The nematode (Ascaris suum) intestine is a location of synergistic anthelmintic effects of Cry5B and levamisole.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes.

An extracellular scaffolding complex confers unusual rectification upon an ionotropic acetylcholine receptor in C. elegans.

Biophysical properties of ligand-gated receptors can be profoundly modified by auxiliary subunits or by the lipid microenvironment of the membrane. Hence, it is sometimes challenging to relate the properties of receptors reconstituted in heterologous expression systems to those of their native counterparts. Here we show that the properties of Caenorhabditis elegans levamisole-sensitive acetylcholine receptors (L-AChRs), the ionotropic acetylcholine receptors targeted by the cholinergic anthelmintic levamisole at neuromuscular junctions, can be profoundly modified by their clustering machinery. We uncovered that L-AChRs exhibit a strong outward rectification in vivo, which was not previously described in heterologous systems. This unusual feature for an ionotropic AChR is abolished by disrupting the interaction of the receptors with the extracellular complex required for their synaptic clustering. When recorded at -60 mV, levamisole-induced currents are similar in the wild type and in L-AChR-clustering-defective mutants, while they are halved in these mutants at more depolarized physiological membrane potentials. Consequently, levamisole causes a strong muscle depolarization in the wild type, which leads to complete inactivation of the voltage-gated calcium channels and to an irreversible flaccid paralysis. In mutants defective for L-AChR clustering, the levamisole-induced depolarization is weaker, allowing voltage-gated calcium channels to remain partially active, which eventually leads to adaptation and survival of the worms. This explains why historical screens for C. elegans mutants resistant to levamisole identified the components of the L-AChR clustering machinery, in addition to proteins required for receptor biosynthesis or efficacy. This work further emphasizes the importance of pursuing ligand-gated channel characterization in their native environment.

Levamisole in childhood idiopathic nephrotic syndrome: new promises, and advocacy for global access.

In the current issue of Kidney International, Sinha et al. present data from an open-label, noninferior, randomized controlled trial comparing 12-months of alternate-day prednisolone, given daily during infection, versus levamisole, in children with frequently relapsing or steroid-dependent nephrotic syndrome. This study suggests that both of these strategies are efficacious and safe. Results of this study should redefine the role of levamisole in future guidelines, and a call for global availability of levamisole should be advocated.

An open label non-inferiority randomized controlled trial evaluated alternate day prednisolone given daily during infections vs. levamisole in frequently relapsing nephrotic syndrome.

Initial therapies for children with frequently relapsing nephrotic syndrome include alternate-day prednisolone that is given daily during infections, or levamisole. In this open label, non-inferiority trial, 160 patients, 2 to 18-years-old with frequent relapses, were randomly assigned to receive either prednisolone (0.5-0.7 mg/kg/alternate-day, given daily during infections), or levamisole (2-2.5 mg/kg/alternate-days) for one-year. Patients with relapses on alternate day prednisolone at over 1 mg/kg, prior use of potent steroid-sparing therapies, eGFR under 60 ml/min/1.73 m2 and significant steroid toxicity were excluded. Primary outcome was the proportion of patients with frequent relapses, defined as three-relapses in one-year, or two-relapses within six-months if associated with significant steroid toxicity or loss to follow up. Eighty patients each were randomized to receive prednisolone and levamisole. Baseline features showed preponderance of young patients presenting within two-years of disease onset. On intention-to-treat analysis, frequent relapses were more common in patients administered prednisolone (40% versus 22.5%; risk difference 17.5%; 95% confidence interval 3.4-31.6%). Prednisolone was not non-inferior to levamisole in preventing frequent relapses. However, the two groups showed similar proportions of patients in sustained remission, comparable frequency of relapses, and low frequency of adverse events. The decline in steroid requirement from baseline was higher in the levamisole group. Per-protocol analysis showed similar results. These results have implications for choice of therapy for frequently relapsing nephrotic syndrome. Although therapy with alternate-day prednisolone was not non-inferior to levamisole in preventing frequent relapses, both therapies were effective in other outcome measures. Thus, levamisole was relatively steroid-sparing and may be preferred in patients at risk of steroid toxicity.

