Antidepressant effect of teriflunomide via oligodendrocyte protection in a mouse model.

Addressing the treatment of depression is crucial; nevertheless, the etiology and pathogenesis remain unelucidated. Therefore, this study investigated the effects of teriflunomide (TF) on corticosterone (CORT)-induced depression-like behaviors in mice. Notably, TF administration resulted in a substantial amelioration of anxiety and depression-like behaviors observed in CORT-treated mice. This was evidenced by behavioral assessments conducted via the sucrose preference test (SPT), open-field test (OFT), novelty-suppressed feeding test (NSFT), forced swimming test (FST), and tail suspension test (TST). The administration of CORT inflicts damage upon oligodendrocytes and neurons within the hippocampus. Our findings indicate that TF offers significant protective effects on oligodendrocytes, mitigating apoptosis both invivo and invitro. Additionally, TF was found to counteract the CORT-induced neuronal loss and synaptic damage, as demonstrated by an increase in Nissl-positive cells across hippocampal regions CA1, CA3, and the dentate gyrus (DG) alongside elevated levels of synapse-related proteins including PSD-95 and synaptophysin. Additionally, TF treatment facilitated a reduction in the levels of apoptosis-related proteins while simultaneously augmenting the levels of Bcl2. Our findings indicate that TF administration effectively mitigates CORT-induced depression-like behaviors and reverses damage to oligodendrocytes and neurons in the hippocampus, suggesting TF as a promising candidate for depression.

Adsorption characteristics and mechanism of ammonia nitrogen and phosphate from biogas slurry by Ca2+-modified soybean straw biochar.

The utilization of biogas slurry is critical for the sustainable development of animal husbandry. Biomass carbon adsorption is a feasible method for the recycling of nutrients from biogas slurry. However, research on the co-adsorption of ammonia nitrogen and phosphate is scarce. Herein, soybean straw was utilized as the raw material to prepare Ca2+-modified biochar (CaSSB), which was investigated for its ammonia nitrogen and phosphate adsorption mechanisms. Compared with natural biochar (SSB), CaSSB possesses a high H/C ratio, larger surface area, high porosity and various functional groups. Ca2+-modified soybean straw biochar exhibited excellent adsorption performance for NH4+-N (103.18 mg/g) and PO43--P (9.75 mg/g) at pH = 6, using an adsorbent dosage of 2 g/L. The experimental adsorption data of ammonia nitrogen by CaSSB corresponded to pseudo-second-order kinetics and the Langmuir isotherm model, suggesting that the adsorption process was homogeneous and that electrostatic attraction might be the primary adsorption mechanism. Meanwhile, the adsorption of phosphate conformed to pseudo-second-order kinetics and the Langmuir-Freundlich model, whose mechanism might be attributed to ligand exchange and chemical precipitation. These results reveal the potential of CaSSBs as a cost-effective, efficient adsorbent for the recovery of ammonium and phosphate from biogas slurry.

Improving Mitochondrial Function in Skeletal Muscle Contributes to the Amelioration of Insulin Resistance by Nicotinamide Riboside.

High-fat diet (HFD)-induced insulin resistance (IR) in skeletal muscle is often accompanied by mitochondrial dysfunction and oxidative stress. Boosting nicotinamide adenine dinucleotide (NAD) using nicotinamide riboside (NR) can effectively decrease oxidative stress and increase mitochondrial function. However, whether NR can ameliorate IR in skeletal muscle is still inconclusive. We fed male C57BL/6J mice with an HFD (60% fat) ± 400 mg/kg·bw NR for 24 weeks. C2C12 myotube cells were treated with 0.25 mM palmitic acid (PA) ± 0.5 mM NR for 24 h. Indicators for IR and mitochondrial dysfunction were analyzed. NR treatment alleviated IR in HFD-fed mice with regard to improved glucose tolerance and a remarkable decrease in the levels of fasting blood glucose, fasting insulin and HOMA-IR index. NR-treated HFD-fed mice also showed improved metabolic status regarding a significant reduction in body weight and lipid contents in serum and the liver. NR activated AMPK in the skeletal muscle of HFD-fed mice and PA-treated C2C12 myotube cells and upregulated the expression of mitochondria-related transcriptional factors and coactivators, thereby improving mitochondrial function and alleviating oxidative stress. Upon inhibiting AMPK using Compound C, NR lost its ability in enhancing mitochondrial function and protection against IR induced by PA. In summary, improving mitochondrial function through the activation of AMPK pathway in skeletal muscle may play an important role in the amelioration of IR using NR.

