Population Pharmacokinetic Modeling of Cotadutide: A Dual Agonist Peptide of Glucagon-Like Peptide and Glucagon Receptors Administered to Participants with Type II Diabetes Mellitus, Chronic Kidney Disease, Obesity and Non-Alcoholic Steatohepatitis.

BACKGROUND: Cotadutide is a dual glucagon-like peptide-1 (GLP-1) and glucagon (GCG) receptor agonist peptide. The objective of this analysis was to develop a population pharmacokinetic (popPK) model of cotadutide, and to identify any potential effect on the PK from intrinsic and extrinsic covariates. METHODS: The popPK analysis utilized a non-linear mixed-effects modeling approach using the data from 10 clinical studies in different participant categories following once-daily subcutaneous dose administration ranging from 20 to 600 µg. Additionally, the covariates affecting cotadutide exposure were quantified, and the model performance was evaluated through the prediction-corrected visual predictive checks. RESULTS: A one-compartment model with first-order absorption and elimination adequately described the data as confirmed via visual predictive check plots and parameter plausibility. The mean values for cotadutide apparent clearance (CL/F), apparent volume of distribution (V/F), absorption rate constant (Ka), and half-life were 1.05 L/h, 20.0 L, 0.38 h-1, and 13.3 hours, respectively. Covariate modeling identified body weight, alanine transaminase, albumin, anti-drug antibody (ADA) titer values, formulation strength and injection device, and participant categories as significant covariates on PK parameters, where ADAs have been identified to decrease cotadutide clearance. The model demonstrated that a 150-kg participant was estimated to have 30% lower for both AUC and Cmax and a 66 kg participant was estimated to have 35% higher for both AUC and Cmax relative to a reference individual with a median weight of 96 kg. CONCLUSIONS: A popPK model was developed for cotadutide with cotadutide clinical data, and the impact of the statistically significant covariates identified was not considered clinically meaningful. The popPK model will be used to evaluate exposure-response relationships for cotadutide clinical data.

A Case Report of a Successful Percutaneous Trial and Surgical Placement of a Spinal Cord Stimulator in a Patient With Severe Thoracolumbar Scoliosis.

Spinal cord stimulation has been demonstrated as a therapeutic option for patients with persistent lumbar radicular pain secondary to failed back surgery syndrome. This case report demonstrates a successful percutaneous spinal cord stimulator (SCS) trial followed by surgical placement of a permanent SCS to treat lumbar radicular pain and axial low back pain in a patient with severe thoracolumbar scoliosis status after laminectomy and spinal fusion surgery. Currently, there is a paucity of literature on this topic.

Can the same controlled attenuation parameter cut-offs be used for M and XL probes for diagnosing hepatic steatosis?

BACKGROUND AND AIM: There are limited studies on controlled attenuation parameter (CAP) using Fibroscan XL probe for the diagnosis of hepatic steatosis grade. The aim of this study was to determine whether previously defined optimal cut-offs for CAP using the M probe could be applied for the XL probe. METHODS: Adult patients with chronic liver disease who had a liver biopsy and examination with both the M and XL probes were included. Previously defined optimal cut-offs for CAP using the M probe were used for the diagnosis of steatosis grades ≥S1, ≥S2, and S3 (248, 268, and 280 dB/m, respectively). RESULTS: Data for 180 patients were analyzed (mean age 53.7 ± 10.8 years; central obesity 84.5%; non-alcoholic fatty liver disease 86.7%). The distribution of steatosis grades was S0, 9.4%; S1, 28.3%; S2, 43.9%, and S3, 18.3%. The sensitivity, specificity, positive predictive value, and negative predictive value of CAP using the M/XL probe for the diagnosis of steatosis grade ≥S1 was 93.9%/93.3%, 58.8%/58.8%, 95.6%/95.6%, and 50.0%/47.6%, respectively. These values were 94.6%/94.6%, 41.2%/44.1%, 72.6%/73.6%, and 82.4%/83.3%, respectively, for ≥S2, and 87.9%/87.9%, 27.2%/27.9%, 21.3%/21.5%, and 90.9%/91.1%, respectively, for S3. CONCLUSION: The same cut-off values for CAP may be used for the M and XL probes for the diagnosis of hepatic steatosis grade.

