Deposition Of Extracellular Proteins Research Articles

Current Therapies and Future Horizons in Cardiac Amyloidosis Treatment.

Cardiac amyloidosis (CA) is a condition characterized by misfolding and extracellular deposition of proteins, leading to organ dysfunction. While numerous forms of CA exist, two subtypes dominate clinical prevalence: Transthyretin amyloid (ATTR) and immunoglobulin light chain amyloid. The current scientific landscape reflects the urgency to advance therapeutic interventions with over 100 ongoing clinical trials. Heart failure treatment is affected by CA phenotype with poor tolerance of otherwise frequently used medications. Treating comorbidities including atrial fibrillation and valvular disease remains a challenge in CA, driven by technical difficulties and uncertain outcomes. Tafamidis is the first ATTR-stabilizer approved with a rapidly growing rate of clinical use. In parallel, various new therapeutic classes are in late-stage clinical trials including silencers, antibodies and genetic therapy. Managing CA is a critical challenge for future heart failure care. This review delineates the current standard-of-care and scientific landscape of CA therapy.

Emerging Role of Scintigraphy Using Bone-Seeking Tracers for Diagnosis of Cardiac Amyloidosis: AJR Expert Panel Narrative Review.

Amyloidoses are a complex group of clinical diseases that result from progressive organ dysfunction due to extracellular protein misfolding and deposition. The two most common types of cardiac amyloidosis are transthyretin amyloidosis (ATTR) and light-chain (AL) amyloidosis. Diagnosis of ATTR cardiomyopathy (ATTR-CM) is challenging owing to its phenotypic similarity to other more common cardiac conditions, the perceived rarity of the disease, and unfamiliarity with its diagnostic algorithms; endomyocardial biopsy was historically required for diagnosis. However, myocardial scintigraphy using bone-seeking tracers has shown high accuracy for detection of ATTR-CM and has become a key noninvasive diagnostic test for the condition, receiving support from professional society guidelines and transforming prior diagnostic paradigms. This AJR Expert Panel Narrative Review describes the role of myocardial scintigraphy using bone-seeking tracers in the diagnosis of ATTR-CM. The article summarizes available tracers, acquisition techniques, interpretation and reporting considerations, diagnostic pitfalls, and gaps in the current literature. The critical need for monoclonal testing of patients with positive scintigraphy results to differentiate ATTR-CM from AL cardiac amyloidosis is highlighted. Recent updates in guideline recommendations that emphasize the importance of a qualitative visual assessment are also discussed.

Ethanol extract of Andrographis paniculata alleviates aluminum-induced neurotoxicity and cognitive impairment through regulating the p62-keap1-Nrf2 pathway

BackgroundAlzheimer’s disease (AD) is the most prevalent neurodegenerative and remains incurable. Aluminum is a potent neurotoxin associated with AD. The main pathological features of AD are extracellular amyloid-β protein deposition and intracellular hyperphosphorylated Tau protein. A body of evidence suggest that oxidative stress and autophagy are involved in the pathogenesis of AD. Andrographis paniculata (AP) is a native plant with anti-inflammatory, anti-oxidative stress, and regulation of autophagy properties. AP significantly alleviated cognitive impairments, reduced Aβ deposition and has neuroprotective effect. However, its effects on aluminum-induced AD model have not been studied much. In this study, we investigated whether AP protect against aluminum-induced neurotoxicity through regulation of p62-Kelch-like ECH-associated protein 1(Keap1)-Nuclear factor E2 related factor 2 (Nrf2) pathway and activation autophagy in vivo and in vitro.MethodsUPLC-ESI-qTOF-MS/MS was used to identify the chemical constituents of AP ethanol extract. The mice with cognitive deficit were established by injecting aluminum chloride and D-galactose, and treated with either AP extract (200, 400, or 600 mg/kg/d) or andrographolide (2 mg/kg/2d).The spatial memory ability was detected by Morris water maze, HE staining were used to detect in brain tissue,Oxidative stress indexs and SOD activity in both serum and brain tissue were detected by kit.The expression of p62-Nrf2 pathway proteins were measured via western blotting. Furthermore, the neurotoxicity model was induced by aluminum maltolate (700 µM) in PC12 cells. Following AP and andrographolide treatment, the cell viability was detected. The relevant mRNA and protein expressions were detected in cells transfected with the p62 siRNA.ResultsThe main active components of AP included andrographolide, neoandrographolide and deoxyandrographolide as identified. AP and andrographolide significantly improved the spatial memory ability of mice, attenuated pathological changes of hippocampal cells, reduced the level of malondialdehyde, and increased superoxide dismutase activity in serum or brain tissue as compared to model control. In addition, the Nrf2, p62 and LC3B-II proteins expression were increased, and p-Tau and Keap1 proteins were decreased in the hippocampus after AP and andrographolide treatment.Furthermore, AP increased aluminum maltolate-induced cell viability in PC12 cells. Silencing p62 could reverse the upregulation expression of Nrf2 and downregulation of Keap1 and Tau proteins induced by AP in aluminum maltolate-treated cells.ConclusionsAP had neuroprotective effects against aluminum -induced cognitive dysfunction or cytotoxicity, which was involved in the activation of the p62-keap1-Nrf2 pathway and may develop as therapeutic drugs for the treatment of AD. However, this study has certain limitations, further optimize the protocol or model and study the molecular mechanism of AP improving AD.