Levamisole suppresses activation and proliferation of human T cells by the induction of a p53-dependent DNA damage response.

Levamisole (LMS) is a small molecule used in the treatment of idiopathic nephrotic syndrome (INS). The pathogenesis of INS remains unknown, but evidence points toward an immunological basis of the disease. Recently, LMS has been shown to increase the relapse-free survival in INS patients. While LMS has been hypothesized to exert an immunomodulatory effect, its mechanism of action remains unknown. Here, we show that LMS decreased activation and proliferation of human T cells. T-cell activation-associated cytokines such as IL-2, TNF-α, and IFN-γ were reduced upon LMS treatment, whereas IL-4 and IL-13 were increased. Gene expression profiling confirmed that the suppressive effects of LMS as genes involved in cell cycle progression were downregulated. Furthermore, genes associated with p53 activation were upregulated by LMS. In agreement, LMS treatment resulted in p53 phosphorylation and increased expression of the p53 target gene FAS. Accordingly, LMS sensitized activated T cells for Fas-mediated apoptosis. LMS treatment resulted in a mid-S phase cell cycle arrest accompanied by γH2AX-foci formation and phosphorylation of CHK1. Our findings indicate that LMS acts as an immunosuppressive drug that directly affects the activation and proliferation of human T cells by induction of DNA damage and the activation of a p53-dependent DNA damage response.

LIPCAR levels in plasma-derived extracellular vesicles is associated with left ventricle remodeling post-myocardial infarction.

BACKGROUND: Long Intergenic noncoding RNA predicting CARdiac remodeling (LIPCAR) is a long noncoding RNA identified in plasma of patients after myocardial infarction (MI) to be associated with left ventricle remodeling (LVR). LIPCAR was also shown to be a predictor of early death in heart failure (HF) patients. However, no information regarding the expression of LIPCAR and its function in heart as well as the mechanisms involved in its transport to the circulation is known. The aims of this study are (1) to characterize the transporter of LIPCAR from heart to circulation; (2) to determine whether LIPCAR levels in plasma isolated-extracellular vesicles (EVs) reflect the alteration of its expression in total plasma and could be used as biomarkers of LVR post-MI. METHODS: Since expression of LIPCAR is restricted to human species and the limitation of availability of cardiac biopsy samples, serum-free conditioned culture media from HeLa cells were first used to characterize the extracellular transporter of LIPCAR before validation in EVs isolated from human cardiac biopsies (non-failing and ischemic HF patients) and plasma samples (patients who develop or not LVR post-MI). Differential centrifugation at 20,000g and 100,000g were performed to isolate the large (lEVs) and small EVs (sEVs), respectively. Western blot and nanoparticle tracking (NTA) analysis were used to characterize the isolated EVs. qRT-PCR analysis was used to quantify LIPCAR in all samples. RESULTS: We showed that LIPCAR is present in both lEVs and sEVs isolated from all samples. The levels of LIPCAR are higher in lEVs compared to sEVs isolated from HeLa conditioned culture media and cardiac biopsies. No difference of LIPCAR expression was observed in tissue or EVs isolated from cardiac biopsies obtained from ischemic HF patients compared to non-failing patients. Interestingly, LIPCAR levels were increased in lEVs and sEVs isolated from MI patients who develop LVR compared to patients who did not develop LVR. CONCLUSION: Our data showed that large EVs are the main extracellular vesicle transporter of LIPCAR from heart into the circulation independently of the status, non-failing or HF, in patients. The levels of LIPCAR in EVs isolated from plasma could be used as biomarkers of LVR in post-MI patients.

Long-term oncological outcomes of robotic versus laparoscopic gastrectomy for gastric cancer: multicentre cohort study.

