Good manufacturing practices production of human placental derived mesenchymal stem cells for therapeutic applications: focus on multiple sclerosis.

BACKGROUND: Among neurological diseases, multiple sclerosis (MS) affects mostly young adults and can cause long-term disability. While most medications with approval from regulatory agencies are very effective in treating MS disease, they are unable to repair the tissue damage found in the central nervous system (CNS). Consequently, Cell-based therapy particularly using mesenchymal stem/stromal cells (MSCs), holds promise for neuroprotection and tissue repair in MS treatment. Furthermore, placenta-derived MSCs (PLMSCs) have shown the potential to treat MS due to their abundance, noninvasive isolation from discarded tissues, no ethical problems, anti-inflammatory, and reparative properties. Accordingly, good manufacturing practices (GMPs) plays a crucial part in clinical SCs manufacturing. The purpose of our article is to discuss GMP-grade PLMSC protocols for treating MS as well as other clinical applications. METHODS AND RESULTS: Placental tissue obtained of a healthy donor during the caesarean delivery and PLMSCs isolated by GMP standards. Flow cytometry was used to assess the expression of the CD markers CD34, CD105, CD90, and CD73 in the MSCs and the mesodermal differentiation ability was evaluated. Furthermore, Genetic evaluation of PLMSCs was done by G-banded karyotyping and revealed no chromosomal instability. In spite of the anatomical origin of the starting material, PLMSCs using this method of culture were maternal in origin. CONCLUSIONS: We hope that our protocol for clinical manufacturing of PLMSCs according to GMP standards will assist researchers in isolating MSCs from placental tissue for clinical and pre-clinical applications.

Characteristics and management of multiple sclerosis patients during the Omicron era: is there a concern about the MS course in the face of the new variant of COVID-19?

INTRODUCTION: The Omicron variant of COVID-19 is highly transmissible, triggering unprecedented infection rates. The present study aimed to investigate the course of multiple sclerosis (MS) in the Omicron era among Iranian patients with MS. METHODS: This observational study was designed on MS patients of the national MS registry of Iran through a self-designed online questionnaire. A questionnaire was prepared as a Google Form for MS patients during the Omicron outbreak from 1 March to 30 April 2022. RESULTS: One hundred seventy-four patients with a mean age of 37.3 ± 9.04 were enrolled. Of the patients, 95.97% used DMT, the most common of which were rituximab and fingolimod. Of the patients, 77.58% were fully vaccinated for COVID-19. Regardless of the COVID-19 vaccination status, 76 patients developed COVID-19, which was mild to moderate. Except for recent corticosteroid therapy and secondary progressive MS (SPMS), other demographic and MS characteristics were not significantly associated with the severity of COVID-19. There was also a marginal association between the Expanded Disability Status Scale (EDSS) and the severity of COVID-19. In addition, 17.10% of patients reported MS relapse following COVID-19 leading to escalation therapy in eight patients. CONCLUSION: Our study demonstrated that in the Omicron era, most patients developed mild COVID-19. Although the predominant COVID-19 variant in this period was Omicron, we could not separate the pathogenic variants. The risk factors for COVID-19 during the Omicron era were not different from other pandemic waves. Our preliminary results revealed that the MS relapse following COVID-19 was higher than in previous waves.

Radiological features of late-onset multiple sclerosis: A systematic review and meta-analysis.

BACKGROUND: Late-onset multiple sclerosis (LOMS) is most commonly defined as the onset of the disease's presentations at age 50 or older. There is still much to discover about the radiological features of LOMS. The current study aims to assess the imaging features of LOMS, as well as the correlation between these findings and the clinical characteristics of these patients. METHOD: This study was conducted following the PRISMA statement. A systematic search was conducted through PubMed, Scopus, and EMBASE databases to identify the studies that have applied magnetic-resonance imaging (MRI) or other imaging methods to investigate the radiological findings, as well as the relationship between them and clinical findings of LOMS patients. The risk of bias was assessed using the Joanna Briggs Institute (JBI) checklists. Meta-analysis was conducted using the third version of the compressive meta-analysis software (CMA3). RESULTS: Our search identified 753 unique titles. Among them, 15 studies, including seven case-control, five case-series, and three cross-sectional studies, met the eligibility criteria. According to the quantitative synthesis, brain lesions were detected among 72.2% of LOMS patients (4 studies; 95% CI: 67.0% - 93.1%). In the context of spinal lesions, overall spinal cord involvement was 64.0% (8 studies; 95% CI: 42.5% - 81.1%). Based on the available evidence, supratentorial involvement was found in 82.7% of cases (3 studies; 95% CI: 17.4% - 99.1%), juxtacortical involvement in 34.1% (3 studies; 95% CI: 26.4% - 42.7%), infratentorial involvement in 51.3% (4 studies; 95% CI: 32.1% - 70.1%), and cerebellar involvement in 18.5% (3 studies; 95% CI: 13.9% - 24.1%). CONCLUSION: Based on the neuroimaging findings, we found that, given the heterogeneity of MS, LOMS patients have a high rate of spinal cord lesions and supratentorial involvement. The limited available evidence suggests that Barkhof criteria are the best compromise for the diagnosis of LOMS. There is still a need for future studies.

