Molecular mechanism of selective substrate engagement and inhibitor disengagement of cysteine synthase.

O-acetyl serine sulfhydrylase (OASS), referred to as cysteine synthase (CS), synthesizes cysteine from O-acetyl serine (OAS) and sulfur in bacteria and plants. The inherent challenge for CS is to overcome 4 to 6 log-folds stronger affinity for its natural inhibitor, serine acetyltransferase (SAT), as compared with its affinity for substrate, OAS. Our recent study showed that CS employs a novel competitive-allosteric mechanism to selectively recruit its substrate in the presence of natural inhibitor. In this study, we trace the molecular features that control selective substrate recruitment. To generalize our findings, we used CS from three different bacteria (Haemophilus, Salmonella, and Mycobacterium) as our model systems and analyzed structural and substrate-binding features of wild-type CS and its ∼13 mutants. Results show that CS uses a noncatalytic residue, M120, located 20 Šaway from the reaction center, to discriminate in favor of substrate. M120A and background mutants display significantly reduced substrate binding, catalytic efficiency, and inhibitor binding. Results shows that M120 favors the substrate binding by selectively enhancing the affinity for the substrate and disengaging the inhibitor by 20 to 286 and 5- to 3-folds, respectively. Together, M120 confers a net discriminative force in favor of substrate by 100- to 858-folds.

Principles of RNA and nucleotide discrimination by the RNA processing enzyme RppH.

All enzymes face a challenge of discriminating cognate substrates from similar cellular compounds. Finding a correct substrate is especially difficult for the Escherichia coli Nudix hydrolase RppH, which triggers 5'-end-dependent RNA degradation by removing orthophosphate from the 5'-diphosphorylated transcripts. Here we show that RppH binds and slowly hydrolyzes NTPs, NDPs and (p)ppGpp, which each resemble the 5'-end of RNA. A series of X-ray crystal structures of RppH-nucleotide complexes, trapped in conformations either compatible or incompatible with hydrolysis, explain the low reaction rates of mononucleotides and suggest two distinct mechanisms for their hydrolysis. While RppH adopts the same catalytic arrangement with 5'-diphosphorylated nucleotides as with RNA, the enzyme hydrolyzes 5'-triphosphorylated nucleotides by extending the active site with an additional Mg2+ cation, which coordinates another reactive nucleophile. Although the average intracellular pH minimizes the hydrolysis of nucleotides by slowing their reaction with RppH, they nevertheless compete with RNA for binding and differentially inhibit the reactivity of RppH with triphosphorylated and diphosphorylated RNAs. Thus, E. coli RppH integrates various signals, such as competing non-cognate substrates and a stimulatory protein factor DapF, to achieve the differential degradation of transcripts involved in cellular processes important for the adaptation of bacteria to different growth conditions.

Study of renal parenchymal changes by diffusion tensor imaging in diabetic nephropathy.

Purpose: To evaluate differences in diffusion imaging parameters, including fractional anisotropy (FA) and the apparent diffusion coefficient (ADC), in control and diabetic subjects, and to assess changes in these parameters to patient's urine albumin/protein levels, estimated glomerular filtration rate (eGFR), and glycated haemoglobin (HbA1c). Material and methods: This is a cross-sectional analytical study involving 100 patients who underwent diffusion imaging including diffusion tensor imaging (DTI) of the kidneys in our hospital from 2019 to 2020. Diffusion imaging parameters (ADC and FA) were obtained from the medulla and cortex of both kidneys using dedicated software. Statistical analysis was done. Results: Out of 100 subjects, 27 were controls and 73 were diabetics (19 normoalbuminuric, 23 microalbuminuric, and 31 proteinuric). The medullary FA (0.419 ± 0.024 vs. 0.346 ± 0.042), cortical FA (0.194 ± 0.035 vs. 0.303 ± 0.067), and cortical ADC (3.307 ± 0.341 vs. 2.309 ± 0.515) values showed significant differences between controls and diabetics. Medullary FA and cortical ADC values showed a decreasing trend with an increasing amount of albumin/protein in the urine, decreasing renal function (reducing eGFR), and increasing HbA1c, whereas the trend was opposite for cortical FA. In addition, on ROC curve analysis a cut-off value for medullary FA of 0.4 had a sensitivity of 64% and specificity of 80.95% to differentiate healthy volunteers and diabetics with normo-albuminuria. Conclusions: DTI has the potential to be a promising non-invasive test for the detection of early renal parenchymal changes in diabetic nephropathy.