BACKGROUND: The aim of this multicentre cohort study was to compare the long-term oncological outcomes of robotic gastrectomy (RG) and laparoscopic gastrectomy (LG) for patients with gastric cancer. METHODS: Patients with gastric cancer who underwent radical gastrectomy by robotic or laparoscopic approaches from 1 March 2010 to 31 December 2018 at 10 high-volume centres in China were selected from institutional databases. Patients receiving RG were matched 1 : 1 by propensity score with patients undergoing LG. The primary outcome was 3-year disease-free survival. Secondary outcomes were overall survival and disease recurrence. RESULTS: Some 2055 patients who underwent RG and 4309 patients who had LG were included. The propensity score-matched cohort comprised 2026 RGs and 2026 LGs. Median follow-up was 41 (i.q.r. 39-58) months for the RG group and 39 (38-56) months for the LG group. The 3-year disease-free survival rates were 80.8% in the RG group and 79.5% in the LG group (log rank P = 0.240; HR 0.92, 95% c.i. 0.80 to 1.06; P = 0.242). Three-year OS rates were 83.9 and 81.8% respectively (log rank P = 0.068; HR 0.87, 0.75 to 1.01; P = 0.068) and the cumulative incidence of recurrence over 3 years was 19.3% versus 20.8% (HR 0.95, 0.88 to 1.03; P = 0.219), with no difference between groups. CONCLUSION: RG and LG in patients with gastric cancer are associated with comparable disease-free and overall survival.

Saliva monitoring of prednisolone in children with first onset steroid-sensitive nephrotic syndrome: Is it possible?

AIMS: Prednisolone is the cornerstone of treatment for idiopathic nephrotic syndrome in children, but is associated with marked side-effects. Therapeutic drug monitoring using saliva would be a patient-friendly option to monitor prednisolone treatment. To assess the feasibility of saliva monitoring, we described the pharmacokinetics (PK) of unbound prednisolone in plasma and saliva of children with first onset steroid-sensitive nephrotic syndrome (SSNS). METHODS: Children (age 2-16 years) with SSNS participating in a randomized, placebo-controlled trial with levamisole were treated with an 18-week tapering schedule of prednisolone. Five serial samples were collected at 4 (saliva) and 8 weeks (saliva and plasma) after first onset. A nonlinear mixed-effects model was used to estimate the PK parameters of unbound prednisolone and the saliva-to-plasma ratio. Monte Carlo simulations were performed to assess the predictive performance of saliva monitoring. RESULTS: From 39 children, 109 plasma and 275 saliva samples were available. Estimates (relative squared error) of unbound plasma clearance and volume of distribution were 93 (5%) L h-1 70 kg-1 and 158 (7%) L 70 kg-1, respectively. Typical saliva-to-plasma ratio was 1.30 (8%). Monte Carlo simulations demonstrated that on basis of 4 saliva samples and a single plasma sample unbound plasma area-under-the-concentration-time curve can be predicted within 20% imprecision in 79% of the patients compared to 87% based on 4 plasma samples. CONCLUSION: Saliva proved to be a reliable and patient-friendly option to determine prednisolone plasma exposure in children with SSNS. This opens opportunities for further PK and pharmacodynamics studies of prednisolone in a variety of paediatric conditions.

Real-time single-base specific detection of the Haemonchus contortus S168T variant associated with levamisole resistance using loop-primer endonuclease cleavage loop-mediated isothermal amplification.

Haemonchus contortus is a parasitic haematophagous nematode that primarily affects small ruminants and causes significant economic loss to the global livestock industry. Treatment of haemonchosis typically relies on broad-spectrum anthelmintics, resistance to which is an important cause of treatment failure. Resistance to levamisole remains less widespread than to other major anthelmintic classes, prompting the need for more effective and accurate surveillance to maintain its efficacy. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) is a recently developed diagnostic method that facilitates multiplex target detection with single nucleotide polymorphism (SNP) specificity and portable onsite testing. In this study, we designed a new LEC-LAMP assay and applied it to detect the levamisole resistance marker S168T in H. contortus. We explored multiplexing probes for both the resistant S168T and the susceptible S168 alleles in a single-tube assay. We then included a generic probe to detect the acr-8 gene in the multiplex assay, which could facilitate the quantification of both resistance markers and overall genetic material from H. contortus in a single step. Our results showed promising application of these technologies, demonstrating a proof-of-concept assay which is amenable to detection of resistance alleles within the parasite population, with the potential for multiplex detection, and point-of-care application enabled by lateral flow end-point detection. However, further optimisation and validation is necessary.

