of NAT: structural aspects.

Arylamine N-acetyltransferases (NATs) catalyze the transfer of an acetyl group from acetyl-CoA to arylhydrazines and to arylamine drugs and carcinogens or to their N-hydroxylated metabolites. NAT plays an important role in detoxification and metabolic activation of xenobiotics and was first identified as the enzyme responsible for inactivation of the antitubercular drug isoniazid, an arylhydrazine. The rate of inactivation was polymorphically distributed in the population: the first example of interindividual pharmacogenetic variation. Polymorphism in NAT activity is primarily due to single nucleotide polymorphisms (SNPs) in the coding region of NAT genes. NAT enzymes are widely distributed in eukaryotes and genome sequences have revealed many homologous members of this enzyme family in prokaryotes. The structures of S almonella typhimurium and Mycobacterium smegmatis NATs have been determined, revealing a unique fold in which a catalytic triad (Cys-His-Asp) forms the active site. Determination of prokaryotic and eukaryotic NAT structures could lead to a better understanding of their role in xenobiotics and endogenous metabolism.

The COOH terminus of arylamine N-acetyltransferase from Salmonella typhimurium controls enzymic activity.

Arylamine N-acetyltransferases (NATs) are a homologous family of enzymes, which acetylate arylamines, arylhydroxylamines, and arylhydrazines by acetyl transfer from acetyl-coenzyme A (Ac-CoA) and are found in many organisms. NAT was first identified as the enzyme responsible for the inactivation of the anti-tubercular drug isoniazid in humans. The three-dimensional structure of NAT from Salmonella typhimurium has been resolved and shown to have three distinct domains and an active site catalytic triad composed of "Cys(69)-His(107)-Asp(122)," which is typical of hydrolytic enzymes such as the cysteine proteases. The crystal unit cell consists of a dimer of tetramers, with the C terminus of individual monomers juxtaposed. To investigate the function of the first two domains of full-length NAT from S. typhimurium and to investigate the role of the C terminus of NAT, truncation mutants were made with either the C-terminal undecapeptide or the entire third domain (85 amino acids) missing. Unlike the full-length NAT protein (281 amino acids), the truncation mutants of NAT from S. typhimurium are toxic when overexpressed intracellularly in Escherichia coli. Full-length NAT hydrolyses Ac-CoA but only in the presence of an arylamine substrate. Both truncation mutants, however, hydrolyze Ac-CoA even in the absence of arylamine substrate, illustrating that the C-terminal undecapeptide controls hydrolysis of Ac-CoA by NAT from S. typhimurium.

Expression and purification of the rifamycin amide synthase, RifF, an enzyme homologous to the prokaryotic arylamine N-acetyltransferases.

The assembly of the polyketide backbone of rifamycin B by the type I rifamycin polyketide synthase, encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. The sequence of the RifF protein from Amycolatopsis mediterranei shows 26% identity and 40% homology with the members of the arylamine N-acetyltransferase (NAT) family of proteins. Based on the homology of the primary structures and the similarity of the reactions catalyzed by the two enzymes, we have compared the RifF protein with members of the NAT family. We have modeled the three-dimensional (3D) structure of RifF using NAT from Salmonella typhimurium and Mycobacterium smegmatis as a template. Proteolytic digestions of RifF revealed accessible regions in the protein which are in agreement with the modeled structure. We have expressed the whole protein and individual domains of the protein based on comparison with NAT from S. typhimurium and have purified the proteins by affinity chromatography using a hexahistidine tag. RifF has been further purified using ion-exchange (Mono Q) chromatography. An antiserum has been generated using the C-terminal nona- and tridecapeptides of RifF and has been shown to recognize RifF uniquely. It does not cross-react with any other member of the NAT family.

Eubacterial arylamine N-acetyltransferases - identification and comparison of 18 members of the protein family with conserved active site cysteine, histidine and aspartate residues.

Arylamine N-acetyltransferases (NATs) are enzymes involved in the detoxification of a range of arylamine and hydrazine-based xenobiotics. NATs have been implicated in the endogenous metabolism of p-aminobenzoyl glutamate in eukaryotes, although very little is known about the distribution and function of NAT in the prokaryotic kingdom. Using DNA library screening techniques and the analysis of data from whole-genome sequencing projects, we have identified 18 nat-like sequences from the Proteobacteria and Firmicutes. Recently, the three-dimensional structure of NAT derived from the bacterium Salmonella typhimurium (PDB accession code 1E2T) was resolved and revealed an active site catalytic triad composed of Cys(69)-His(107)-Asp(122). These residues have been shown to be conserved in all prokaryotic and eukaryotic NAT homologues together with three highly conserved regions which are found proximal to the active site triad. The characterization of prokaryotic NATs and NAT-like enzymes is reported. It is also predicted that prokaryotic NATs, based on gene cluster composition and distribution amongst genomes, participate in the metabolism of xenobiotics derived from decomposition of organic materials.