Associations between Adipokines and Metabolic Dysfunction-Associated Fatty Liver Disease Using Three Different Diagnostic Criteria.

BACKGROUND: A panel of experts proposed a new definition of metabolic dysfunction-associated fatty liver disease (MAFLD) in 2020. To date, the associations between adipokines, such as adiponectin, adipsin, and visfatin and MAFLD remain unclear. Therefore, we aimed to evaluate the associations between each of these three adipokines and MAFLD using different diagnostic criteria. METHODS: In total, 221 participants were included in our study based on medical examination. Detailed questionnaire information, physical examination, abdominal ultrasound, and blood-biochemical-test indexes were collected. The levels of adipokines were tested by using an enzyme immunoassay. Logistic regression models were used to assess the associations of the adipokines with MAFLD. RESULTS: In total, 122 of the participants were diagnosed with MAFLD. Higher levels of adipsin and lower levels of adiponectin were found in the MAFLD group than in the non-MAFLD group (all p < 0.05). According to the logistic regression analysis, the ORs were 0.11 (95% CI: 0.05-0.23) for adiponectin, 4.46 (95% CI: 2.19-9.12) for adipsin, and 0.51 (95% CI: 0.27-0.99) for visfatin when comparing the highest tertile with the lowest tertile (all p-trend < 0.05). The inverse association between adiponectin and MAFLD was strongest when T2DM was used as the diagnostic criterion alone, and the positive association between adipsin and MAFLD was strongest when BMI was used as the diagnostic criterion alone. There was no significant association between visfatin and MAFLD, regardless of whether each of BMI, T2DM, or metabolic dysregulation (MD) was used as the diagnostic criterion for MAFLD alone. CONCLUSION: Adipsin levels were positively associated with MAFLD and adiponectin levels were inversely associated with MAFLD. The strength of these associations varied according to the different diagnostic criteria for MAFLD.

Lipopolysaccharide-binding protein expression is increased by stress and inhibits monoamine synthesis to promote depressive symptoms.

Monoamine insufficiency is suggested to be associated with depressive features such as sadness, anhedonia, insomnia, and cognitive dysfunction, but the mechanisms that cause it are unclear. We found that the acute-phase protein lipopolysaccharide-binding protein (LBP) inhibits monoamine biosynthesis by acting as an endogenous inhibitor of dopamine-ß-hydroxylase (DBH) and aromatic-L-amino-acid-decarboxylase (DDC). LBP expression was increased in individuals with depression and by diverse stress challenges in mice. LBP antibodies and LBP knockdown inhibited monoamine insufficiency and depression-like features in mice, which worsened with LBP overexpression or administration. Monoamine insufficiency and depression-like symptoms were not induced by stressful stimuli in LBP-deficient mice, further highlighting a role for LBP in stress-induced depression, and a peptide we designed that blocks LBP-DBH and LBP-DDC interactions showed anti-depression effects in mice. This study reveals an important role for LBP in regulating monoamine biosynthesis and suggests that targeting LBP may have potential as a treatment for some individuals with depression.

Increased Serum Adipsin Correlates with MAFLD and Metabolic Risk Abnormalities.

Purpose: A panel of international experts proposed a new definition of fatty liver in 2020, namely metabolic dysfunction-associated fatty liver disease (MAFLD). As an adipokine, adipsin is closely related to metabolic-related diseases. In this study, we aimed to evaluate the relationship among MAFLD, serum adipsin, and metabolic risk abnormalities. Methods: Our study was a cross-sectional study based on the first follow-up of the Guangzhou Nutrition and Health Study (GNHS). A total of 908 patients with hepatic steatosis were involved in our study. Detailed data of patients were collected based upon questionnaire information, physical examination, and blood biochemical test. Results: Among the 908 patients, 789 patients were diagnosed with MAFLD. The levels of serum adipsin in the MAFLD group and non-MAFLD group were (3543.00 (3187.94-3972.50) ng/mL) and (3095.33 (2778.71-3354.77) ng/mL) (P < 0.001), respectively. After adjusting for potential confounders, adipsin levels were found to be associated with MAFLD. The OR was 3.46 (95% CI: 1.57-7.64) for adipsin when comparing subjects in the highest tertile with those in the lowest tertile. With the increase in the number of metabolic risk abnormalities, both the levels of serum adipsin and the proportion of moderate to severe fatty liver increased (all p-trend < 0.001). Conclusion: Increased serum adipsin correlates with MAFLD. Both adipsin levels as well as fatty liver severity increase with higher numbers of metabolic risk abnormalities.