Antineutrophil cytoplasmic antibody in children with nephrotic syndrome treated with levamisole: a cross-sectional cohort study.

BACKGROUND: Levamisole is a commonly used steroid-sparing agent (SSA), but the reported incidence of antineutrophil cytoplasmic antibody (ANCA) positivity has been concerning. METHODS: Observational cross-sectional study wherein children aged 2 to 18 years with frequently relapsing/steroid dependent nephrotic syndrome (FRNS/SDNS) on levamisole for ≥ 12 months were tested for ANCA. RESULTS: A total of 210 children (33% female), median age of 7.3 (IQR: 5.6-9.6) years, and a median duration of levamisole exposure of 21 (IQR: 15-30) months were tested. ANCA was positive in 18% (n = 37): 89% (n = 33) perinuclear ANCA (pANCA), 3% (n = 1) cytoplasmic ANCA (cANCA), and 8% (n = 3) both. Of ANCA-positive children, none had reduced eGFR or abnormal urinalysis. The majority of these children were asymptomatic (81%, n = 30). Rash was more common among ANCA-positive children [6/37 (16%) vs. 3/173 (2%), p = 0.0001]. On multivariate analysis, higher age (OR = 1.02, [95th CI: 1.01 to 1.03], p = 0.007) and longer duration of levamisole exposure (OR = 1.05, [95th CI: 1.02 to 1.08], p = 0.0007) were associated with ANCA positivity. Levamisole was stopped in ANCA-positive children with the resolution of any clinical manifestations if present. Repeat ANCA testing was performed in 54% (20/37), and all were ANCA negative by 18 months. CONCLUSIONS: Children with FRNS/SDNS on longer duration of levamisole were associated with increasing prevalence of ANCA positivity, but most of these children were clinically asymptomatic. Prospective studies are required to determine the chronology of ANCA positivity and its clinical implication.

Survival outcomes of patients with stage III colorectal cancer aged ≥ 80 years who underwent curative resection: the HiSCO-04 prospective cohort study.

BACKGROUND: The efficacy of adjuvant chemotherapy in elderly patients aged ≥ 80 years with stage III colorectal cancer remains unclear. In parallel with a multicenter prospective phase II trial evaluating the efficacy of uracil-tegafur and leucovorin as adjuvant chemotherapy (HiSCO-03), we conducted a prospective observational study of these patients to assess survival outcomes, including those ineligible for chemotherapy. METHODS: This multi-institutional prospective cohort study included 17 institutions in Hiroshima, Japan. Patients aged ≥ 80 years with stage III colorectal cancer who underwent curative resection were enrolled. The primary endpoint was 3-year disease-free survival, and the secondary endpoints were 3-year overall and relapse-free survival. Propensity score matching was used to assess the effects of adjuvant chemotherapy on survival outcomes. RESULTS: A total of 214 patients were analyzed between 2013 and 2018, including 99 males and 115 females with a median age of 84 years (range 80-101 years). Recurrence occurred in 58 patients and secondary cancers were observed in 17. The 3-year disease-free, overall, and relapse-free survival rates were 63.3%, 76.9%, and 62.9%, respectively. Adjuvant chemotherapy was administered to 65 patients with a completion rate of 52%. In a study of 80 patients that adjusted for background factors using propensity score matching, patients who completed the planned treatment showed improved disease-free survival (3-year disease-free survival: completed, 80.0%; not received, 65.5%; and discontinued, 56.3%; p = 0.029). CONCLUSIONS: Completion of adjuvant chemotherapy may improve the prognosis of patients with colorectal cancer aged ≥ 80 years, although the number of patients who would benefit from it is limited.

Optimization and validation of high-performance liquid chromatography using modified QuEChERS to determine anthelmintic drugs in mutton.