Depression, anxiety, and fear of COVID-19 in patients with multiple sclerosis in pandemic era: a cross-sectional study.

BACKGROUND: Depression and anxiety are the two important factors determining quality of life of patients with multiple sclerosis (PWMS). In COVID-19 pandemic era, several factors can provoke mental issues of people and patients. In this cross-sectional study, we aim to estimate the new prevalence of anxious and depressive symptoms and their relating factors in PWMS. METHODS: In this cross-sectional study, we include PWMS who are recruited in the MS clinic of Sina Hospital, Tehran, and are joined in our channel of Telegram media. A self-designed online questionnaire consisted of 4 parts handed out between patients: demographic and clinical data, Beck depression inventory, Beck anxiety inventory, and Fear of COVID-19 Scale. Univariate and multiple logistic regression analyses were performed to find the relating factors of expression of depressive and anxious symptoms in PWMS. RESULTS: Of a total of 282 participants with the mean age of 35.66 (30.75-40) years, had been suffering from multiple sclerosis for 7.36 (3-10) years, 81.7% were women and 69.1% classified as relapsing-remitting MS. Mean score of BDI was 17.13 ± 11.51 which is classified as minimal-moderate depressive symptoms. 48.6% of patients did not express depressive symptoms (BDI-II ≤ 14) and the others reported some degrees of depression. In the univariate analysis employment (p = 0.015), marital status (p = 0.022), level of education (p = 0.004), number of hospitalization due to MS attacks (p = 0.048), and fear of COVID-19 (p ≤ 0.0001) associated significantly with presence of depressive symptoms. After entering these factors in a binary logistic regression model, level of education (p = 0.019), marital status (p = 0.044), number of hospital admissions due to MS relapses (ß = 1.10, p = 0.02), and fear of COVID-19 (ß = 1.07, p ≤ 0001) remained significant as relating factors. Mean score of the anxiety calculated 14.54 ± 9.75 and just 3.2% of patients had severe anxiety. Employment (p = 0.045), EDSS score (p = 0.004), and fear of COVID-19 (p ≤ 0.0001) reported relating to anxious symptoms significantly in the univariate analysis. After entering in the logistic regression analysis, EDSS (ß = 1.30, p = 0.001) and fear of COVID-19 (ß = 1.13, p ≤ 0.0001) remained as significant relating factors of anxious symptoms. CONCLUSION: The overall prevalence of depressive symptoms in PWMS in our MS clinic is 51.4% which is obviously higher than other world's centers which could be due to fear of COVID-19. In addition to fear of COVID-19, presence of depressive symptoms in PWMS is related significantly with level of education, number of hospital admissions due to MS relapses, and marital status. Other side, the patients classified as suffering from anxious symptoms had more severe problems on fear of COVID-19. But it is recommended for future studies to compare patients score in the COVID-19 era with their score before this pandemic.

[18F]FDG brain PET and clinical symptoms in different autoantibodies of autoimmune encephalitis: a systematic review.

