Developing a robust in vitro release method for a polymeric nanoparticle: Challenges and learnings.

Nanomedicines have emerged as a promising approach for targeted therapeutic delivery and specifically as a beneficial alternative to conventional cancer therapies as they can deliver higher concentrations of chemotherapeutic agents at the tumour site compared to healthy tissue, thus providing improved drug efficacy and lower systemic toxicity. Long acting injectables are increasingly becoming the focus of pharmaceutical research, as they can reduce dosing frequency and improve the life quality of patients. Development of an in vitro release (IVR) method for modified release nanomedicines is challenging because of the uniqueness and range of different formulation design approaches, as well as the complex nature of drug release mechanisms which may result in inherent variability. Regulatory guidance on the development of dissolution or release methods for parenteral products is limited relative to oral products. This article details the extensive in vitro release method development work conducted on a polymeric nanoparticle to develop the release media composition and selection of suitable apparatus and sampling technique to separate the released drug from the formulation. The aim was to develop a suitably robust analytical method that generated clinically relevant in vitro release data.

Risk of Neonatal Sepsis With Rescue Steroids in Preterm Premature Rupture of Membranes.

Objective To evaluate whether a rescue course of corticosteroids, when given at least 14 days after the initial course, is associated with an increased risk of neonatal sepsis after preterm premature rupture of membranes (PPROM). Methods We performed a retrospective, descriptive cohort study of women with singleton gestations from 23+0 to 34+0 weeks of gestation who received a rescue course of corticosteroids within the Indiana University Health Network from January 2009 through October 2016. Patients were separated into three groups based on amniotic membrane status at the time of each corticosteroid administration: Group 1 (intact membranes at initial/intact membranes at rescue), Group 2 (intact membranes at initial/PPROM at rescue), and Group 3 (PPROM at initial/PPROM at rescue). The primary outcome (neonatal sepsis) was compared between the groups. Patient characteristics and neonatal outcomes were analyzed with Fisher's exact test for categorical variables and ANOVA for continuous variables. Relative risk (RR) was calculated by comparing those with ruptured membranes to those with intact membranes at the time of rescue course administration. Results A total of 143 patients were eligible. Neonatal sepsis occurred in 6.8% of patients in Group 1, 21.1% of patients in Group 2, and 23.8% of patients in Group 3. Groups 2 and 3 had a statistically significant higher rate of neonatal sepsis than Group 1 (p = 0.021). The RR of neonatal sepsis after a rescue course in patients with PPROM (Groups 2 and 3) was 3.31 (95% CI = 1.32, 8.29) compared to those with intact membranes at the time of rescue course administration (Group 1). Conclusion A rescue course of corticosteroids in women with PPROM at the time of rescue administration was associated with an increased risk of neonatal sepsis. This increased risk was seen in women with intact membranes as well as ruptured membranes during their initial course of steroids. Larger studies are needed to further investigate this association.

Oxygen treatment reduces neurological deficits and demyelination in two animal models of multiple sclerosis.

AIMS: The objective of the study is to explore the importance of tissue hypoxia in causing neurological deficits and demyelination in the inflamed CNS, and the value of inspiratory oxygen treatment, using both active and passive experimental autoimmune encephalomyelitis (EAE). METHODS: Normobaric oxygen treatment was administered to Dark Agouti rats with either active or passive EAE, compared with room air-treated, and naïve, controls. RESULTS: Severe neurological deficits in active EAE were significantly improved after just 1 h of breathing approximately 95% oxygen. The improvement was greater and more persistent when oxygen was applied either prophylactically (from immunisation for 23 days), or therapeutically from the onset of neurological deficits for 24, 48, or 72 h. Therapeutic oxygen for 72 h significantly reduced demyelination and the integrated stress response in oligodendrocytes at the peak of disease, and protected from oligodendrocyte loss, without evidence of increased oxidative damage. T-cell infiltration and cytokine expression in the spinal cord remained similar to that in untreated animals. The severe neurological deficit of animals with passive EAE occurred in conjunction with spinal hypoxia and was significantly reduced by oxygen treatment initiated before their onset. CONCLUSIONS: Severe neurological deficits in both active and passive EAE can be caused by hypoxia and reduced by oxygen treatment. Oxygen treatment also reduces demyelination in active EAE, despite the autoimmune origin of the disease.