The aim of this study was to rapidly determine the presence of anthelmintic drugs in sheep meat using the optimized high-performance liquid chromatography-ultraviolet (HPLC-UV) method with modified QuEChERS (quick, easy, cheap, effective, rugged, safe) technology. Fifty fresh sheep meat samples from different slaughterhouses were collected. A double extraction procedure (QuEChERS/HPLC-UV technology) was used to extract the target analytes. A multilevel calibration curve from 1 to 1000 g/kg was used to establish instrument linearity for rafoxanide, albendazole, and closantel, whereas 0.1-100 µg/kg was used for ivermectin, levamisole, and oxyclozanide to find the lowest concentration, maximum residue limit (MRL), and occupied range for targeted analytes. The concentration levels were used to investigate the linearity, whereas several certified reference materials were applied to determine accuracy. The process was linear for all combinations, from the limit of quantification (LOQ) to the maximum concentration. The LOQ was established at 0.5 µg/kg for ivermectin, levamisole, and oxyclozanide and 10 µg/kg for rafoxanide, albendazole, and closantel. Recovery values were 70%-120%, and repeatability/reproducibility stated in relative standard deviation was obtained at less than 20%. QuEChERS method revealed that most meat samples contained anthelmintic drug residues, of which the majority exceeded the MRLs. Thus, the drugs should be used correctly in animals to avoid residues in food for human consumption.

Concurrent use of two dual-combination drenches containing monepantel/abamectin and oxfendazole/levamisole in sheep: effect on marker residues 21 and 28 days after administration.

AIMS: To determine the concentration, in comparison with the maximum residue limit (MRL), of anthelmintic marker residues in the target tissues (liver and fat) of sheep treated concurrently with two oral drenches, one containing monepantel and abamectin and the other oxfendazole and levamisole. METHODS: On day 0 of the study, 12 sheep (six male and six female; 8-9-months old) were dosed according to individual body weight determined the day prior. Zolvix Plus (dual-active oral drench containing 25 g/L monepantel and 2 g/L abamectin) was administered to all animals prior to administration of Scanda (dual-active oral drench containing 80 g/L levamisole hydrochloride and 45.3 g/L oxfendazole). Six sheep (three male and three female) were slaughtered 21 and 28 days after treatment and renal fat and liver samples were collected.Using validated methods, analyses for monepantel sulfone, abamectin, levamisole and oxfendazole (expressed as total fenbendazole sulfone following conversion of the combined concentrations of oxfendazole, fenbendazole and fenbendazole sulfone) were performed on liver samples while renal fat specimens were analysed for monepantel sulfone and abamectin residues only. Detected concentrations were compared to the established MRL in sheep for each analyte determined by the Ministry for Primary Industries. RESULTS: All residues detected in samples of liver and fat collected 21 and 28 days after treatment were below the MRL for each analyte. All liver samples collected on day 21 had detectable monepantel sulfone (mean 232 (min 110, max 388) µg/kg) and oxfendazole (mean 98.7 (min 51.3, max 165) µg/kg) residues below the MRL (5,000 and 500 µg/kg, respectively). Monepantel sulfone (mean 644 (min 242, max 1,119) µg/kg; MRL 7,000 µg/kg) residues were detected in 6/6 renal fat samples. Levamisole residues were detected in 3/6 livers (mean 40.0 (min 14.3, max 78.3) µg/kg; MRL 100 µg/kg), and abamectin residues in 1/6 livers (0.795 µg/kg; MRL 25 µg/kg) and 2/6 fat samples, (mean 0.987 (min 0.514, max 1.46) µg/kg; MRL 50 µg/kg) 21 days after treatment. CONCLUSION AND CLINICAL RELEVANCE: These results suggest that concurrent administration of Zolvix Plus and Scanda to sheep is unlikely to result in an extended residue profile for any of the active ingredients, with all analytes measured being under the approved New Zealand MRL 21 days after treatment. This work was not completed in line with guidance for establishing official residue profiles, nor is it sufficient to propose a new withholding period.

Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Cholinergic signaling at the body wall neuromuscular junction distally inhibits feeding behavior in Caenorhabditis elegans.