INTRODUCTION: Autoimmune encephalitis (AE) is caused by the antibodies that target receptors and intracellular or surface proteins. To achieve the appropriate therapeutic results, early and proper diagnosis is still the most important issue. In this review, we provide an overview of FDG-PET imaging findings in AE patients and possible relation to different subtypes and clinical features. METHODS: PubMed, Web of Science, and Scopus were searched in August 2021 using a predefined search strategy. RESULTS: After two-step reviewing, 22 studies with a total of 332 participants were entered into our qualitative synthesis. In anti-NMDAR encephalitis, decreased activity in the occipital lobe was present, in addition, to an increase in frontal, parietal, and specifically medial temporal activity. Anti-VGKC patients showed altered metabolism in cortical and subcortical regions such as striata and cerebellum. Abnormal metabolism in patients with anti-LGI1 has been reported in diverse areas of the brain including medial temporal, hippocampus, cerebellum, and basal ganglia all of which had hypermetabolism. Hypometabolism in parietal, frontal, occipital lobes, temporal, frontal, and hippocampus was observed in AE patients with anti-GAD antibodies. CONCLUSION: Our results indicate huge diversity in metabolic patterns among different AE subtypes and it is hard to draw a firm conclusion. Moreover, the timing of imaging, seizures, and acute treatments can alter the PET patterns strongly. Further prospective investigations with specific inclusion and exclusion criteria should be carried out to identify the metabolic defect in different AE subtypes.

Exome sequencing reveals novel rare variants in Iranian familial multiple sclerosis: The importance of POLD2 in the disease pathogenesis.

The prevalence of familial multiple sclerosis (FMS) is increasing worldwide which endorses the heritability of the disease. Given that many genome variations are ethnicity-specific and consanguineous marriage could affect genetic diseases, hereditary disease gene analysis among FMS patients from Iran, a country with high rates of parental consanguinity, could be highly effective in finding mutations underlying disease pathogenesis. To examine rare genetic mutations, we selected three Iranian FMS cases with ≥3 MS patients in more than one generation and performed whole exome sequencing. We identified a homozygous rare missense variant in POLD2 (p. Arg141Cys; rs372336011). Molecular dynamics analysis showed reduced polar dehydration energy and conformational changes in POLD2 mutant. Further, we found a heterozygote rare missense variant in NBFP1 (p. Gly487Asp; rs778806175). Our study revealed the possible role of novel rare variants in FMS. Molecular dynamic simulation provided the initial evidence of the structural changes behind POLD2 mutant.

The possible role of Interleukin-6 as a regulator of insulin sensitivity in patients with neuromyelitis optica spectrum disorder.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is associated with inflammatory mediators that may also trigger downstream signaling pathways leading to reduce insulin sensitivity. METHODS: We aimed to determine the risk association of hyperinsulinemia in NMOSD patients with seropositive AQP4-IgG and the serum levels of interleukin (IL)-6 and IL-17A compared with the control group. Serum levels of metabolic (Insulin, Fasting Blood Sugar (FBS), lipid profile) and inflammatory (IL-6 and IL-17) markers were assessed in 56 NMOSD patients and 100 controls. RESULTS: Hyperinsulinemia was more prevalent in NMOSD patients independent of age, sex and body mass index (BMI) (48.2% vs. 26%, p = 0.005) compared to control group. After adjusting age, sex and BMI, there was significant association between lower insulin sensitivity (IS) and NMOSD risk (95% CI: Beta = 0.73, 0.62 to 0.86, p = 0.0001). Circulating levels of IL-6 and IL-17 were higher in NMOSD patients, and only IL-6 had an effect modifier for the association between lower insulin sensitivity and NMOSD risk. CONCLUSIONS: Our data suggests that inflammatory pathogenesis of NMOSD leads to hyperinsulinemia and increases the risk of insulin resistance.

Encephalopathy associated with COVID-19 in a patient with multiple sclerosis.

From the beginning of COVID-19 pandemics, the involvement of patient's nervous system with this virus is increasingly reporting. Although various reports are published on affliction of multiple sclerosis (MS) patients with SARS-CoV-2, no report has been published on brain involvement by this virus in MS patients so far. Herein, a 34-year-old patient with MS who experienced the decreased level of consciousness and encephalopathy following COVID-19 involvement has been reported.

Convergent effects of a functional C3 variant on brain atrophy, demyelination, and cognitive impairment in multiple sclerosis.