Opening a Window into Accessory Pathway Mapping in Children with Wolff-Parkinson-White Syndrome?

We discuss the use of an open-window mapping technique to define the accessory pathway location in a child presenting with symptomatic Wolff-Parkinson-White (WPW) syndrome. This technique may have important applications for children with WPW syndrome and can be carried out using conventional mapping catheters.

Spore exines increase vitamin D clinical bioavailability by mucoadhesion and bile triggered release.

Sporopollenin exine capsules (SpECs) are microcapsules derived from the outer shells (exines) of plant spore and pollen grains. This work reports the first clinical study on healthy volunteers to show enhanced bioavailability of vitamin D encapsulated in SpECs from Lycopodium clavatum L. spore grains vs vitamin D alone, and the first evidence (in vitro, ex vivo and in vivo) of mechanisms to account for the enhancement and release of the active in the small intestine. Evidence for mucoadhesion of the SpECs contributing to the mechanism of the enhancement is based on: (i) release profile over time of vitamin D in a double blind cross-over human study showing significant release in the small intestine; (ii) in vivo particle counting data in rat showing preferred retention of SpECs vs synthetic beads; (iii) ex vivo99mTc labelling and counting data using rat small intestine sections showing preferred retention of SpECs vs synthetic beads; (iv) in vitro mucoadhesion data. Triggered release by bile in the small intestine was shown in vitro using solid state NMR and HPLC.

Organ-on-a-chip: current gaps and future directions.

As an emerging hot topic of the last decade, Organ on Chip (OoC) is a new technology that is attracting interest from both basic and translational scientists. The Biochemical Society, with its mission of supporting the advancement of science, with addressing grand challenges that have societal impact, has included OoC into their agenda to review the current state of the art, bottlenecks and future directions. This conference brought together representatives of the main stakeholders in the OoC field including academics, end-users, regulators and technology developers to discuss and identify requirements for this new technology to deliver on par with the expectations and the key challenges and gaps that still need to be addressed to achieve robust human-relevant tools, able to positively impact decision making in the pharmaceutical industry and reduce overreliance on poorly predictive animal models.

Exploring the predictors of financial impairment in Huntington's disease using the Enroll-HD dataset.

OBJECTIVES: Huntington's disease (HD) is a neurodegenerative disease in which cognitive and behavioural symptoms impair the performance of instrumental activities of daily living, including the handling of finances. We sought to determine the prevalence of financial dysfunction in HD, and the demographic and clinical predictors of such impairments. METHODS: We analysed longitudinal data for pre-manifest gene carriers and HD patients from the Enroll-HD dataset. Financial dysfunction was determined by finance-related items in the Total Functional Capacity (TFC) and Functional Assessment (FA) scales. A binary logistical regression model was used to investigate the predictive value of demographic and clinical factors for the development of financial dysfunction. RESULTS: Financial impairment was found to be common in HD gene carriers, and over half required financial assistance within 5 years from diagnosis. Cognitive impairment, apathy, unemployment and disease severity predicted financial dysfunction in manifest patients. For pre-manifest patients, the predictors were proximity to disease onset and depression. CONCLUSIONS: Loss of financial autonomy is common in HD, and cognitive and psychiatric factors are important in its development. Clinicians must be vigilant to identify patients that may be vulnerable to financial exploitation.

Reduced expression of dopamine D2 receptors on astrocytes in R6/1 HD mice and HD post-mortem tissue.

Dysfunction of the central dopaminergic system is thought to contribute to some of the clinical features of Huntington's disease (HD), and dopamine (DA) receptor antagonists are commonly used to good effect in its treatment. It is well established that there is an early significant reduction in neuronal D2 receptors in HD, considered to be a compensatory response to increased dopaminergic activity. However, no studies have examined the expression of D2 receptors on astrocytes which is important given that these cells have been shown to play a role in the pathogenesis of HD, as well as express dopamine receptors and modulate DA homeostasis in the normal brain. We therefore sought to investigate the expression of D2 receptors on astrocytes in HD, and found them to be reduced in both the R6/1 HD mouse model, and in human post-mortem brain in comparison to controls, suggesting that astrocytes may be important in DA-dependent aspects of HD. Further studies are needed to determine the functional significance of this finding.