Complex biological functions within organisms are frequently orchestrated by systemic communication between tissues. In the model organism Caenorhabditis elegans, the pharyngeal and body wall neuromuscular junctions are two discrete structures that control feeding and locomotion, respectively. Separate, the well-defined neuromuscular circuits control these distinct tissues. Nonetheless, the emergent behaviors, feeding and locomotion, are coordinated to guarantee the efficiency of food intake. Here, we show that pharmacological hyperactivation of cholinergic transmission at the body wall muscle reduces the rate of pumping behavior. This was evidenced by a systematic screening of the effect of the cholinesterase inhibitor aldicarb on the rate of pharyngeal pumping on food in mutant worms. The screening revealed that the key determinants of the inhibitory effect of aldicarb on pharyngeal pumping are located at the body wall neuromuscular junction. In fact, the selective stimulation of the body wall muscle receptors with the agonist levamisole inhibited pumping in a lev-1-dependent fashion. Interestingly, this response was independent of unc-38, an alpha subunit of the nicotinic receptor classically expressed with lev-1 at the body wall muscle. This implies an uncharacterized lev-1-containing receptor underpins this effect. Overall, our results reveal that body wall cholinergic transmission not only controls locomotion but simultaneously inhibits feeding behavior.

GMMAD: a comprehensive database of human gut microbial metabolite associations with diseases.

BACKGROUND: The natural products, metabolites, of gut microbes are crucial effect factors on diseases. Comprehensive identification and annotation of relationships among disease, metabolites, and microbes can provide efficient and targeted solutions towards understanding the mechanism of complex disease and development of new markers and drugs. RESULTS: We developed Gut Microbial Metabolite Association with Disease (GMMAD), a manually curated database of associations among human diseases, gut microbes, and metabolites of gut microbes. Here, this initial release (i) contains 3,836 disease-microbe associations and 879,263 microbe-metabolite associations, which were extracted from literatures and available resources and then experienced our manual curation; (ii) defines an association strength score and a confidence score. With these two scores, GMMAD predicted 220,690 disease-metabolite associations, where the metabolites all belong to the gut microbes. We think that the positive effective (with both scores higher than suggested thresholds) associations will help identify disease marker and understand the pathogenic mechanism from the sense of gut microbes. The negative effective associations would be taken as biomarkers and have the potential as drug candidates. Literature proofs supported our proposal with experimental consistence; (iii) provides a user-friendly web interface that allows users to browse, search, and download information on associations among diseases, metabolites, and microbes. The resource is freely available at http://guolab.whu.edu.cn/GMMAD . CONCLUSIONS: As the online-available unique resource for gut microbial metabolite-disease associations, GMMAD is helpful for researchers to explore mechanisms of disease- metabolite-microbe and screen the drug and marker candidates for different diseases.

Hydroxy levamisole and its phase II conjugates as potential indicators of levamisole doping in thoroughbred horses.

RATIONALE: According to previous research, aminorex is the major metabolite of levamisole; however, in the screening of levamisole-positive racehorse urine and plasma samples, aminorex could only be detected in trace amounts or not at all. In forensic laboratories, hydroxy levamisole and its phase II conjugates make it easier to confirm levamisole misuse and to differentiate between the abuse of levamisole and aminorex. This study aimed to identify the major levamisole metabolites that can be detected along with the parent drug. METHODS: The study describes levamisole and its metabolites in thoroughbred horses following oral administration and in vitro with equine liver microsomes. The plausible structures of the detected metabolites were postulated using liquid chromatography combined with high-resolution mass spectrometry. RESULTS: Under the experimental conditions 26 metabolites (17 phase I, 2 phase II, and 7 conjugates of phase I metabolites) were detected (M1-M26). The major phase I metabolites identified were formed by hydroxylation. In phase II, the glucuronic acid conjugates of levamisole and hydroxy levamisole were detected as the major metabolites. In plasma, the parent drug and major metabolites are detectable for up to eight days, while in urine, they are detectable for up to twenty days. Levamisole levels rapidly increased at 45 min following administration, then declined gradually until detectable levels were reached approximately 8 days after administration, according to a pharmacokinetics study. CONCLUSIONS: A prolonged elimination profile and relatively high concentration of hydroxy metabolites suggest that the detection of hydroxy metabolites is imperative for investigating levamisole doping in horses.