Two Distinct Assembly States of the Cysteine Regulatory Complex of Salmonella typhimurium Are Regulated by Enzyme-Substrate Cognate Pairs.

Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), which catalyze the last two steps of cysteine biosynthesis, interact and form the cysteine regulatory complex (CRC). The current model of Salmonella typhimurium predicts that CRC is composed of one [SAT]hexamer unit and two molecules of [OASS]dimer. However, it is not clear why [SAT]hexamer cannot engage all of its six high-affinity binding sites. We examined the assembly state(s) of CRC by size exclusion chromatography, analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) approaches. We show that CRC exists in two major assembly states, low-molecular weight (CRC1; 1[SAT]hexamer + 2[OASS]dimer) and high-molecular weight (CRC2; 1[SAT]hexamer + 4[OASS]dimer) states. Along with AUC results, ITC and SPR studies show that [OASS]dimer binds to [SAT]hexamer in a stepwise manner but the formation of fully saturated CRC3 (1[SAT]hexamer + 6[OASS]dimer) is not favorable. The fraction of CRC2 increases as the [OASS]dimer/[SAT]hexamer ratio increases to >4-fold, but CRC2 can be selectively dissociated into either CRC1 or free enzymes, in the presence of OAS and sulfide, in a concentration-dependent manner. Together, we show that CRC is a regulatable multienzyme assembly, sensitive to OASS-substrate(s) levels but subject to negative cooperativity and steric hindrance. Our results constitute the first report of the dual-assembly-state nature of CRC and suggest that physiological conditions, which limit sulfate uptake, would favor CRC1 over CRC2.

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (104-106) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

Mechanistic insights on the ortho-hydroxylation of aromatic compounds by non-heme iron complex: a computational case study on the comparative oxidative ability of ferric-hydroperoxo and high-valent Fe(IV)═O and Fe(V)═O intermediates.

ortho-Hydroxylation of aromatic compounds by non-heme Fe complexes has been extensively studied in recent years by several research groups. The nature of the proposed oxidant varies from Fe(III)-OOH to high-valent Fe(IV)═O and Fe(V)═O species, and no definitive consensus has emerged. In this comprehensive study, we have investigated the ortho-hydroxylation of aromatic compounds by an iron complex using hybrid density functional theory incorporating dispersion effects. Three different oxidants, Fe(III)-OOH, Fe(IV)═O, and Fe(V)═O, and two different pathways, H-abstraction and electrophilic attack, have been considered to test the oxidative ability of different oxidants and to underpin the exact mechanism of this regiospecific reaction. By mapping the potential energy surface of each oxidant, our calculations categorize Fe(III)-OOH as a sluggish oxidant, as both proximal and distal oxygen atoms of this species have prohibitively high barriers to carry out the aromatic hydroxylation. This is in agreement to the experimental observation where Fe(III)-OOH is found not to directly attack the aromatic ring. A novel mechanism for the explicit generation of non-heme Fe(IV)═O and Fe(V)═O from isomeric forms of Fe(III)-OOH has been proposed where the O···O bond is found to cleave via homolytic (Fe(IV)═O) or heterolytic (Fe(V)═O) fashion exclusively. Apart from having favorable formation energies, the Fe(V)═O species also has a lower barrier height compared to the corresponding Fe(IV)═O species for the aromatic ortho-hydroxylation reaction. The transient Fe(V)═O prefers electrophilic attack on the benzene ring rather than the usual aromatic C-H activation step. A large thermodynamic drive for the formation of a radical intermediate is encountered in the mechanistic scene, and this intermediate substantially diminishes the energy barrier required for C-H activation by the Fe(V)═O species. Further spin density distribution and the frontier orbitals of the computed species suggest that the Fe(IV)═O species has a substantial barrier height for this reaction, as the substrate is coordinated to the metal atoms. This coordination restricts the C-H activation step by Fe(IV)═O species to proceed via the π-type pathway, and thus the usual energy lowering due to the low-lying quintet state is not observed here.

Advances in multi-omics based quantitative microbial risk assessment in the dairy sector: A semi-systematic review.