Advanced glycation end products induce senescence of atrial myocytes and increase susceptibility of atrial fibrillation in diabetic mice.

Diabetes mellitus (DM) is a common chronic metabolic disease caused by significant accumulation of advanced glycation end products (AGEs). Atrial fibrillation (AF) is a common cardiovascular complication of DM. Here, we aim to clarify the role and mechanism of atrial myocyte senescence in the susceptibility of AF in diabetes. Rapid transesophageal atrial pacing was used to monitor the susceptibility of mice to AF. Whole-cell patch-clamp was employed to record the action potential (AP) and ion channels in single HL-1 cell and mouse atrial myocytes. More importantly, anti-RAGE antibody and RAGE-siRNA AAV9 were used to investigate the relationship among diabetes, aging, and AF. The results showed that elevated levels of p16 and retinoblastoma (Rb) protein in the atrium were associated with increased susceptibility to AF in diabetic mice. Mechanistically, AGEs increased p16/Rb protein expression and the number of SA-ß-gal-positive cells, prolonged the action potential duration (APD), reduced protein levels of Cav1.2, Kv1.5, and current density of ICa,L , IKur in HL-1 cells. Anti-RAGE antibody or RAGE-siRNA AAV9 reversed these effects in vitro and in vivo, respectively. Furthermore, downregulating p16 or Rb by siRNA prevented AGEs-mediated reduction of Cav1.2 and Kv1.5 proteins expression. In conclusion, AGEs accelerated atrial electrical remodeling and cellular senescence, contributing to increased AF susceptibility by activating the p16/Rb pathway. Inhibition of RAGE or the p16/Rb pathway may be a potential therapeutic target for AF in diabetes.

The Effect of Physical Exercise on the Elderly's Anxiety: Based on Systematic Reviews and Meta-Analysis.

Purpose: Based on meta-analysis to explore the effect of physical exercise on relieving the anxiety of the elderly. Methods: The retrieval time was published in the domestic and foreign literatures on the effect of physical exercise on the anxiety of the elderly published from 2005 to 2021. The random effects model was used to evaluate the mean standard deviation of the scores of the intervention group on reducing the anxiety level of the elderly before and after the test. According to the inclusion and exclusion criteria, the articles were screened, quality evaluated, and data extracted, and the literature was meta-analyzed by RevMan5.3. Results: In meta-analysis and systematic review, 17 papers finally met the inclusion criteria. After sensitivity analysis, the random effects model (MD = 8.00, 95% CI (6.90, 9.10), Z = 14.23 (P < 0.00001)) and the fixed effects model (MD = 7.71, 95% CI (6.98, 8.43), Z = 20.72 (P < 0.00001)) show that physical exercise has a positive and significant effect on the anxiety of the elderly. Conclusion: Physical exercise plays an important role in reducing the anxiety of the elderly. Therefore, regular physical exercise can be regarded as part of the elderly pension plan, but more high-quality research is needed to further explore the impact of physical exercise on elderly anxiety.

A Strategy for Efficient Preparation of Genus-Specific Diagnostic Antibodies for Snakebites.

As said by former United Nations Secretary-General Kofi Annan, "Snakebite is the most important tropical disease you've never heard of." Listed as a priority neglected tropical disease by the World Health Organization, snakebite envenoming (SBE) kills in excess of 125,000 people per year. However, due to the complexity and overlap of snake venom compositions, few reliable venom diagnostic methods for genus-/species-specific identification, which is crucial for successful SBE therapy, are available. Here, we develop a strategy to select and prepare genus-specific snake venom antibodies, which allows rapid and efficient clinical diagnosis of snakebite. Multi-omics approaches are used to choose candidate antigens from snake venoms and identify genus-specific antigenic epitope peptide fragments (GSAEPs) with ideal immunogenicity, specificity, and spatial accessibility. Double-antibody sandwich ELISA kit was established by matching a polyclonal antibody against a natural antigen and a monoclonal antibody that was prepared by natural protein as antigen and can specifically target the GSAEPs. The kit shows the ability to accurately identify venoms from similar genera of Trimeresurus and Protobothrops with a detection limit of 6.25 ng/ml on the snake venoms and a little cross-reaction, thus proving high feasibility and applicability.