The microRNA miR-1 regulates a MEF-2-dependent retrograde signal at neuromuscular junctions.

We show that miR-1, a conserved muscle-specific microRNA, regulates aspects of both pre- and postsynaptic function at C. elegans neuromuscular junctions. miR-1 regulates the expression level of two nicotinic acetylcholine receptor (nAChR) subunits (UNC-29 and UNC-63), thereby altering muscle sensitivity to acetylcholine (ACh). miR-1 also regulates the muscle transcription factor MEF-2, which results in altered presynaptic ACh secretion, suggesting that MEF-2 activity in muscles controls a retrograde signal. The effect of the MEF-2-dependent retrograde signal on secretion is mediated by the synaptic vesicle protein RAB-3. Finally, acute activation of levamisole-sensitive nAChRs stimulates MEF-2-dependent transcriptional responses and induces the MEF-2-dependent retrograde signal. We propose that miR-1 refines synaptic function by coupling changes in muscle activity to changes in presynaptic function.

Novel insights on the therapeutic effect of levamisole on the chronic toxoplasmosis in mice model.

Latent toxoplasmosis mostly reactivates which could result in acute encephalitis. Chronic toxoplasmosis treatments are severely constrained by Toxoplasma cyst resistance. Novel therapeutic approaches are therefore becoming more essential. In this study, the effects of levamisole (LEVA) and spiramycin on the early and late stages of experimental toxoplasmosis are investigated. MATERIALS AND METHODS: Seventy-five Me49 Toxoplasma gondii infected Swiss albino mice were divided into five groups; (GI): noninfected control group; (GII): infected untreated control group; (GIII): infected- LEVA treated group; (GIV): infected and received combination of spiramycin and LEVA and (GV): infected-spiramycin treated group. The impact was assessed through brain cyst count by Quantitative Real-Time Polymerase Chain Reaction (PCR), interferon gamma (IFN-γ) assay, histopathological study, and total blood counts. RESULTS: The progression of chronic toxoplasmosis could only be partially controlled by using either levamisole or spiramycin as a separate drug. The combined spiramycin and levamisole treatment significantly decreased the burden of Toxoplasma brain cyst, increased IFN-γ level, total blood parameters and improved the histopathological features especially at the late stage of infection. IN CONCLUSION: Levamisole effectively modulated Toxoplasma-induced immune responses, resulting in chronic toxoplasmosis remission. Further clinical trials will be needed to study the effect of these combination in HIV/AIDS (human immunodeficiency virus) patients with toxoplasmosis.

Levamisole hydrochloride as an anthelmintic against Neoechinorhynchus buttnerae in juvenile Colossoma macropomum.

Acanthocephalosis is caused by the endoparasite Neoechynorhyncus buttnerae and affects fish farmed in the Amazon. This study assessed the efficacy of therapeutic levamisole hydrochloride (LVC) baths against N. buttnerae and its effects on juvenile tambaqui blood parameters. In vitro and in vivo tests were carried out, the latter employing two experimental therapeutic LVC bath protocols. Concerning in vitro efficacy, the T75 (75 mg.L-1 LVC) and T100 (mg.L-1 LVC) treatments were 100% effective in 15 min, while the T50 (50 mg.L-1 LVC) and T25 (25 mg.L-1 LVC) treatments required parasite exposure for 45 and 60 min, respectively. During exposure, the parasites displayed reduced motility, proboscis retraction, coiling into a spiral shape, body rigidity and swelling. The LVC LC50-72h for juvenile tambaqui was 115 mg.L-1 . Regarding in vivo efficacy for Protocol I (8-h bath), the T125 resulted in 82% effectiveness, while in Protocol II (two 8-h baths with a 24-h interval), the T115 treatment (115 mg.L-1 LVC) achieved 95.6% effectiveness without clinical intoxication signs, despite behavioural changes. No significant changes were observed in fish blood parameters. LVC was, therefore, highly effective both in vitro and in vivo in controlling the acanthocephalan N. buttnerae without compromising tambaqui juvenile homeostasis.