Development and characterization of a prototypic pan-amyloid clearing agent - a novel murine peptide-immunoglobulin fusion.

Systemic amyloidosis is a progressive disorder characterized by the extracellular deposition of amyloid fibrils and accessory proteins in visceral organs and tissues. Amyloid accumulation causes organ dysfunction and is not generally cleared by the immune system. Current treatment focuses on reducing amyloid precursor protein synthesis and slowing amyloid deposition. However, curative interventions will likely also require removal of preexisting amyloid deposits to restore organ function. Here we describe a prototypic pan-amyloid binding peptide-antibody fusion molecule (mIgp5) that enhances macrophage uptake of amyloid. The murine IgG1-IgG2a hybrid immunoglobulin with a pan amyloid-reactive peptide, p5, fused genetically to the N-terminal of the immunoglobulin light chain was synthesized in HEK293T/17 cells. The binding of the p5 peptide moiety was assayed using synthetic amyloid-like fibrils, human amyloid extracts and amyloid-laden tissues as substrates. Binding of radioiodinated mIgp5 with amyloid deposits in vivo was evaluated in a murine model of AA amyloidosis using small animal imaging and microautoradiography. The bioactivity of mIgp5 was assessed in complement fixation and in vitro phagocytosis assays in the presence of patient-derived amyloid extracts and synthetic amyloid fibrils as substrates and in the presence or absence of human serum. Murine Igp5 exhibited highly potent binding to AL and ATTR amyloid extracts and diverse types of amyloid in formalin-fixed tissue sections. In the murine model of systemic AA amyloidosis, 125I-mIgp5 bound rapidly and specifically to amyloid deposits in all organs, including the heart, with no evidence of non-specific uptake in healthy tissues. The bioactivity of the immunoglobulin Fc domain was uncompromised in the context of mIgp5 and served as an effective opsonin. Macrophage-mediated uptake of amyloid extract and purified amyloid fibrils was enhanced by the addition of mIgp5. This effect was exaggerated in the presence of human serum coincident with deposition of complement C5b9. Immunostimulatory, amyloid-clearing therapeutics can be developed by incorporating pan-amyloid-reactive peptides, such as p5, as a targeting moiety. The immunologic functionality of the IgG remains intact in the context of the fusion protein. These data highlight the potential use of peptide-antibody fusions as therapeutics for all types of systemic amyloidosis.

Cyclic di-peptide in situ inhibited protein-aggregation

An increasing number of neurodegenerative diseases seem to be associated with protein misfolding that often leads to misfolded protein aggregates with a β-sheet conformation and accumulation in the brain which directly contributes to or modulates the associated pathology. Protein aggregation diseases like Huntington’s disease results from the deposition of aggregated huntingtin proteins within the nucleus, transmissible prion encephalopathies occur due to extracellular deposition of pathogenic prion proteins whereas Alzheimer’s disease from the accumulation of both extracellular β-amyloid and intracellular hyperphosphorylated tau protein aggregates. In the generalized purpose, we have taken the core sequence of amyloid-β (responsible for its aggregation) as the aggregating peptide (AP). Among the various emerging therapeutic approaches against aggregation-related degenerative diseases such as diminishing the monomeric precursor protein, inhibiting aggregation, or blocking aggregation-induced cellular toxicity pathways, we focussed on the strategy based on the inhibition of protein aggregation using rationally designed peptide inhibitors comprising both the recognition and β-breaking component in the sequence. The “O → N acyl migration” concept was used to form cyclic peptide in situ for the generation of a bent unit which may act as disruption moiety for the inhibition process. The kinetics of aggregation was characterized by various biophysical tools (ThT-assay, TEM, CD, and FTIR). Results implied that the designed inhibitor peptides (IP) might be valuable to inhibit all the related aggregated peptides.