BACKGROUND: Complement system activation products are present in areas of neuroinflammation, demyelination, and neurodegeneration in brains of patients with multiple sclerosis (MS). C3 is a central element in the activation of complement cascades. A common coding variant in the C3 gene (rs2230199, C3R102G) affects C3 activity. OBJECTIVES: To assess the effects of rs2230199 on MS severity using clinical, cognitive, and imaging measures. METHODS: In total, 161 relapse-onset MS patients (Expanded Disability Status Scale (EDSS) ≤ 6) underwent physical assessments, cognitive tests (Paced Auditory Serial Addition Test (PASAT), Symbol Digit Modalities Test (SDMT), and California Verbal Learning Test (CVLT)), and magnetic resonance imaging (MRI). Lesion volumes were quantified semi-automatically. Voxel-wise analyses were performed to assess the effects of rs2230199 genotype on gray matter (GM) atrophy ( n = 155), white matter (WM) fractional anisotropy (FA; n = 105), and WM magnetization transfer ratio (MTR; n = 90). RESULTS: While rs2230199 minor-allele dosage (C3-102G) showed no significant effect on EDSS and Multiple Sclerosis Functional Composite (MSFC), it was associated with worse cognitive performance ( p = 0.02), lower brain parenchymal fraction ( p = 0.003), and higher lesion burden ( p = 0.02). Moreover, voxel-wise analyses showed lower GM volume in subcortical structures and insula, and lower FA and MTR in several WM areas with higher copies of rs2230199 minor allele. CONCLUSION: C3-rs2230199 affects white and GM damage as well as cognitive impairment in MS patients. Our findings support a causal role for complement system activity in the pathophysiology of MS.

Therapeutic Use of Intrathecal Mesenchymal Stem Cells in patients with Multiple Sclerosis: A Pilot Study with Booster Injection.

BACKGROUND: Mesenchymal stem cells (MSCs) are a potential therapy for various diseases. Here, the results of intrathecal injection of MSCs in multiple sclerosis (MS) patients are reported. MATERIALS AND METHODS: Four patients were enrolled in the study. Three were male and one was female. There were three secondary-progressive MS patients and one relapsing-remitting MS patient. An amount of 50-80 ml of bone marrow was collected from the patients. MSC cultures were collected for each microbiological examination at each change of medium and passage as well as at the time of sample injection. Then, MSCs were injected into the patients by the intrathecal method. In two patients, the injection was replicated in 1 year. RESULTS: All the patients were followed up for 2 years. Three patients who had secondary progression did not show disease progress after the injection, and the disease entered a stable state. A degree of recovery was observed in two patients. The relapsing-remitting patient suffered an attack that led to corticosteroid injection. None of the patients reported side effects. In terms of magnetic resonance imaging, there were no new plaques or enhanced plaques. CONCLUSION: This study suggests that injection of MSC can be a suitable method, especially for secondary-progressive patients. It seems that reinjection of these stem cells can be safe and sustaining it positively increases the effects of this therapeutic approach.

Sleep Disorder as a Triggering Factor for Relapse in Multiple Sclerosis.

BACKGROUND/AIMS: To determine the effect of sleep disturbances on predisposing multiple sclerosis (MS) patients for acute relapse. METHODS: This case-control study was conducted on 80 MS patients including 40 patients in the remission phase and 40 in the relapse phase. Patients were asked to fill in the Pittsburgh Sleep Quality Index to determine their sleep quality during the previous month. Individuals with scores of 5 or less were considered having normal sleep quality. RESULTS: Mean ± SD ages were 32.5 ± 7.7 and 30.2 ± 7.2 years among patients with and without acute relapses, respectively (p > 0.05). The mean disease duration and disease severity (according to Expanded Disability Status Scale [EDSS]) were comparable across the groups (p > 0.05). Among those with and without acute exacerbations, 87.5 and 50% had poor sleep quality, respectively (p = 0.0001), with OR of 1.75 (95% CI 1.25-2.43). The age, gender, EDSS, and disease duration did not associate with sleep quality in either groups (p > 0.05). CONCLUSIONS: This study showed that sleep disturbance might be a trigger for an acute MS exacerbation. Increasing the awareness of specialists and routine screening of sleep disorders in MS patients are warranted, as treatment of these disorders might decrease the likelihood of acute relapses.

Beyond the division of multiple sclerosis into different subgroups: The Concept of Connectomopathy.