Detection of a local Mycobacterium bovis reservoir using cattle surveillance data.

The incidence of bovine tuberculosis (TB, caused by Mycobacterium bovis) in cattle has been associated with TB in badgers (Meles meles) in parts of England. The aim was to identify badger-associated M. bovis reservoirs in the Edge Area, between the High- and Low-Risk Areas for cattle TB. Data from badger TB surveys were sparse. Therefore, a definition for a local M. bovis reservoir potentially shared by cattle and badgers was developed using cattle TB surveillance data. The performance of the definition was estimated through Latent Class Analysis using badger TB survey data. Spatial units (25 km2 ) in the Edge Area were classified as having a reservoir if they had (i) at least one TB incident in at least three of the previous 7 years, (ii) at least one TB incident in a cattle herd confirmed by post-mortem tests as due to M. bovis infection and not attributable to cattle movements in the previous 2 years and (iii) more confirmed TB incidents than un-confirmed in the previous 2 years. Approximately 20% of the Edge Area was classified as having a local M. bovis reservoir using the cattle-based definition. Assuming 15% TB prevalence in Edge Area badgers, sensitivity for the local M. bovis reservoir definition varied from 25.7% [95% credible interval (CrI): 10.7%-85.1%] to 64.8% (95% CrI: 48.1%-88.0%). Specificity was 91.9% (CrI: 83.6%-97.4%). Over 90% of the local reservoir was in stable endemic TB areas identified through previous work and its spatial distribution was largely consistent with local veterinary knowledge. Uncertainty in the reservoir spatial distribution was explored through its recalculation in spatial units shifted in different directions. We recommend that the definition is re-evaluated as further data on badger infection with M. bovis become available.

Distinct subcellular autophagy impairments in induced neurons from patients with Huntington's disease.

Huntington's disease is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling Huntington's disease is challenging, as rodent and cellular models poorly recapitulate the disease as seen in ageing humans. To address this, we generated induced neurons through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics. Huntington's disease induced neurons (HD-iNs) displayed profound deficits in autophagy, characterized by reduced transport of late autophagic structures from the neurites to the soma. These neurite-specific alterations in autophagy resulted in shorter, thinner and fewer neurites specifically in HD-iNs. CRISPRi-mediated silencing of HTT did not rescue this phenotype but rather resulted in additional autophagy alterations in control induced neurons, highlighting the importance of wild-type HTT in normal neuronal autophagy. In summary, our work identifies a distinct subcellular autophagy impairment in adult patient derived Huntington's disease neurons and provides a new rationale for future development of autophagy activation therapies.

Octanoate is differentially metabolized in liver and muscle and fails to rescue cardiomyopathy in CPT2 deficiency.

Long-chain fatty acid oxidation is frequently impaired in primary and systemic metabolic diseases affecting the heart; thus, therapeutically increasing reliance on normally minor energetic substrates, such as ketones and medium-chain fatty acids, could benefit cardiac health. However, the molecular fundamentals of this therapy are not fully known. Here, we explored the ability of octanoate, an eight-carbon medium-chain fatty acid known as an unregulated mitochondrial energetic substrate, to ameliorate cardiac hypertrophy in long-chain fatty acid oxidation-deficient hearts because of carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). CPT2 converts acylcarnitines to acyl-CoAs in the mitochondrial matrix for oxidative bioenergetic metabolism. In Cpt2M-/- mice, high octanoate-ketogenic diet failed to alleviate myocardial hypertrophy, dysfunction, and acylcarnitine accumulation suggesting that this alternative substrate is not sufficiently compensatory for energy provision. Aligning this outcome, we identified a major metabolic distinction between muscles and liver, wherein heart and skeletal muscle mitochondria were unable to oxidize free octanoate, but liver was able to oxidize free octanoate. Liver mitochondria, but not heart or muscle, highly expressed medium-chain acyl-CoA synthetases, potentially enabling octanoate activation for oxidation and circumventing acylcarnitine shuttling. Conversely, octanoylcarnitine was oxidized by liver, skeletal muscle, and heart, with rates in heart 4-fold greater than liver and, in muscles, was not dependent upon CPT2. Together, these data suggest that dietary octanoate cannot rescue CPT2-deficient cardiac disease. These data also suggest the existence of tissue-specific mechanisms for octanoate oxidative metabolism, with liver being independent of free carnitine availability, whereas cardiac and skeletal muscles depend on carnitine but not on CPT2.