Extracellular protein homeostasis: The dawning of a new age for human disease therapies?

Extracellular protein homeostasis: The dawning of a new age for human disease therapies?

Arrhythmias in patients with cardiac amyloidosis

Cardiac amyloidosis is characterized by the extracellular deposition of fibrillar proteins in various heart parts, which can lead to restrictive heart failure and cardiac arrhythmias. In many cases, the incidence of both atrial and ventricular arrhythmias and conduction disorders is high and underestimated. The clinical picture often manifests itself only at the time of extensive involvement of the muscular and conduction system of the heart. Secondary to amyloid infiltration, bradyarrhythmias, and conduction disorders can occur, however, the timing of pacemaker implantation remains unclear. Many commonly used drugs may be poorly tolerated by patients with cardiac amyloidosis.

Diagnostic Challenges and Solutions in Systemic Amyloidosis

Amyloidosis refers to a clinically heterogeneous group of disorders characterized by the extracellular deposition of amyloid proteins in various tissues of the body. To date, 42 different amyloid proteins that originate from normal precursor proteins and are associated with distinct clinical forms of amyloidosis have been described. Identification of the amyloid type is essential in clinical practice, since prognosis and treatment regimens both vary according to the particular amyloid disease. However, typing of amyloid protein is often challenging, especially in the two most common forms of amyloidosis, i.e., the immunoglobulin light chain amyloidosis and transthyretin amyloidosis. Diagnostic methodology is based on tissue examinations as well as on noninvasive techniques including serological and imaging studies. Tissue examinations vary depending on the tissue preparation mode, i.e., whether it is fresh-frozen or fixed, and they can be carried out by ample methodologies including immunohistochemistry, immunofluorescence, immunoelectron microscopy, Western blotting, and proteomic analysis. In this review, we summarize current methodological approaches used for the diagnosis of amyloidosis and discusses their utility, advantages, and limitations. Special attention is paid to the simplicity of the procedures and their availability in clinical diagnostic laboratories. Finally, we describe new methods recently developed by our team to overcome limitations existing in the standard assays used in common practice.

What's New in Hand and Wrist Surgery.

What's New in Hand and Wrist Surgery.

Amyloid Beta Alters the Expression of microRNAs Regulating HMGCR and ABCA1 Genes in Astrocytes of C57BL/6J Mice.

Dysregulation of brain cholesterol homeostasis causes the accumulation of extracellular protein deposits called amyloid plaques in the hippocampus which eventually leads to neuronal death, memory and learning deficits. The aim of the present study was to investigate the effect of beta amyloid on miRNAs regulating HMGCR and ABCA1 as cholesterol synthesis and homeostasis genes. Primary astrocytes were isolated from C57BL/6J mice, and were treated with 0.5 μM amyloid beta (Aβ). Expression levels of genes and miRNAs were measured by real-time PCR. In comparison to control, Aβ treatment resulted in a significant decrease in miR-96-5p expression as a positive and negative regulator of HMGCR and ABCA1, respectively. There was no significant increase in miR-27a-3p expression as a negative regulator of HMGCR. miR- 106b- 5p and miR-143-3p expressions were also dramatically decreased as ABCA1 negative regulators. Amyloid beta can alter the expression of major genes in the cholesterol homeostasis pathway via their regulatory miRNAs.