Multiple Sclerosis (MS) pathophysiologically is a dynamic and progressive disease that involves all parts of central nervous system. This widespread involvement of the CNS has paved the way for proposing a new theory in MS in which MS is considered as a connectomopathy. Connectomopathy is a new concept describing the diseases in which not only the brain connectome is completely and extensively damaged, but the defective connectome itself can also become a breeding ground for the disease's progression. Connectomopathy provides a dynamic picture of MS. Since each person's connectome is unique to him/herself, so MS patients' connectomopathy varies from one to another. This variety not only challenges the classification of MS into different phenotypes, but also emphasizes the need for providing a personalized approach for the treatment of these patients.

Chronic periodontitis in a patient with multiple sclerosis-A case report.

Multiple sclerosis (MS) is one of the common central nervous system diseases, but it can cause dysfunction in other organs such as periodontal tissues. However, it has not been as noticeable. This report aimed to present a 44-year-old patient with severe chronic periodontitis, and sudden teeth loss since the diagnosis of MS.

Focal central neurological disorder in patients with autoimmune thyroid disease without encephalopathy.

The report underscores the necessity for a comprehensive evaluation in patients with suggestive laboratory findings or AITD history. Prompt diagnosis and appropriate management are imperative in averting long-term complications.

Effects of robotic rehabilitation on fatigue experience, disability, and quality of life in patients with multiple sclerosis (MS): A systematic review and meta-analysis.

Background: Rehabilitation plays an important role in improving symptoms in patients with multiple sclerosis (MS). There are studies evaluating the effects of robotic rehabilitation in patients with MS, but the results varied between the studies. So, we designed this systematic review and meta-analysis to estimate pooled effects of robotic rehabilitation on fatigue, disability, and quality of life in subjects with MS. Methods: We systematically searched PubMed, Scopus, EMBASE, Web of Science, Google Scholar, and also gray literature including references of the included studies, and also conference abstracts on October 1th 2022. Data regarding the total number of participants, first author, publication year, country of origin, mean age, EDSS, and results of fatigue and quality of life were recorded. Results: The first literature search revealed 6878 results, after deleting duplicates, 5019 studies remained. Two researchers, evaluated the titles and abstracts, and finally 77 full texts were assessed. For meta-analysis, we included 11 studies. The pooled Standardized Mean Difference (SMD) of Kurtzke Expanded Disability Status Scale (EDSS) (after-before) estimated as -0.56 (95%CI: -0.89,-0.23). The pooled SMD of Fatigue Severity Scale (FSS) estimated as -0.54(95%CI: -1.06, -0.01) (I2=66.7%, P=0.01). The pooled SMD of physical health subscale of multiple sclerosis quality of life (MSQOL-54) estimated as 0.36(95%CI:-0.23, 0.96) (I2=51.4%, P=0.1). The pooled SMD of mental health subscale of MSQOL54 estimated as 0.48 (95%CI: 0.07, 0.88) (I2=0%, P=0.6). Conclusions: The results of this systematic review and meta-analysis show that robotic rehabilitation has positive effects on fatigue, and disability in patients with MS.

The hidden link: How oral and respiratory microbiomes affect multiple sclerosis.

BACKGROUND: Extensive research has explored the role of gut microbiota in multiple sclerosis (MS). However, the impact of microbial communities in the oral cavity and respiratory tract on MS is an emerging area of investigation. PURPOSE: We aimed to review the current literature related to the nasal, oral, and lung microbiota in people with MS (PwMS). METHODS: We conducted a narrative review of clinical and preclinical original studies on PubMed that explored the relationship between the bacterial or viral composition of the nasal, lung, and oral microbiota and MS. Additionally, to find relevant studies not retrieved initially, we also searched for references in related review papers, as well as the references cited within the included studies. RESULTS AND CONCLUSIONS: Thirteen studies were meticulously reviewed in three sections; oral microbiota (n = 8), nasal microbiota (n = 3), and lung microbiota (n = 2), highlighting considerable alterations in the oral and respiratory microbiome of PwMS compared to healthy controls (HCs). Genera like Aggregatibacter and Streptococcus were less abundant in the oral microbiota of PwMS compared to HCs, while Staphylococcus, Leptotrichia, Fusobacterium, and Bacteroides showed increased abundance in PwMS. Additionally, the presence of specific bacteria, including Streptococcus sanguinis, within the oral microbiota was suggested to influence Epstein-Barr virus reactivation, a well-established risk factor for MS. Studies related to the nasal microbiome indicated elevated levels of specific Staphylococcus aureus toxins, as well as nasal glial cell infection with human herpes virus (HHV)-6 in PwMS. Emerging research on lung microbiome in animal models demonstrated that manipulating the lung microbiome towards lipopolysaccharide-producing bacteria might suppress MS symptoms. These findings open avenues for potential therapeutic strategies. However, further research is crucial to fully understand the complex interactions between the microbiome and MS. This will help identify the most effective timing, bacterial strains, and modulation techniques.