Comparison of 10 Control hPSC Lines for Drug Screening in an Engineered Heart Tissue Format.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are commercially available, and cardiac differentiation established routine. Systematic evaluation of several control hiPSC-CM is lacking. We investigated 10 different control hiPSC-CM lines and analyzed function and suitability for drug screening. Five commercial and 5 academic hPSC-CM lines were casted in engineered heart tissue (EHT) format. Spontaneous and stimulated EHT contractions were analyzed, and 7 inotropic indicator compounds investigated on 8 cell lines. Baseline contractile force, kinetics, and rate varied widely among the different lines (e.g., relaxation time range: 118-471 ms). In contrast, the qualitative correctness of responses to BayK-8644, nifedipine, EMD-57033, isoprenaline, and digoxin in terms of force and kinetics varied only between 80% and 93%. Large baseline differences between control cell lines support the request for isogenic controls in disease modeling. Variability appears less relevant for drug screening but needs to be considered, arguing for studies with more than one line.

Blinded, Multicenter Evaluation of Drug-induced Changes in Contractility Using Human-induced Pluripotent Stem Cell-derived Cardiomyocytes.

Animal models are 78% accurate in determining whether drugs will alter contractility of the human heart. To evaluate the suitability of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for predictive safety pharmacology, we quantified changes in contractility, voltage, and/or Ca2+ handling in 2D monolayers or 3D engineered heart tissues (EHTs). Protocols were unified via a drug training set, allowing subsequent blinded multicenter evaluation of drugs with known positive, negative, or neutral inotropic effects. Accuracy ranged from 44% to 85% across the platform-cell configurations, indicating the need to refine test conditions. This was achieved by adopting approaches to reduce signal-to-noise ratio, reduce spontaneous beat rate to ≤ 1 Hz or enable chronic testing, improving accuracy to 85% for monolayers and 93% for EHTs. Contraction amplitude was a good predictor of negative inotropes across all the platform-cell configurations and of positive inotropes in the 3D EHTs. Although contraction- and relaxation-time provided confirmatory readouts forpositive inotropes in 3D EHTs, these parameters typically served as the primary source of predictivity in 2D. The reliance of these "secondary" parameters to inotropy in the 2D systems was not automatically intuitive and may be a quirk of hiPSC-CMs, hence require adaptations in interpreting the data from this model system. Of the platform-cell configurations, responses in EHTs aligned most closely to the free therapeutic plasma concentration. This study adds to the notion that hiPSC-CMs could add value to drug safety evaluation.

Antidopaminergic treatment is associated with reduced chorea and irritability but impaired cognition in Huntington's disease (Enroll-HD).

OBJECTIVES: Alterations in dopamine neurotransmission underlie some of the clinical features of Huntington's disease (HD) and as such are a target for therapeutic intervention, especially for the treatment of chorea and some behavioural problems. However, justification for such an intervention is mainly based on case reports and small open label studies and the effects these drugs have on cognition in HD remain unclear. METHODS: In this study, we used the Enroll-HD observational database to assess the effects of antidopaminergic medication on motor, psychiatric and cognitive decline, over a 3-year period. We first looked at the annual rate of decline of a group of HD patients taking antidopaminergic medication (n=466) compared with an untreated matched group (n=466). The groups were matched on specified clinical variables using propensity score matching. Next, we studied a separate group of HD patients who were prescribed such medications part way through the study (n=90) and compared their rate of change before and after the drugs were introduced and compared this to a matched control group. RESULTS: We found that HD patients taking antidopaminergic medication had a slower progression in chorea and irritability compared with those not taking such medications. However, this same group of patients also displayed significantly greater rate of decline in a range of cognitive tasks. CONCLUSION: In conclusion we found that antidopaminergic treatment is associated with improvements in the choreic movements and irritability of HD but worsens cognition. However, further research is required to prospectively investigate this and whether these are causally linked, ideally in a double-blind placebo-controlled trial.