Recent advancement in drugs of Alzheimer's disease

Alzheimer's disease (AD), a multifaceted neurological ailment that progresses over time, is the most common cause of dementia in older people. Intracellular neurofibrillary tangles and extracellular amyloidal protein deposits contribute to senile plaques on a pathological level. AD symptoms vary depending on the stage of the disease. Depending on the severity of cognitive impairment, AD is classified as preclinical or presymptomatic, mild, or dementia-stage. In addition, the condition is influenced by a number of risk factors, including advancing age, hereditary factors, head injuries, vascular diseases, infections, and environmental variables. There are currently only two types of licenced medications to treat AD: inhibitors of the cholinesterase enzyme such as donepezil, rivastigmine, galantamine and antagonists of N-methyl d-aspartate (NMDA) such as memantine, both of which are only effective in treating the symptoms of the disease and do not cure or prevent it.

A rare case of systemic AL-amyloidosis under the mask of myocardial infarction in the absence of myocardial hypertrophy: case report

Systemic amyloidosis is a group of diseases associated with extracellular deposition of fibrillar proteins, resulting in the loss of normal organ structure and function. AL-amyloidosis occurs when amyloid is deposited, consisting of full-length lambda or kappa immunoglobulin light chains, or fragments thereof. This article describes the complexity of timely diagnosis of systemic AL-amyloidosis with a predominant heart lesion in the absence of myocardial hypertrophy on the example of an elderly patient in whom the leading manifestation of the disease was heart failure with a preserved left ventricular ejection fraction.

S2203 A Rare Case of Amyloid-Induced Colonic Pseudo-Obstruction

Introduction: Colonic pseudo-obstruction is a rare complication of AL Amyloidosis that is described as a diffuse colonic dilation without underlying mechanical cause. In AL Amyloidosis, extracellular protein deposits disrupt tissue structure and function. Manifestations of colonic involvement of AL Amyloidosis include chronic diarrhea, weight loss, abdominal pain, and pseudo-obstruction. Here we present a patient with worsening abdominal pain and distention, found to have colonic pseudo-obstruction likely from AL Amyloidosis. Her symptoms improved with colonic decompression and with the utility of prokinetic agents. Case Description/Methods: A 79-year-old female presented with 3 days of progressively worsening abdominal pain and distention, with associated constipation. Her past medical history was significant for anemia, AL-Amyloidosis diagnosed 4 months prior to arrival, and PEG placement 3 weeks prior to arrival due to worsening dysphagia secondary to macroglossia. Vital signs were normal. She did not appear in acute distress and was hemodynamically stable. Pertinent examination findings included abdominal distention, decreased bowel sounds, and diffuse abdominal tenderness. Pertinent labs included WBC 5.6 k/uL, albumin 2.1 g/dL, AST 39 U/L, ALT 43 U/L, ALP 196 U/L, and total bilirubin 0.3 mg/dL. Clostridium difficile, giardia lamblia, salmonella, and stool occult blood were negative. CT abdomen and pelvis revealed gaseous distention of the transverse colon and diffuse colonic wall thickening, as displayed in figure 1. Colonoscopy revealed diffuse dilation of the transverse colon, with components of inflammation including friability and loss of vascularity. Decompression was successfully performed through the colonoscope. The patient tolerated the procedure well and had marked improvement of abdominal distention and pain. Her percutaneous tube feeds were reinitiated and tolerated well. (Figure) Discussion: Colonic pseudo-obstruction is a rare complication of AL Amyloidosis that is caused by deposits of protein in extracellular tissue. AL Amyloidosis is a multisystem disease that rarely presents with colonic manifestations. However, colonic manifestations may be the sole presentation of AL Amyloidosis. Colonic pseudo-obstruction remains a clinical challenge and can lead to bacterial overgrowth and malnutrition. Here we demonstrate successful air decompression of a colonic pseudo-obstruction due to AL Amyloidosis. We hope to increase awareness of AL Amyloidosis as an etiology colonic pseudo-obstruction.Figure 1.: CT Abdomen Pelvis reveals diffuse gaseous distention of the transverse colon with bowel wall thickening.

A Case of Laryngeal Amyloidosis: Role of US and US-CNB for Preoperative Diagnosis

Amyloidosis is a rare benign disease characterized by the extracellular deposition of nonsoluble fibrillar proteins (amyloids) within organs. Laryngeal amyloidosis (LA) accounts for only 9%-15% of all cases of amyloidosis. Since clinical manifestations and laryngoscopic findings often overlap with those of laryngeal cancer, it is challenging to differentiate LA from laryngeal cancer prior to surgical biopsy. We report a case of LA mimicking laryngeal cancer, in which the diagnosis was facilitated by preoperative ultrasonography (US) and US-guided core-needle biopsy (US-CNB) prior to surgical biopsy. The US findings of this case were distinguishable from those of laryngeal cancer, which enabled us to consider a diagnosis other than laryngeal cancer. Amyloidosis was diagnosed preoperatively using office-based percutaneous US-CNB, avoiding general anesthesia needed for suspension laryngoscopic examination. This case suggests that US and US-CNB could be used as supplementary diagnostic modalities to evaluate suspicious laryngeal masses mimicking laryngeal cancer.

