Quantification of Thiram in Honeybees: Development of a Chemiluminescent ELISA

International audienceA Chemiluminescence Enzyme-Linked Immuno-Sorbent Assay (CL-ELISA) for determination and quantification of the fungicide thiram in honeybees was developed in an indirect competitive format. The assay was optimized by determining: the optimal coating conjugate concentration and anti-thiram antiserum dilution, the effect of the incubation time on the competitive step, the tolerance to organic solvents. The IC50 and the limit of detection (LOD) values were 60 ng mL-1 and 9 ng mL -1, respectively, similar to those of colorimetric ELISA with a calibration range of 9 – 15,000 ng mL-1. Cross reactivity of some related compounds such as some dithiocarbamates, a thiocarbamate, the ethylenethiourea and the tetramethylthiourea were tested. The assay was then applied to honeybees sample extracts obtained by using the liquid-liquid extraction or the graphitized carbon-based solid phase extraction. The calibration curves in honeybee extracts from liquid-liquid procedure gave an IC50 of 141 ng mL-1 and a LOD of 17 ng mL-1. In case of extracts obtained by SPE these values were 139 ng mL-1 and 15 ng mL-1, respectively. The average recovery value from honeybee extracts spiked with 75 ng mL-1 of thiram was 72% for SPE, higher than for liquid-liquid extraction (60%). On the opposite, when the honeybees were directly spiked with 2 and 10 ppm the average recovery was higher for liquid-liquid extraction (54%), than for SPE (31%). Finally, the assay was applied to honeybee samples collected during monitoring activities in Italy and Russia

Case report: p.Glu134del SOD1 mutation in two apparently unrelated ALS patients with mirrored phenotype

With upcoming personalized approaches based on genetics, it is important to report new mutations in amyotrophic lateral sclerosis (ALS) genes in order to understand their pathogenicity and possible patient responses to specific therapies. SOD1 mutations are the second most frequent genetic cause of ALS in European populations. Here, we describe two seemingly unrelated Italian patients with ALS carrying the same SOD1 heterozygous c.400_402 deletion (p.Glu134del). Both patients had spinal onset in their lower limbs, progressive muscular weakness with respiratory involvement, and sparing bulbar function. In addition to the clinical picture, we discuss the possible pathogenic role of this unfamiliar SOD1 mutation

The β-lactamase Inhibitor Boronic Acid SM23 as a new anti-Pseudomonas aeruginosa Biofilm Compound

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often causative agent of severe device-related infections, given its great ability to produce biofilm. P. aeruginosa finely regulates the expression of numerous virulence factors, including biofilm production, by Quorum Sensing (QS), an intercellular communication mechanism used by many bacteria. Biofilm formation can enhance bacterial resistance to antimicrobial agents due to a decreased penetration of the antibiotic and a reduced rate of bacterial cells in biofilm. Selective inhibition of biofilm formation may thus represent a novel promising strategy to overcome the well-known and widespread drug-resistance of P. aeruginosa. METHODS: In this study, we investigated the effects of SM23, a boronic acid derivate specifically designed as β-lactamase inhibitor, on biofilm formation and production of virulence factors, using the P. aeruginosa bioluminescent strain P1242. RESULTS: Our results indicated that SM23: a) inhibited the development of biofilm and the production of the virulence factors pyoverdine, elastase and pyocyanin, without affecting bacterial growth; b) decreased the levels of P. aeruginosa QS-related Autoinducers molecules 3-oxo-C12-HSL and C4-HSL by dampened lasR/lasI system gene expression in the biofilm; c) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; d) reduced both biofilm formation and pyoverdine production by P. aeruginosa onto endotracheal tubes, as assessed by a new in vitro model, closely mimicking the clinical settings. CONCLUSION: Taken together, our results indicate that, besides inhibiting β-lactamase, SM23 can also act as potent inhibitor of P. aeruginosa biofilm, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections

SerpinA1 levels in amyotrophic lateral sclerosis patients: An exploratory study

Background: SerpinA1, a serine protease inhibitor, is involved in the modulation of microglial-mediated inflammation in neurodegenerative diseases. We explored SerpinA1 levels in cerebrospinal fluid (CSF) and serum of amyotrophic lateral sclerosis (ALS) patients to understand its potential role in the pathogenesis of the disease. Methods: SerpinA1, neurofilament light (NfL) and heavy (NfH) chain, and chitinase-3-like protein-1 (CHI3L1) were determined in CSF and serum of ALS patients (n = 110) and healthy controls (n = 10) (automated next-generation ELISA), and correlated with clinical parameters, after identifying three classes of progressors (fast, intermediate, slow). Biomarker levels were analyzed for diagnostic power and association with progression and survival. Results: SerpinA1serum was significantly decreased in ALS (median: 1032 μg/mL) compared with controls (1343 μg/mL) (p = 0.02). SerpinA1CSF was elevated only in fast progressors (8.6 μg/mL) compared with slow (4.43 μg/mL, p = 0.01) and intermediate (4.42 μg/mL, p = 0.03) progressors. Moreover, SerpinA1CSF correlated with neurofilament and CHI3L1 levels in CSF. Contrarily to SerpinA1CSF , neurofilament and CHI3L1 concentrations in CSF correlated with measures of disease progression in ALS, while SerpinA1serum mildly related with time to generalization (rho = 0.20, p = 0.04). In multivariate analysis, the ratio between serum and CSF SerpinA1 (SerpinA1 ratio) and NfHCSF were independently associated with survival. Conclusions: Higher SerpinA1CSF levels are found in fast progressors, suggesting SerpinA1 is a component of the neuroinflammatory mechanisms acting upon fast-progressing forms of ALS. Both neurofilaments or CHI3L1CSF levels outperformed SerpinA1 at predicting disease progression rate in our cohort, and so the prognostic value of SerpinA1 alone as a measure remains inconclusive