Corrigendum: The Mechanisms and Boundary Conditions of Drug Memory Reconsolidation.

[This corrects the article DOI: 10.3389/fnins.2021.717956.].

The Mechanisms and Boundary Conditions of Drug Memory Reconsolidation.

Drug addiction can be seen as a disorder of maladaptive learning characterized by relapse. Therefore, disrupting drug-related memories could be an approach to improving therapies for addiction. Pioneering studies over the last two decades have revealed that consolidated memories are not static, but can be reconsolidated after retrieval, thereby providing candidate pathways for the treatment of addiction. The limbic-corticostriatal system is known to play a vital role in encoding the drug memory engram. Specific structures within this system contribute differently to the process of memory reconsolidation, making it a potential target for preventing relapse. In addition, as molecular processes are also active during memory reconsolidation, amnestic agents can be used to attenuate drug memory. In this review, we focus primarily on the brain structures involved in storing the drug memory engram, as well as the molecular processes involved in drug memory reconsolidation. Notably, we describe reports regarding boundary conditions constraining the therapeutic potential of memory reconsolidation. Furthermore, we discuss the principles that could be employed to modify stored memories. Finally, we emphasize the challenge of reconsolidation-based strategies, but end with an optimistic view on the development of reconsolidation theory for drug relapse prevention.

Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis.

A large number of protease inhibitors have been found from leeches, which are essential in various physiological and biological processes. In the curret study, a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis, which was named HMEI-A. Primary structure analysis showed that HMEI-A belonged to a new family of proteins. HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant (Ki) of 1.69 × 10-8 mol·L-1. Further study showed that HMEI-A inhibited the formation of neutrophil extracellular trap (NET). These results suggested that HMEI-A from the leech of H. manillensis is a novel elastase inhibitor which can suppress NET formation. It may play a significant role in blood-sucking of leeches and is a potential candidate as an anti-inflammatory agent.

Atractylenolide III reduces depressive- and anxiogenic-like behaviors in rat depression models.

Atractylenolide III, a major component of the atractylodes macrocephala Koidz, derived from the rhizoma atractylodes, has been reported to produce various pharmacological effects including anti-aging, anti-inflammation, anti-tumor, and other effects. Growing evidence suggests that proinflammatory cytokines, such as interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-α, are increased in depressed patients. The present study was aimed at investigating the antidepressant- and anxiolytic-like effects of atractylenolide III in lipopolysaccharide (LPS) challenge and chronic unpredictable mild stress (CUMS) rat model. We found that 30 mg/kg of atractylenolide III administered by oral gavage for 14 days, significantly reduced the immobility time in a forced swimming test (FST), but did not alter the number of crossings in an open field test (OFT), respectively. The results indicated that atractylenolide III has an antidepressant-like effect without affecting locomotor activity. We then used the LPS-induced depression model to assess the effects of atractylenolide III on behaviors in FST, sucrose preference test (SPT), and novelty-suppressed feeding test (NSFT). Interestingly, in addition to the antidepressant-like effects, 30 mg/kg of atractylenolide III also produced an anxiolytic-like effect. To further identify the antidepressant- and anxiolytic-like effects of atractylenolide III, we used the CUMS model with 28 consecutive days of the atractylenolide III treatment, followed by the SPT, FST, and NSFT. Atractylenolide III prevented CUMS-induced depressive- and anxiety-like behaviors in rats. To illustrate the underlying possible mechanisms of action of atractylenolide III, we measured the proinflammatory cytokines levels. The results showed that atractylenolide III decreased the proinflammatory cytokines levels in the hippocampus of CUMS exposed rats. In summary, our findings demonstrated that atractylenolide III produces antidepressant- and anxiolytic-like effects in rats, and these effects appear to be mediated by inhibition of hippocampal neuronal inflammation.