Roles of NRF2 in Fibrotic Diseases: From Mechanisms to Therapeutic Approaches.

Fibrosis is a persistent inflammatory response that causes scarring and tissue sclerosis by stimulating myofibroblasts to create significant quantities of extracellular matrix protein deposits in the tissue. Oxidative stress has also been linked to the development of fibrosis in several studies. The nuclear erythroid 2-related factor 2 (NRF2) transcription factor controls the expression of several detoxification and antioxidant genes. By binding to antioxidant response elements, NRF2 is activated by oxidative or electrophilic stress and promotes its target genes, resulting in a protective effect on cells. NRF2 is essential for cell survival under oxidative stress conditions. This review describes Kelch-like epichlorohydrin-associated protein 1 (KEAP1)/NRF2 signaling mechanisms and presents recent research advances regarding NRF2 and its involvement in primary fibrotic lesions such as pulmonary fibrosis, hepatic fibrosis, myocardial fibrosis, and renal fibrosis. The related antioxidant substances and drugs are described, along with the mechanisms by which KEAP1/NRF2 regulation positively affects the therapeutic response. Finally, the therapeutic prospects and potential value of NRF2 in fibrosis are summarized. Further studies on NRF2 may provide novel therapeutic approaches for fibrosis.

Rare cause of thyroid enlargement: Localized AA amyloid goiter - A case report.

IntroductionAmyloidosis is extracellular deposition of fibrillary amyloid proteins in various organs. Amyloid infiltration in thyroid is common; however, the occurrence of clinically enlarged thyroid, subsequently leading to goiter, is a rare phenomenon.Case presentation36 years old female presented to our OPD with multinodular goiter. She subsequently underwent total thyroidectomy. Thyroidectomy specimen revealed amyloid deposition with characteristic congophilia and birefringence. Further evaluation revealed it to be AA amyloidosis. There were no features of systemic amyloidosis.DiscussionAmyloidosis is classified on the type of amyloid protein. Treatment of individual types of amyloidosis is diverse, and hence identification of the protein subtype is paramount.ConclusionAA amyloidosis localized primarily to thyroid is infrequent, as it usually occurs with chronic inflammatory conditions or infections. Currently, there are no guidelines for the treatment of localized AA amyloid goiter. We have evidence demonstrating the successful treatment of such a condition with no recurrence noted.

Neuromuscular Complications of Systemic Amyloidosis

Systemic amyloidosis is characterized by extracellular deposition of insoluble fibrillar proteins in multiple tissues, frequently at a distance from the site of synthesis. The 2 most common forms, light chain (AL) and transthyretin (ATTR) amyloidosis can cause peripheral neuropathy and, rarely, myopathy. Diagnosis can be challenging, and abundant suspicion is required to identify patients. As neurological manifestations of amyloidosis may precede involvement of other organs by several years, recognizing amyloid neuropathy and myopathy are crucial, especially in this new and exciting era of effective therapies for AL and ATTR neuropathy. This review will focus on the neuromuscular manifestations of AL and ATTR amyloidosis, diagnostic approaches, and recent advances in the treatment of amyloid neuropathy.

Renal Amyloidosis: A Clinicopathological Study From a Tertiary Care Hospital in Pakistan.