Ability of postoperative delirium to predict intermediate-term postoperative cognitive function in patients undergoing elective surgery at an academic medical centre: protocol for a prospective cohort study.

INTRODUCTION: Postoperative delirium (POD) is a common complication in elderly patients, characterised by a fluctuating course of altered consciousness, disordered thinking and inattention. Preliminary research has linked POD with persistent cognitive impairment and decreased quality of life. However, these findings maybe confounded by patient comorbidities, postoperative complications and frailty. Our objective is to determine whether POD is an independent risk factor for persistent impairments in attention and executive function after elective surgery. Our central hypothesis is that patients with POD are more likely to have declines in cognition and quality of life 1 year after surgery compared with patients without POD. We aim to clarify whether these associations are independent of potentially confounding factors. We will also explore the association between POD and incident dementia. METHODS AND ANALYSIS: This study will recruit 200 patients from the ongoing Electroencephalography Guidance of Anesthesia to Alleviate Geriatric Syndromes (ENGAGES) study. Patients who live ≤45 miles from the study centre or have a planned visit to the centre 10-16 months postoperatively will be eligible. Patients with POD, measured by the Confusion Assessment Method, will be compared with patients without delirium. The primary outcome of cognitive function and secondary outcomes of quality of life and incident dementia will be compared between cohorts. Cognition will be measured by Trails A and B and Stroop Color and Word Test, quality of life with Veteran's RAND 12-item Health Survey and incident dementia with the Short Blessed Test. Multivariable regression analyses and a Cox proportional hazards analysis will be performed. All results will be reported with 95% CIs and α=0.05. ETHICS AND DISSEMINATION: The study has been approved by the Washington University in St. Louis Institutional Review Board (IRB no 201601099). Plans for dissemination include scientific publications and presentations at scientific conferences. TRIAL REGISTRATION NUMBER: NCT02241655.

Hepatitis C virus induced up-regulation of microRNA-27: a novel mechanism for hepatic steatosis.

UNLABELLED: MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly linked with diseased states, including hepatitis C virus (HCV) infection. Herein, we demonstrate that HCV replication induces the expression of miR-27 in cell culture and in vivo HCV infectious models. Overexpression of the HCV proteins core and NS4B independently activates miR-27 expression. Furthermore, we establish that miR-27 overexpression in hepatocytes results in larger and more abundant lipid droplets, as observed by coherent anti-Stokes Raman scattering (CARS) microscopy. This hepatic lipid droplet accumulation coincides with miR-27b's repression of peroxisome proliferator-activated receptor (PPAR)-α and angiopoietin-like protein 3 (ANGPTL3), known regulators of triglyceride homeostasis. We further demonstrate that treatment with a PPAR-α agonist, bezafibrate, is able to reverse the miR-27b-induced lipid accumulation in Huh7 cells. This miR-27b-mediated repression of PPAR-α signaling represents a novel mechanism of HCV-induced hepatic steatosis. This link was further demonstrated in vivo through the correlation between miR-27b expression levels and hepatic lipid accumulation in HCV-infected SCID-beige/Alb-uPa mice. CONCLUSION: Collectively, our results highlight HCV's up-regulation of miR-27 expression as a novel mechanism contributing to the development of hepatic steatosis.

Upregulation and expression patterns of the angiogenic transcription factor ets-1 in Alzheimer's disease brain.