Visual hallucinations in neurological and ophthalmological disease: pathophysiology and management.

Visual hallucinations are common in older people and are especially associated with ophthalmological and neurological disorders, including dementia and Parkinson's disease. Uncertainties remain whether there is a single underlying mechanism for visual hallucinations or they have different disease-dependent causes. However, irrespective of mechanism, visual hallucinations are difficult to treat. The National Institute for Health Research (NIHR) funded a research programme to investigate visual hallucinations in the key and high burden areas of eye disease, dementia and Parkinson's disease, culminating in a workshop to develop a unified framework for their clinical management. Here we summarise the evidence base, current practice and consensus guidelines that emerged from the workshop.Irrespective of clinical condition, case ascertainment strategies are required to overcome reporting stigma. Once hallucinations are identified, physical, cognitive and ophthalmological health should be reviewed, with education and self-help techniques provided. Not all hallucinations require intervention but for those that are clinically significant, current evidence supports pharmacological modification of cholinergic, GABAergic, serotonergic or dopaminergic systems, or reduction of cortical excitability. A broad treatment perspective is needed, including carer support. Despite their frequency and clinical significance, there is a paucity of randomised, placebo-controlled clinical trial evidence where the primary outcome is an improvement in visual hallucinations. Key areas for future research include the development of valid and reliable assessment tools for use in mechanistic studies and clinical trials, transdiagnostic studies of shared and distinct mechanisms and when and how to treat visual hallucinations.

Huntington's disease patients display progressive deficits in hippocampal-dependent cognition during a task of spatial memory.

BACKGROUND: Cognitive disturbances occur early in Huntington's disease (HD) and place a significant burden on the lives of patients and family members. Whilst these impairments are typically attributed to deterioration of the frontal-striatal pathways, accumulating evidence suggests that hippocampal dysfunction may also contribute to such impairments. Here, we employ a novel spatial memory task that has previously been shown to elicit impairments in individuals with focal hippocampal lesions, as a means to further investigate the role of hippocampal dysfunction in HD. METHOD: Sixty-four individuals participated in the study, including 32 healthy controls, 11 patients with diagnosed HD and 16 premanifest HD gene carriers. We also included an additional control group of 5 individuals with focal unilateral basal ganglia lesions. Participants undertook a task that measured perception and short-term spatial memory using computer-generated visual scenes. RESULTS: HD patients experienced significant impairments in spatial perception and memory, which strongly correlated with disease burden score (DBS). Premanifest gene carriers performed at a similar level to healthy controls throughout all aspects of the task indicating that the effects seen in the HD patients represent a deterioration in function. Interestingly, basal ganglia lesion patients were not impaired in any aspects of the task. CONCLUSION: There is evidence of significant deficits in hippocampal-dependent spatial cognition in HD that cannot be explained as a function of degeneration to the basal ganglia. The impairments were greatest in individuals with higher DBSs, suggesting that deficits relate to the disease process in HD.

Nanofibrous Scaffolds Support a 3D in vitro Permeability Model of the Human Intestinal Epithelium.

Advances in drug research not only depend on high throughput screening to evaluate large numbers of lead compounds but also on the development of in vitro models which can simulate human tissues in terms of drug permeability and functions. Potential failures, such as poor permeability or interaction with efflux drug transporters, can be identified in epithelial Caco-2 monolayer models and can impact a drug candidate's progression onto the next stages of the drug development process. Whilst monolayer models demonstrate reasonably good prediction of in vivo permeability for some compounds, more developed in vitro tools are needed to assess new entities that enable closer in vivo in vitro correlation. In this study, an in vitro model of the human intestinal epithelium was developed by utilizing nanofibers, fabricated using electrospinning, to mimic the structure of the basement membrane. We assessed Caco-2 cell response to these materials and investigated the physiological properties of these cells cultured on the fibrous supports, focusing on barrier integrity and drug-permeability properties. The obtained data illustrate that 2D Caco-2 Transwell® cultures exhibit artificially high trans-epithelial electrical resistance (TEER) compared to cells cultured on the 3D nanofibrous scaffolds which show TEER values similar to ex vivo porcine tissue (also measured in this study). Furthermore, our results demonstrate that the 3D nanofibrous scaffolds influence the barrier integrity of the Caco-2 monolayer to confer drug-absorption properties that more closely mimic native gut tissue particularly for studying passive epithelial transport. We propose that this 3D model is a suitable in vitro model for investigating drug absorption and intestinal metabolism.