Blockade of ß-Adrenergic Receptors by Propranolol Disrupts Reconsolidation of Drug Memory and Attenuates Heroin Seeking.

Persistent traces of drug reward memories contribute to intense craving and often trigger relapse. A number of pharmacological interventions on drug-associated memories have shown significant benefits in relapse prevention at a preclinical level but their translational potential is limited due to deleterious side effects. Propranolol, a non-specific ß-adrenergic receptors antagonist, is known for its ability to erase maladaptive memories associated with nicotine or cocaine in rodents and humans. However, little is known about its effect on reconsolidation of heroin memory and heroin seeking. In the present study, rats with a history of intravenous heroin self-administration received the propranolol treatment (10 mg/kg; i.p.) at different time windows with or without CS (conditioned stimulus) exposure. Our results showed that propranolol, when administered immediately after CS exposure but not 6 h later, can significantly attenuate cue-induced and drug-primed reinstatement of heroin seeking, suggesting that propranolol has the ability to disrupt heroin memory and reduce relapse. The propranolol treatment without retrieval of drug memory had no effect on subsequent reinstatement of heroin seeking, suggesting that its interfering effects are retrieval-dependent. Importantly, the effects of propranolol were long lasting as rats showed diminished drug seeking even 28 days after the treatment. Altogether, our study suggests that propranolol can interfere with reconsolidation of heroin memory and reduce subsequent drug seeking, making it an attractive therapeutic candidate for the treatment of opioid addiction and relapse prevention.

Integrated genomic analysis reveals regulatory pathways and dynamic landscapes of the tRNA transcriptome.

tRNAs and tRNA-derived RNA fragments (tRFs) play various roles in many cellular processes outside of protein synthesis. However, comprehensive investigations of tRNA/tRF regulation are rare. In this study, we used new algorithms to extensively analyze the publicly available data from 1332 ChIP-Seq and 42 small-RNA-Seq experiments in human cell lines and tissues to investigate the transcriptional and posttranscriptional regulatory mechanisms of tRNAs. We found that histone acetylation, cAMP, and pluripotency pathways play important roles in the regulation of the tRNA gene transcription in a cell-specific manner. Analysis of RNA-Seq data identified 950 high-confidence tRFs, and the results suggested that tRNA pools are dramatically distinct across the samples in terms of expression profiles and tRF composition. The mismatch analysis identified new potential modification sites and specific modification patterns in tRNA families. The results also show that RNA library preparation technologies have a considerable impact on tRNA profiling and need to be optimized in the future.

Berberine Facilitates Extinction and Prevents the Return of Fear.

Exposure to a catastrophic event or intense stimulation can trigger fear memories, and the threatening memories persist even over a lifetime. Exposure therapy is based on extinction learning and is widely used to treat fear-related disorders, but its effect on remote fear memory is modest. Berberine, an isoquinoline alkaloid derived from Coptis chinensis or Berberis spp., has been recently reported to exert a diversity of pharmacological effects on the central nervous system, such as facilitating extinction of drug memory. Here, we explored the effect of berberine on extinction of fear memory using a classical contextual fear conditioning (CFC) paradigm, which is Pavlovian conditioning, can rapidly create fear memories related to contexts. Twenty-four hours or 30 days after CFC training, mice were subjected to context extinction (10 days) to extinguish their behaviors and treated with 12.5 or 25 mg/kg berberine intragastrically 1 or 6 h after each extinction session, followed by reinstatement and spontaneous recovery tests. The results showed that intragastric administration of 25 mg/kg berberine 1 h after extinction significantly promoted the extinction of recent and remote fear memories and prevented reinstatement and spontaneous recovery of extinguished fear in mice. These findings indicate that berberine combined with extinction training could serve as a promising novel avenue for the treatment of fear-related disorders.

Potential Role of Platelet-Activating C-Type Lectin-Like Proteins in Viper Envenomation Induced Thrombotic Microangiopathy Symptom.