Investigating the Correlation Between Cognitive Function and Fasting Blood Sugar, Fasting Insulin Level and Insulin Sensitivity in Patients With Multiple Sclerosis.

INTRODUCTION: There has been a surge in interest in identifying the factors that impact cognitive impairment (CI) in patients with multiple sclerosis (MS). The purpose of our study was to examine the correlation between fasting blood sugar (FBS), fasting insulin level, as well as insulin sensitivity and cognitive function in patients with MS. MATERIAL AND METHODS: A total of 85 patients with MS enrolled in this cross-sectional study. Insulin sensitivity (IS) was determined using the quantitative insulin sensitivity check index (Quicki) formula. Cognitive function was evaluated using the Persian version of the Brief International Cognitive Assessment for MS (BICAMS). Spearman correlation test was employed to examine the correlation between cognition and FBS, insulin and IS. RESULTS: The mean ± SD age of the participants was 39.4 ± 10.2 years, and 62 (72.9%) were female. The participants had a FBS level of 87.05 ± 11.73 mg/dL, insulin level of 10.14 ± 7.57 µU/mL and a Quicki index of 0.36 ± 0.05. A higher score on the BVMT-R and BVMT-R-Delayed subtests showed a significant negative correlation with FBS (r: -0.32; p: 0.003 and r: -0.31; p: 0.004, respectively). Conversely, a significant negative correlation (r: -0.24; p: 0.031) was observed between higher fasting insulin levels and the CVLT_II score. IS showed a positive correlation with the CVLT-II (r: 0.24; p: 0.027) and BVMT_R (r: 0.21; p: 0.054) subtests. CONCLUSION: Our data indicate that elevated fasting glucose, developed fasting insulin levels and reduced insulin sensitivity may serve as potential predictors for CI in patients with MS.

Effects of non-pharmacological interventions on gait and balance of persons with Multiple Sclerosis: A narrative review.

BACKGROUND: Multiple Sclerosis (MS) is among the most common reasons for disability in young adults. Mobility impairment, primarily related to gait and balance, is ranked as the preeminent concern among persons with MS (PwMS). Gait and balance dysfunction can directly affect the quality of life and activities of daily life in PwMS, hence the importance of effective treatment strategies. Previous studies have demonstrated the positive effect of various non-pharmacological rehabilitation methods, including physiotherapy and electrical stimulation, on gait and mobility in PwMS. Non-pharmacological methods can be tailored to the individual needs and abilities of each patient, allowing healthcare providers to create personalized training programs. Furthermore, these methods typically result in minimal or no side effects. PURPOSE: This review provides a comprehensive overview of an array of non-pharmacological treatment approaches aimed at enhancing ambulatory performance in PwMS. METHODS: We performed a narrative review of the original papers available in PubMed, investigating the effects of different nonmedical approaches on the gait and balance performance of the PwMS. Reviewed treatment approaches include "exercise, physical rehabilitation, dual-task (DT) rehabilitation, robot-assisted rehabilitation, virtual reality-assisted rehabilitation, game training, electrical stimulation devices, auditory stimulation, visual feedback, and shoe insoles". RESULTS AND CONCLUSIONS: Eighty articles were meticulously reviewed. Our study highlights the positive effects of non-pharmacological interventions on patients' quality of life, reducing disability, fatigue, and muscle spasticity. While some methods, including exercise and physiotherapy, showed substantial promise, further research is needed to evaluate whether visual biofeedback and auditory stimulation are preferable over conventional approaches. Additionally, approaches such as functional electrical stimulation, non-invasive brain stimulation, and shoe insoles demonstrate substantial short-term benefits, prompting further investigation into their long-term effects. Non-pharmacological interventions can serve as a valuable complement to medication-based approaches.