Antimicrobial Resistance Diversity Suggestive of Distinct Salmonella Typhimurium Sources or Selective Pressures in Food-Production Animals.

Salmonella enterica subsp. enterica serovar Typhimurium is a common cause of enterocolitis in humans globally, with multidrug resistant (MDR) strains posing an enhanced threat. S. Typhimurium is also a pathogen in food-production animals, and these populations can act as reservoirs of the bacterium. Therefore, surveillance and control measures within food-production animal populations are of importance both to animal and human health and have the potential to be enhanced though improved understanding of the epidemiology of S. Typhimurium within and between food-production animal populations. Here, data from Scotland and national surveillance England and Wales data for isolates from cattle (n = 1115), chickens (n = 248) and pigs (n = 2174) collected between 2003 and 2014 were analyzed. Ecological diversity analyses and rarefaction curves were used to compare the diversity of observed antimicrobial resistance (AMR) profiles between the host species, and within host species populations. Higher AMR profile diversity was observed in isolates from pigs compared to chickens across diversity measures and isolates from cattle for three of four diversity measures. Variation in AMR profile diversity between production sectors was noted, with higher AMR diversity of isolates from broiler compared to layer chickens, breeder compared to rearer and finisher pigs and beef compared to dairy cattle. Findings indicate variation in AMR profile diversity both within and between food-production animal host species. These observations suggest alternate sources of AMR bacteria and/or variation in selective evolutionary pressures within and between food-production animal host species populations.

The effect of cushioning materials on musculoskeletal discomfort and fatigue during prolonged standing at work: A systematic review.

This systematic review updates the current state of evidence on the effectiveness of softer flooring and cushioned shoe insoles on reducing musculoskeletal discomfort amongst workers who are required to stand for prolonged periods to work and the impact of factors such as age and gender on the outcomes. A systematic search identified 10 unique studies that met the eligibility criteria. The heterogeneity of study designs impacted on the strength of evidence. A moderate level of evidence was found in support of using cushioned materials in reducing discomfort/fatigue among standing workers. A limited level of evidence exists in favour of using insoles over anti-fatigue mats. Insufficient information exists for the impact of gender or age. Larger, good quality prospective intervention trials based in real workplaces that consider the impact of psychosocial and organisational factors on musculoskeletal discomfort whilst standing at work are required to inform industry recommendations.

A pragmatic approach to monitor and evaluate implementation and impact of differentiated ART delivery for global and national stakeholders.

INTRODUCTION: The World Health Organization's (WHO) recommendation of "Treat All" has accelerated the call for differentiated antiretroviral therapy (ART) delivery, a method of care that efficiently uses limited resources to increase access to HIV treatment. WHO has further recommended that stable individuals on ART receive refills every 3 to 6 months and attend clinical visits every 3 to 6 months. However, there is not yet consensus on how to ensure that the quality of services is maintained as countries strive to meet these standards. This commentary responds to this gap by defining a pragmatic approach to the monitoring and evaluation (M&E) of the scale up of differentiated ART delivery for global and national stakeholders. DISCUSSION: Programme managers need to demonstrate that the scale up of differentiated ART delivery is achieving the desired effectiveness and efficiency outcomes to justify continued support by national and global stakeholders. To achieve this goal, the two existing global WHO HIV treatment indicators of ART retention and viral suppression should be augmented with two broad aggregate measures. The addition of indicators measuring the frequency of (1) clinical and (2) refill visits by PLHIV per year will allow evaluation of the pace of scale up while monitoring its overall effect on the quality and efficiency of services. The combination of these four routinely collected aggregate indicators will also facilitate the comparison of outcomes among facilities, regions or countries implementing different models of ART delivery. Enhanced monitoring or additional assessments will be required to answer other critical questions on the process of implementation, acceptability, effectiveness and efficiency. CONCLUSIONS: These proposed outcomes are useful markers for the effectiveness and efficiency of the health system's attempts to deliver quality treatment to those who need it-and still reserve as much of the available resource pool as possible for other key elements of the HIV response.