Immunohistochemical staining has been used to determine expression patterns of the angiogenic transcription factor, Ets-1, in the brains of Alzheimer's disease (AD) individuals. Brain tissue from non-demented controls showed little expression of Ets-1 whereas in AD brain tissue, Ets-1 was ubiquitously expressed in cortex and hippocampus. Double immunostaining with von Willerbrand factor demonstrated prominent Ets-1 intravascular immunoreactivity (ir) in AD cortical microvessels. In addition, Ets-1 also exhibited extravascular expression characterized by a diffuse pattern of Ets-1 ir in AD brain. Double staining also showed Ets-1 colocalization in microvasculature with the potent angiogenic agent, vascular endothelial growth factor (VEGF). Cell-associated tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine with pro-angiogenic activity, was primarily associated with diffuse extravascular Ets-1 ir. Clusters of HLA-DR positive microglia, resident immune cells of brain which release TNF-α, were also localized with diffuse Ets-1. Intravascular Ets-1 ir was maximally co-localized with soluble amyloid-ß peptide (Aß), Aß1-40, in microvasculature whereas diffuse extravascular Ets-1 ir appeared in proximity to Aß plaques in brain parenchyma. Similar overall results were obtained for patterns of Ets-1 staining in AD hippocampal tissue. This work provides novel findings on expression of the angiogenic transcription factor, Ets-1, in vascular remodeling and its association with pro-angiogenic factors, reactive microglia, and Aß deposition in AD brain.

Three-mode electrochemical sensing of ultralow microRNA levels.

MicroRNAs (miRNAs) are an emerging class of biomarkers that are frequently deregulated in cancer cells and have shown great promise for cancer classification and prognosis. In this work, we developed a three-mode electrochemical sensor for detection and quantitation of ultralow levels of miRNAs in a wide dynamic range of measured concentrations. The sensor facilitates three detection modalities based on hybridization (H-SENS), p19 protein binding (P-SENS), and protein displacement (D-SENS). The combined three-mode sensor (HPD-SENS) identifies as low as 5 aM or 90 molecules of miRNA per 30 µL of sample without PCR amplification, and can be operated within the dynamic range from 10 aM to 1 µM. The HPD sensor is made on a commercially available gold nanoparticles-modified electrode and is suitable for analyzing multiple miRNAs on a single electrode. This three-mode sensor exhibits high selectivity and specificity and was used for sequential analysis of miR-32 and miR-122 on one electrode. In addition, the H-SENS can recognize miRNAs with different A/U and G/C content and distinguish between a fully matched miRNA and a miRNA comprising either a terminal or a middle single base mutation. Furthermore, the H- and P-SENS were successfully employed for direct detection and profiling of three endogenous miRNAs, including hsa-miR-21, hsa-miR-32, and hsa-miR-122 in human serum, and the sensor results were validated by qPCR.

Assessing the human lid margin epithelium using impression cytology.

PURPOSE: To establish if impression cytology combined with histochemical and immunocytochemical staining can be used to assess epithelium of the human upper lid margin. METHODS: Following an initial eye examination of 40 healthy subjects (19 soft contact lens wearers and 21 non-contact lens wearers, aged 18-57 years), lid margin staining was assessed with lissamine green using slit lamp biomicroscopy and graded (grade 0-3). Impression cytology of the upper lid margin of both eyes was collected, fixed and stained with periodic acid Schiff (PAS) and haematoxylin for cell morphology analysis (Nelson grade) or for immunocytochemistry (keratinization-related proteins: filaggrin, transglutaminase1 (TGase1) and cytokeratin 1/10). RESULTS: In 57% of all subjects, grade 0 lissamine green staining showed a thin line (the Marx line), just posterior to the meibomian gland ducts. Grade 2 or 3 lissamine green staining was observed in 17% of all subjects. There was no difference between contact lens and non-contact lens wearers for lid margin staining or Nelson grade (p = 0.4, Fisher's exact test). PAS/haematoxylin staining and immunocytochemistry showed transition in epithelial cell morphology, with marginal conjunctival epithelium, mucocutaneous junction and squamous epithelium, adjacent to meibomian gland ducts. This transition in epithelium was associated with differential expression of keratinization-related proteins (filaggrin, cytokeratin 1/10 and TGase1). CONCLUSION: Lid margin epithelium can be successfully sampled using impression cytology and further characterized using histochemistry and immunocytochemistry staining techniques. This approach can be applied to assess lid margin changes in conditions such as dry eye and meibomian gland dysfunction.