With the increasing consumption of packaged and ready-to-eat food products, the risk of foodborne illness has drastically increased and so has the dire need for proper management. The conventional Microbial Risk Assessment (MRA) investigations require prior knowledge of process flow, exposure, and hazard assessment throughout the supply chain. These data are often generated using conventional microbiological approaches based either on shelf-life studies or specific spoilage organisms (SSOs), frequently overlooking crucial information such as antimicrobial resistance (AMR), biofilm formation, virulence factors and other physiological variations coupled with bio-chemical characteristics of food matrix. Additionally, the microbial risks in food are diverse and heterogenous, that might be an outcome of growth and activity of multiple microbial populations rather than a single species contamination. The uncertainty on the microbial source, time as well as point of entry into the food supply chain poses a constraint to the efficiency of preventive approaches and conventional MRA. In the last few decades, significant breakthroughs in molecular methods and continuously progressing bioinformatics tools have opened up a new horizon for risk analysis-based approaches in food safety. Real time polymerase chain reaction (qPCR) and kit-based assays provide better accuracy and precision with shorter processing time. Despite these improvements, the effect of complex food matrix on growth environment and recovery of pathogen is a persistent problem for risk assessors. The dairy industry is highly impacted by spoilage and pathogenic microorganisms. Therefore, this review discusses the evolution and recent advances in MRAmethodologies equipped with predictive interventions and "multi-omics" approach for robust MRA specifically targeting dairy products. It also highlights the limiting gap area and the opportunity for improvement in this field to ensure precision food safety.

Moonlighting Biochemistry of Cysteine Synthase: A Species-specific Global Regulator.

Cysteine Synthase (CS), the enzyme that synthesizes cysteine, performs non-canonical regulatory roles by binding and modulating functions of disparate proteins. Beyond its role in catalysis and regulation in the cysteine biosynthesis pathway, it exerts its moonlighting effect by binding to few other proteins which possess a C-terminal "CS-binding motif", ending with a terminal ILE. Therefore, we hypothesized that CS might regulate many other disparate proteins with the "CS-binding motif". In this study, we developed an iterative sequence matching method for mapping moonlighting biochemistry of CS and validated our prediction by analytical and structural approaches. Using a minimal protein-peptide interaction system, we show that five previously unknown CS-binder proteins that participate in diverse metabolic processes interact with CS in a species-specific manner. Furthermore, results show that signatures of protein-protein interactions, including thermodynamic, competitive-inhibition, and structural features, highly match the known CS-Binder, serine acetyltransferase (SAT). Together, the results presented in this study allow us to map the extreme multifunctional space (EMS) of CS and reveal the biochemistry of moonlighting space, a subset of EMS. We believe that the integrated computational and experimental workflow developed here could be further modified and extended to study protein-specific moonlighting properties of multifunctional proteins.

Inhibitors of bacterial H2S biogenesis targeting antibiotic resistance and tolerance.

Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.

Robust marker detection and high precision measurement for real-time anatomical registration using Taguchi method.

BACKGROUND: Accurate autonomous marker detection and measurement is essential for high precision anatomical registration. The measurement should be in real-time, accurate, and robust to the varied conditions of the operation theatre. METHODS: The purpose is to design and implement a robust real-time algorithm to measure the coordinates of the point on the marker for robot-based autonomous registration and surgery. The algorithm is built in two parts based on the recursive Taguchi method. The first part deals with the detection of markers. In the second part, the center of the marker is located, and the coordinates are measured by fitting the concentric ellipse. RESULTS: Three case studies are presented where the algorithm is tested for extreme conditions of uneven lighting, distorted color, surface distortions, and significant random orientation of the marker. The robustness of the algorithm in successfully detecting and measuring in real-time is presented. CONCLUSION: The algorithm is successfully implemented for real-time detection and coordinate measurement of the markers.

Robot-Based Autonomous Neuroregistration and Neuronavigation: Implementation and Case Studies.

BACKGROUND: To conduct an autonomous robot-based neurosurgical procedure in the least time with high accuracy. Further, to analyze and validate the method. METHODS: The coordinates of the markers and the region of interest in the medical image space are measured. Subsequently, in the real patient space, a set of algorithms plans the robot motion. The surgical robot attached with a camera autonomously navigates toward the fiducial markers to measure the coordinates and conduct the neuroregistration. The robot also facilitates the precise constrained guiding path to navigate the surgical tool to reach the region of interest (target) selected in the image. RESULTS: The phantom was registered within 1 mm accuracy in all cases for all poses. High-precision navigation to the target in all poses was shown. CONCLUSIONS: The robot is successful in conducting hands-off neuroregistration and neuronavigation. The accuracy is considerably higher, and the time taken is lesser relative to the manual procedure.

Validation of High Precision Robot-Assisted Methods for Intracranial Applications: Preliminary Study.