IntroductionSystemic amyloidosis can affect any organ in the body, but the kidney is the most commonly involved site. It is characterized by the extracellular deposition of insoluble fibrillar proteins. Amyloid deposits can be identified histologically by Congo red stain, which gives apple-green birefringence under polarized light. Typing of renal amyloidosis is done by direct immunofluorescence on frozen tissue. The most common types of amyloidosis seen in renal tissue are amyloid light chain (AL) primary amyloidosis and amyloid A (AA) secondary amyloidosis. Although primary amyloidosis is considered the most common type in western countries, however, in the subcontinent region, secondary amyloidosis is more common.The spectrum of signs and symptoms in renal amyloidosis is variable including isolated proteinuria, nephrotic syndrome, hypertension, hypotension, and renal insufficiency. The present study aims to evaluate the incidence and aetiology of various types of renal amyloidosis, determine their distribution within the kidney, and study various clinicopathological features.ObjectiveThe present study aims to evaluate the aetiology and clinicopathological profile of renal amyloidosis, determine its various types, and their distribution within the kidney.Materials and methodsThis retrospective cross-sectional study was conducted from 1st January 2013 to 31st December 2020 at the Department of Histopathology, Shifa International Hospital (SIH), Islamabad. All renal biopsies diagnosed as renal amyloidosis were included in the study. Data were analysed using SPSS version 23 (IBM Corp., Armonk, NY). Frequency and percentages were calculated for qualitative variables, and mean and standard deviation were calculated for quantitative variables.ResultsA total of 131 cases were diagnosed with renal amyloidosis during the study period of eight years (from 1st January 2013 to 31st December 2020) at SIH. The age range varied from 17 to 82 years. The mean age of the patients was 45 ± 16.33 years. Out of 131 patients, 82 (62.6%) were males and 49 (37.4%) were females. Amongst them, 72 (54%) cases were diagnosed with secondary AA amyloidosis and 16 (12%) cases were diagnosed with primary AL amyloidosis. The rest of the cases 43 (34%) were of indeterminate type. The associated conditions in secondary amyloidosis were tuberculosis in 41 (57%), rheumatoid arthritis in 16 (22%), ankylosing spondylitis in five (7%), lymphoma in three (4%), diabetes in two (3%), and chronic osteomyelitis, chronic heart disease, hepatitis, and vasculitis in one case each (1.7%). Out of 16 cases reported with AL amyloidosis, 10 cases (62.5%) had a history of multiple myeloma. The most common clinical presentation was nephrotic syndrome followed by subnephrotic proteinuria, renal failure, and hypertension.ConclusionThe findings of the present study show underlying etiological factors and clinicopathological characteristics of renal amyloidosis. AA amyloidosis is the most common type of renal amyloidosis in our study and tuberculosis is the most common aetiological factor. AL amyloidosis is less frequent in our population.

Carcinoma de lábio mimetizando queratoacantoma: relato de casos

ABSTRACT Oral amyloidosis is a disease characterized by extracellular and irreversible deposition of amorphous and fibrillar proteins in the oral cavity, being strongly associated with Multiple Myeloma. The objective of this study is to report a case of a 62-year-old woman diagnosed with Multiple Myeloma who, approximately 2 years after starting treatment for the underlying disease, presented a lesion on the lateral border of the tongue with exophytic growth, pinkish color, vascularized, painless, measuring 3cm in its largest diameter. After histopathological analysis through incisional biopsy, a final diagnosis of amyloidosis was obtained. As a local treatment, we opted for complete excision of the lesion. The patient evolved to death due to Multiple Myeloma influenced by the diagnosis of systemic amyloidosis. Oral amyloidosis is usually associated with the systemic presentation of the disease, making it necessary to conduct a thorough investigation of other organs. Its diagnosis is important since the prognosis is directly related and can negatively influence survival rates and treatment of the underlying disease.

Amiloidose lingual associada a mieloma múltiplo: relato de caso

ABSTRACT Oral amyloidosis is a disease characterized by extracellular and irreversible deposition of amorphous and fibrillar proteins in the oral cavity, being strongly associated with Multiple Myeloma. The objective of this study is to report a case of a 62-year-old woman diagnosed with Multiple Myeloma who, approximately 2 years after starting treatment for the underlying disease, presented a lesion on the lateral border of the tongue with exophytic growth, pinkish color, vascularized, painless, measuring 3cm in its largest diameter. After histopathological analysis through incisional biopsy, a final diagnosis of amyloidosis was obtained. As a local treatment, we opted for complete excision of the lesion. The patient evolved to death due to Multiple Myeloma influenced by the diagnosis of systemic amyloidosis. Oral amyloidosis is usually associated with the systemic presentation of the disease, making it necessary to conduct a thorough investigation of other organs. Its diagnosis is important since the prognosis is directly related and can negatively influence survival rates and treatment of the underlying disease.