Enhanced specificity of the viral suppressor of RNA silencing protein p19 toward sequestering of human microRNA-122.

Tombusviruses express a 19 kDa protein (p19) that, as a dimeric protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The p19 protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the p19 binding surface with the aim of creating p19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe p19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant p19, p19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type p19. Overall, our results suggest that p19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The p19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver.

Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes for labeling human cancer cells.

Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes proceed with rate constants up to 3.38 ± 0.31 M(-1) s(-1) in CD(3)CN, or 59 times faster than the analogous reaction of azides. This highly specific modular labeling strategy can be applied for direct labeling of proteins and for live cell imaging of cancer cells.

Quantitative analysis of microRNA in blood serum with protein-facilitated affinity capillary electrophoresis.

MicroRNAs (miRNAs) are small (∼22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by p19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30 000 miRNA molecules in 1 mL of serum as a potential source of naïve miRNAs.

An enzyme-linked assay for the rapid quantification of microRNAs based on the viral suppressor of RNA silencing protein p19.

MicroRNAs (miRNAs) are endogenous posttranscriptional regulators found in all metazoa and play crucial roles in virtually all cellular processes. Their aberrant expression has been linked to several diseased states; therefore, techniques capable of sensitive and specific profiling of the miRNA milieu will have significant application in prognostics, diagnostics, and therapeutics. Here we present a method for rapid quantification of miRNA levels using p19, a tombusvirus-encoded suppressor of RNA interference with sequence-independent and size-selective affinity toward 19-bp RNA duplexes. We present a surface plasmon resonance (SPR)-based miRNA sensing method where RNA probes are immobilized on gold surfaces demonstrating p19's utility in recognition of miRNA-bound probes. This allows detection of miRNAs in the low nanomolar range. To increase the sensitivity, a bead-based enzyme immunoassay was performed, and this technique displays a lower detection limit of 1fmol and a linear dynamic range from 1pmol to 1fmol.

FLEth RNA intercalating probe is a convenient reporter for small interfering RNAs.

Here we report that the phenanthridine derivative covalently linked to a fluorescein moiety (FLEth) can act as a fluorescence based probe for duplex short interfering RNA (siRNA) and that this probe can also be used to report on protein-RNA interactions. A fluorescence resonance energy transfer (FRET) signal that is observed at 600 nm occurs when FLEth is complexed with siRNA. At least 2 molecules of FLEth can bind to 21 nt duplex siRNA, and the dissociation constants for these interactions are reported. We find that FLEth can also report on the interaction of siRNAs with the Carnation Italian ringspot viral suppressor of RNA silencing p19. FLEth does not bind to the siRNA-p19 complex nor can p19 bind to the siRNA-FLEth complex; rather FLEth can report on the fraction of siRNA that is unbound. FLEth can also bind siRNA in delivery systems such as liposomes. Once the siRNA reaches the interior of Huh 7.5 cells, FLEth dissociates from the siRNA and is found in the nucleoli suggesting that FLEth cannot bind to siRNAs that are associated with the RNA silencing machinery.

Cysteine residues of Carnation Italian Ringspot virus p19 suppressor of RNA silencing maintain global structural integrity and stability for siRNA binding.