BACKGROUND: This work attempts to simulate a robot-based autonomous targeted neurosurgical procedure such as biopsy on a vegetable specimen. The objective of the work is to validate the robot-based autonomous neuroregistration and neuronavigation for neurosurgery in terms of stereotactic navigation and target accuracy. CASE DESCRIPTION: A vegetable (carrot) fixed in a tray was used as a model. The tray was affixed with multiple markers. The robot autonomously registers the subject precisely and subsequently accesses the target. The navigation trajectory closely follows the path from the entry point to the target point, as specified in the medical image. The replication of procedures reveals that the target accuracies are within 1 mm. The results based on the case studies are presented. Intricate cases in terms of entry hole size, depth, and size of the target are considered for both phantom and vegetable trials. CONCLUSIONS: The results of the case studies show enhanced and consistent performance characteristics in terms of accuracy, precision, and repeatability with the added advantage of the economy of time. The case studies serve as validation for a high precision robot-assisted neuroregistration and neuronavigation task for neurosurgery and pave the way for further animal and human trials.

Prognostic value of dynamic MRI in assessing post-traumatic femoral head vascularity.

OBJECTIVE: The vascular status of femoral heads in the post-traumatic period of intracapsular femoral neck fracture (ICFNF) remains uncertain until the patient actually develops avascular necrosis (AVN). Several methods for predicting the viability of femoral head have been reported, that are not effective or widely used because of unreliability, potential complications, and technical difficulties. The present study involved the use of Dynamic MRI (DMRI) in assessing femoral head vascularity to predict AVN. MATERIALS AND METHODS: The role of DMRI was studied prospectively in 30 patients with 31 ICFNF. Fractures were divided in to three types (Type A, B, or C) based on the femoral head vascularity shown by dynamic curve patterns on MRI evaluation. Type A was preserved vascularity, Type B was some decrease in vascularity but still viable while Type C was significantly reduced vascularity. These were followed-up for 6 months to 2 years to observe the final outcome in terms of union, non-union, or AVN. RESULTS: We found that Type A curves correlate well with vascular status and Type C curves correlate well with poor vascularity of the femoral heads. No AVN was seen in any of Type A (13/31) or Type B (eight out of 31). Five cases showed AVN and all of them were of Type C dynamic curves. CONCLUSION: Dynamic MRI is a reliable tool to evaluate vascularity of femoral heads and thus reduces the uncertainty of outcome of treatment of ICFNFs. DMRI can be a useful tool to formulate a treatment algorithm in management of ICFNF.

Autonomous neuro-registration for robot-based neurosurgery.

PURPOSE: Neuro-registration is of primary importance as it has a bearing on the accuracy of neurosurgery. Although the accuracy of surgical robots is within the acceptable medical standards, the overall surgical accuracy is dictated by the errors in the neuro-registration process. The purpose of this work is to automate the neuro-registration process to improve the overall accuracy of the robot-based neurosurgery. METHOD: A highly accurate 6-degree-of-freedom Parallel Kinematic Mechanism (6D-PKM) robot is used for both neuro-registration and neurosurgery. In neuro-registration, after measurement of points in the medical image space, the end-platform of the 6D-PKM surgical robot carrying the camera will autonomously navigate towards the fiducial markers to measure its coordinates in the real patient space. An accurate relationship between the medical image space and the real patient space is established, and the same robot will navigate the surgical tool to the target. RESULTS: In order to validate the proposed method for autonomous neuro-registration, experiments are performed using four phantoms. The four phantoms are as follows: PVC skull model, two acrylic blocks and a glass jar with coaxial shells. These phantoms are specifically designed to simulate the neurosurgical process. All the phantoms are registered successfully using the above-stated method. After autonomous neuro-registration, the coordinates of the target point are determined. Neurosurgery validation is carried out by attaching a 1-mm-diameter needle to the robot platform, which is autonomously traversed to reach the target point passing through the two 2-mm-diameter coaxial holes. The experiments are repeated, and the results reveal very good repeatability. CONCLUSION: A method for autonomous neuro-registration has been developed. The robot has been successfully registered using the above method. After successful neuro-registration the overall accuracy of the robot-based neurosurgery is considerably improved. The other benefits of the above method are as follows: elimination of line-of-sight problem, no need of extra unit for neuro-registration, less time for registration, intraoperative registration, human error reduction and low cost.

The C-terminal extension of Mycobacterium tuberculosis Hsp16.3 regulates its oligomerization, subunit exchange dynamics and chaperone function.