Envenomation by viperid snakes may lead to severe bleeding, consumption coagulopathy, and thrombotic microangiopathy symptoms. The exact etiology or toxins responsible for thrombotic microangiopathy symptoms after snake envenomation remain obscure. Snake C-type lectin-like proteins (snaclecs) are one of the main non-enzymatic protein constituents in viper venoms, of which a majority are considered as modulators of thrombosis and hemostasis. In this study, we demonstrated that two snaclecs (mucetin and stejnulxin), isolated and identified from Protobothrops mucrosquamatus and Trimeresurus stejnegeri venoms, directly induced platelet degranulation and clot-retraction in vitro, and microvascular thrombosis has been confirmed in various organs in vivo. These snaclecs reduced cerebral blood flow and impaired motor balance and spatial memories in mice, which partially represent the thrombotic microangiopathy symptoms in some snakebite patients. The functional blocking of these snaclecs with antibodies alleviated the viper venom induced platelet activation and thrombotic microangiopathy-like symptoms. Understanding the pathophysiology of thrombotic microangiopathy associated with snake envenoming may lead to emerging therapeutic strategies.

Abnormal Ca2+ handling contributes to the impairment of aortic smooth muscle contractility in Zucker diabetic fatty rats.

Vascular dysfunction is a common pathological basis for complications in individuals affected by diabetes. Previous studies have established that endothelial dysfunction is the primary contributor to vascular complications in type 2 diabetes (T2DM). However, the role of vascular smooth muscle cells (VSMCs) in vascular complications associated with T2DM is still not completely understood. The aim of this study is to explore the potential mechanisms associated with Ca2+ handling dysfunction and how this dysfunction contributes to diabetic vascular smooth muscle impairment. The results indicated that endothelium-dependent vasodilation was impaired in diabetic aortae, but endothelium-independent vasodilation was not altered. Various vasoconstrictors such as phenylephrine, U46619 and 5-HT could induce vasoconstriction in a concentration-dependent manner, such that the dose-response curve was parallel shifted to the right in diabetic aortae, compared to the control. Vasoconstrictions mediated by L-type calcium (Cav1.2) channels were attenuated in diabetic aortae, but effects mediated by store-operated calcium (SOC) channels were enhanced. Intracellular Ca2+ concentration ([Ca2+]i) in VSMCs was detected by Fluo-4 calcium fluorescent probes, and demonstrated that SOC-mediated Ca2+ entry was increased in diabetic VSMCs. VSMC-specific knockout of STIM1 genes decreased SOC-mediated and phenylephrine-induced vasoconstrictive response in mice aortae. Additionally, Orai1 expression was up-regulated, Cav1.2 expression was downregulated, and the phenotypic transformation of diabetic VSMCs was determined in diabetic aortae. The overexpression of Orai1 markedly promoted the OPN expression of VSMCs, whereas SKF96365 (SOC channel blocker) reversed the phenotypic transformation of diabetic VSMCs. Our results demonstrated that the vasoconstriction response of aortic smooth muscle was weakened in type 2 diabetic rats, which was related to the downregulation of the Cav1.2 channel and the up-regulation of the SOC channel signaling pathway.

BCREval: a computational method to estimate the bisulfite conversion ratio in WGBS.

BACKGROUND: Whole genome bisulfite sequencing (WGBS) also known as BS-seq has been widely used to measure the methylation of whole genome at single-base resolution. One of the key steps in the assay is converting unmethylated cytosines into thymines (BS conversion). Incomplete conversion of unmethylated cytosines can introduce false positive methylation call. Developing a quick method to evaluate bisulfite conversion ratio (BCR) is benefit for both quality control and data analysis of WGBS. RESULTS: Here we provide a computational method named "BCREval" to estimate the unconverted rate (UCR) by using telomeric repetitive DNA as native spike-in control. We tested the method by using public WGBS data and found that it is very stable and most of BS conversion assays can achieve> 99.5% efficiency. The non-CpG DNA methylation at telomere fits a binomial model and may result from a random process with very low possibility (the ratio < 0.4%). And the comparison between BCREval and Bismark (Krueger and Andrews, Bioinformatics 27:1571-1572, 2011), a widely used BCR evaluator, suggests that our algorithm is much faster and more efficient than the latter. CONCLUSION: Our method is a simple but robust method to QC and speculates BCR for WGBS experiments to make sure it achieves acceptable level. It is faster and more efficient than current tools and can be easily integrated into presented WGBS pipelines.