Carnation Italian Ringspot virus (CIRV) has evolved a protein called p19 that acts as a suppressor of RNA silencing in the host cell and aids in viral persistence. This protein has been shown to be sensitive to cysteine alkylation resulting in a reduction in its ability to bind to short-interfering RNA (siRNA). To determine the sites within the protein that are sensitive to alkylation, we systematically tested the functional role of each cysteine residue using site-directed mutagenesis. Variants of the p19 protein were created at locations C110, C134 and C160 where the cysteines were replaced by an inert amino acid such as serine or isoleucine. The results from activity measurements of the purified mutant p19 proteins indicate that the mutants maintain the ability to bind siRNAs with nanomolar affinity, however, their stabilities, as measured by circular dichroism (CD), vary. Functional studies in the presence of the cysteine alkylating agent N-ethylmaleimide (NEM) indicated that p19's ability to bind siRNAs and act as a suppressor of RNA silencing is sensitive to alkylation at all three cysteine residues with the maximum effects occurring when C110 and C134 are both alkylated. These results suggest that the role of the cysteine amino acid conservation is likely to preserve the overall structural integrity of p19 for optimal thermostability and subsequent siRNA-binding activity. We find that p19 function is maximally compromised at high levels of thiol alkylation or in an oxidizing environment.

Activation of peroxisome proliferator-activated receptor alpha by dietary fish oil attenuates steatosis, but does not prevent experimental steatohepatitis because of hepatic lipoperoxide accumulation.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease is the result of an imbalance in hepatic lipid partitioning that favors fatty acid synthesis and storage over fatty acid oxidation and triglyceride secretion. The progressive, inflammatory disorder of steatohepatitis can be prevented or reversed by correcting this lipid imbalance by activating peroxisome proliferator-activated receptor (PPAR) alpha, a transcription factor which regulates fatty acid oxidation. n-3 polyunsaturated fatty acids (PUFA), such as those found in fish oil (FO), are naturally occurring PPARalpha ligands which also suppress lipid synthesis. METHODS: We tested the role of dietary activation of PPARalpha by feeding mice a n-3 PUFA-enriched FO diet in the methionine and choline deficient (MCD) model of steatohepatitis. Results were compared with mice fed the corresponding diet supplemented with monounsaturated fatty acids as olive oil (OO). RESULTS: As expected, FO feeding led to robust hepatic PPARalpha activation in control mice, and decreased expression of genes involved with fatty acid synthesis. Such lipolytic gene expression profile was also clearly evident in FO MCD-fed mice, and was associated with reduced hepatic lipid accumulation in comparison with mice fed OO MCD diet. FO feeding in control mice also caused marked hepatic accumulation of lipoperoxides compared with OO and chow-fed mice. This was further exacerbated in FO MCD-fed animals, which developed steatohepatitis characterized by mild steatosis and moderate inflammation in comparison with OO MCD-fed mice; such inflammatory recruitment was not related to NF-kappaB activation or enhanced cyclooxygenase-2 activity. CONCLUSIONS: Feeding an n-3 PUFA-enriched diet activated PPARalpha and suppressed hepatic de novo lipogenesis, but failed to prevent development of steatohepatitis in the presence of methionine and choline deficiency. Instead, the very high levels of hepatic lipoperoxides may have abrogated the protection that would otherwise be conferred by PPARalpha activation, and could also be responsible for lipotoxic hepatocellular injury and inflammatory recruitment.

Stabilized recombinant suppressors of RNA silencing: functional effects of linking monomers of Carnation Italian Ringspot virus p19.

Eukaryotes have evolved complex cellular responses to double-stranded RNA including the RNA silencing pathway. Tombusviruses have adapted a mechanism to evade RNA silencing that involves a 19 kDa dimeric protein (p19) that is a suppressor of RNA silencing. In order to develop stabilized p19 proteins, linked versions of p19 from the Carnation Italian Ringspot virus (CIRV) were constructed that joined the C-terminus of one subunit to the N-terminus of the second subunit. Like the native CIRV p19, these linked p19 proteins were able to bind to double-stranded siRNAs with nanomolar affinity and discriminate siRNA according to length. In addition, the interdomain linker improved both the stability and binding properties of the p19 dimer. The observed binding properties support the idea that the semi-rigid cross-link favors the folded, binding-competent state of p19. The cross-linked recombinant CIRV-p19s represent novel stabilized suppressors of RNA silencing and may be useful in future biophysical, immunological and cell biology studies.