Mycobacterium tuberculosis is a human pathogen that secretes a major immunodominant antigen, namely Hsp16.3, throughout the course of infection. Hsp16.3 belongs to the small heat shock protein family and exhibits a molecular chaperone function that is important for the growth and survival of M. tuberculosis in host cell macrophages. The importance of the N-terminal region for the structure and chaperone function of Hsp16.3 is well understood. However, the effect of the C-terminal region on these properties is far from clear. Therefore, we cloned, over-expressed and purified wild-type and seven C-terminal-truncated mutant proteins of Hsp16.3. Mutants with deletions of one and two C-terminal extension (CTE) residues had a structure and chaperone function similar to wild-type protein. Intriguingly, deletion of three residues from the CTE triggered perturbation of the tertiary structure, dissociation of the oligomeric assembly (dodecamer to octamer and dimer), enhancement of subunit exchange dynamics and improvement in the chaperone function of Hsp16.3. Interestingly, these structural modulations (except oligomeric dissociation) as well as chaperoning strength reached their apex upon truncation of the entire CTE (141 RSTN144 ). Further deletions from the C-terminal region beyond the CTE increased only the degree of oligomeric dissociation, and the complete removal of this region made the protein into a dimer. Overall, our study suggests a 'new structural element' in the C-terminal region, i.e. the C-terminal extension, which plays an important role in the oligomerization, subunit exchange dynamics and chaperone function of Hsp16.3.

Crystal Structure of Fad35R from Mycobacterium tuberculosis H37Rv in the Apo-State.

Fad35R from Mycobacterium tuberculosis binds to the promoter site of Fad35 operon and its DNA binding activities are reduced in the presence of tetracycline and palmitoyl-CoA. We resolved the crystal structure of Fad35R using single-wavelength anomalous diffraction method (SAD). Fad35R comprises canonical DNA binding domain (DBD) and ligand binding domain (LBD), but displays several distinct structural features. Two recognition helices of two monomers in the homodimer are separated by ~ 48 Å and two core triangle-shaped ligand binding cavities are well exposed to solvent. Structural comparison with DesT and QacR structures suggests that ligand binding-induced movement of α7, which adopts a straight conformation in the Fad35R, may be crucial to switch the conformational states between repressive and derepressive forms. Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers. Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible. Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline. We present the first structure of a FadR homologue from mycobacterium and the structure reveals DNA and ligand binding features of Fad35R and also provides a view on alternative quaternary states that mimic open and closed forms of the regulator.

To study the role of dynamic magnetic resonance imaging in assessing the femoral head vascularity in intracapsular femoral neck fractures.

Intracapsular femoral neck fractures remain unsolved fractures even after improvement in techniques of diagnosis and internal fixation. Individuals who sustain displaced femoral neck fractures are at high risk of developing avascular necrosis and non-union. Although several methods for predicting the viability of femoral head have been reported, they are not effective or widely used because of unreliability, potential complications and technical difficulties. Dynamic MRI was introduced in the recent past as a simple, non-invasive technique to predict the femoral head viability after the femoral neck fractures. In this study role of dynamic MRI was studied in 30 patients with 31 intracapsular femoral neck fractures. Fractures were divided in to three types according to dynamic curve patterns on MRI evaluation and were followed up for 6 months to 2 years to observe the final outcome. Sensitivity, Specificity and the Accuracy of dynamic MRI in predicting vascularity after femoral neck fracture are 87%, 88% and 87%, respectively. Type A or Type B curve pattern is a positive factor to successful osteosynthesis with p value <0.0001 (Chi-square test). This is a statistically significant value. From this finding it can be suggested that the reliability of dynamic curves A and B in predicting maintained vascularity of femoral head is high. This investigation can be used to predict the vascularity of femoral head after intracapsular femoral neck fractures. There was a good correlation between the outcomes of fractures and dynamic MRI curves done within 48 h of injury. This signifies the role of dynamic MRI in predicting the vascularity of femoral head as early as 48 h. A treatment algorithm can be suggested on the basis of dynamic MRI curves. The fractures with Type C dynamic curve should be considered as fractures with poor vascularity of femoral head and measures to enhance the vascularity of femoral head along with rigid internal fixation should be undertaken to promote revascularization process and better healing of fractures. Patients with these fractures should be on longer non-weight bearing ambulation than other patients. To conclude, the dynamic MRI seems to be reliable, non-invasive, sensitive, specific and accurate method of assessing the femoral head vascularity after intracapsular femoral neck fractures as early as 48 h of injury and to predict the outcome of fractures and may be used as a guideline for management of intracapsular femoral neck fractures.