Apoptosis of viral-infected airway epithelial cells limit viral production and is altered by corticosteroid exposure.

BACKGROUND: Effects of respiratory viral infection on airway epithelium include airway hyper-responsiveness and inflammation. Both features may contribute to the development of asthma. Excessive damage and loss of epithelial cells are characteristic in asthma and may result from viral infection. OBJECTIVE: To investigate apoptosis in Adenoviral-infected Guinea pigs and determine the role of death receptor and ligand expression in the airway epithelial response to limit viral infection. METHODS: Animal models included both an Acute and a Chronic Adeno-infection with ovalbumin-induced airway inflammation with/without corticosteroid treatment. Isolated airway epithelial cells were cultured to study viral production after infection under similar conditions. Immunohistochemistry, western blots and viral DNA detection were used to assess apoptosis, death receptor and TRAIL expression and viral release. RESULTS: In vivo and in vitro Adeno-infection demonstrated different apoptotic and death receptors (DR) 4 and 5 expression in response to corticosteroid exposure. In the Acute Adeno-infection model, apoptosis and DR4/5 expression was coordinated and were time-dependent. However, in vitro Acute viral infection in the presence of corticosteroids demonstrated delayed apoptosis and prolonged viral particle production. This reduction in apoptosis in Adeno-infected epithelial cells by corticosteroids exposure induced a prolonged virus production via both DR4 and TRAIL protein suppression. In the Chronic model where animals were ovalbumin-sensitized/challenged and were treated with corticosteroids, apoptosis was reduced relative to adenovirus-infected or corticosteroid alone. CONCLUSION: Our data suggests that apoptosis of infected cells limits viral production and may be mediated by DR4/5 and TRAIL expression. In the Acute model of Adeno-infection, corticosteroid exposure may prolong viral particle production by altering this apoptotic response of the infected cells. This results from decreased DR4 and TRAIL expression. In the Chronic model treated with corticosteroids, a similar decreased apoptosis was observed. This data suggests that DR and TRAIL modulation by corticosteroids may be important in viral infection of airway epithelium. The prolonged virus release in the setting of corticosteroids may result from reduced apoptosis and suppressed DR4/TRAIL expression by the infected cells.

Conformation of the cell division regulator MinE: evidence for interactions between the topological specificity and anti-MinCD domains.

Symmetric division of Gram-negative bacteria depends on the combined action of three proteins that ensure correct positioning of the cell division septum, namely, MinC, MinD, and MinE. To achieve this function, MinC and MinD form a membrane-bound complex that blocks cell division at all potential sites. Opposing this inhibition is MinE, which interacts with MinD via its N-terminal anti-MinCD domain to site-specifically counter the action of the MinCD complex. The anti-MinCD domain has been proposed to bind MinD in a helical conformation; however, little is actually known about the structure of this functionally critical region. To understand how MinE can perform its anti-MinCD function, we have therefore investigated the conformation of the full-length MinE from Neisseria gonorrhoeae by solution NMR. Although solubility considerations required the use of sample conditions that limit the observation of amide resonances to regions that are protected from solvent exchange, backbone chemical shifts from both N- and C-terminal domains could be assigned. In contrast to previous models, secondary chemical shift analysis of these solvent-protected regions shows that parts of the N-terminal anti-MinCD domain are stably folded with many functionally important residues localizing to a beta-structure. In addition, this N-terminal domain may be interacting with the C-terminal topological specificity domain, since mutations made in one domain led to NMR spectral changes in both domains. The nonfunctional MinE mutant L22D showed even larger evidence of structural perturbations in both domains, with significant destabilization of the entire MinE structure. Overall, these results suggest that there is an intimate structural association between the anti-MinCD and topological specificity domains, allowing the functional properties of the two domains to be modulated through this interaction.