Multibody Computer Model of the Entire Equine Forelimb Simulates Forces Causing Catastrophic Fractures of the Carpus during a Traditional Race

Simple Summary Palios are traditional horseraces held in the main square of few Italian cities. Due to peculiar features of such circuits, adapted to the square architecture and thus characterized by tight curves and unconventional footing surface, horses involved are at particular risk of accidents. Prevention of catastrophic musculoskeletal injuries is a significant issue and matter of debate during these events. In particular, the negotiation of the curves in the city circuits is a significative concern. An experiment was set up to build a model of entire forelimb at the point of failure in the context of a turn comparable to that in a Palio circuit. The model was informed by live data and the output compared to post-mortem findings obtained from a horse that sustained a catastrophic fracture of the carpus during this competition. The objective of this study is to determine the magnitude and distribution of internal forces generated across the carpus under which the catastrophic injury has occurred and describe related post-mortem findings. A catastrophic fracture of the radial carpal bone experienced by a racehorse during a Palio race was analyzed. Computational modelling of the carpal joint at the point of failure informed by live data was generated using a multibody code for dynamics simulation. The circuit design in a turn, the speed of the animal and the surface characteristics were considered in the model. A macroscopic examination of the cartilage, micro-CT and histology were performed on the radio-carpal joint of the limb that sustained the fracture. The model predicted the points of contact forces generated at the level of the radio-carpal joint where the fracture occurred. Articular surfaces of the distal radius, together with the proximal articular surface of small carpal bones, exhibited diffuse wear lines, erosions of the articular cartilage and subchondral bone exposure. Even though the data in this study originated from a single fracture and further work will be required to validate this approach, this study highlights the potential correlation between elevated impact forces generated at the level of contact surfaces of the carpal joint during a turn and cartilage breakdown in the absence of pre-existing pathology. Computer modelling resulted in a useful tool to inversely calculate internal forces generated during specific conditions that cannot be reproduced in-vivo because of ethical concerns

Colchicine treatment in amyotrophic lateral sclerosis: safety, biological and clinical effects in a randomized clinical trial

: In preclinical studies, the anti-inflammatory drug colchicine, which has never been tested in amyotrophic lateral sclerosis, enhanced the expression of autophagy factors and inhibited accumulation of transactive response DNA-binding protein 43 kDa, a known histopathological marker of amyotrophic lateral sclerosis. This multicentre, randomized, double-blind trial enrolled patients with probable or definite amyotrophic lateral sclerosis who experienced symptom onset within the past 18 months. Patients were randomly assigned in a 1:1:1 ratio to receive colchicine at a dose of 0.005 mg/kg/day, 0.01 mg/kg/day or placebo for a treatment period of 30 weeks. The number of positive responders, defined as patients with a decrease lesser than 4 points in the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised total score during the 30-week treatment period, was the primary outcome. Disease progression, survival, safety and quality of life at the end of treatment were the secondary clinical outcomes. Secondary biological outcomes included changes from baseline to treatment end of stress granule and autophagy responses, transactive response DNA-binding protein 43 kDa, neurofilament accumulation and extracellular vesicle secretion, between the colchicine and placebo groups. Fifty-four patients were randomized to receive colchicine (n = 18 for each colchicine arm) or placebo (n = 18). The number of positive responders did not differ between the placebo and colchicine groups: 2 out of 18 patients (11.1%) in the placebo group, 5 out of 18 patients (27.8%) in the colchicine 0.005 mg/kg/day group (odds ratio = 3.1, 97.5% confidence interval 0.4-37.2, P = 0.22) and 1 out of 18 patients (5.6%) in the colchicine 0.01 mg/kg/day group (odds ratio = 0.5, 97.5% confidence interval 0.01-10.2, P = 0.55). During treatment, a slower Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised decline was detected in patients receiving colchicine 0.005 mg/kg/day (mean difference = 0.53, 97.5% confidence interval 0.07-0.99, P = 0.011). Eight patients experienced adverse events in placebo arm (44.4%), three in colchicine 0.005 mg/kg/day (16.7%) and seven in colchicine 0.01 mg/kg/day arm (35.9%). The differences in adverse events were not statistically significant. In conclusion, colchicine treatment was safe for amyotrophic lateral sclerosis patients. Further studies are required to better understand mechanisms of action and clinical effects of colchicine in this condition

Withdrawal of mechanical ventilation in amyotrophic lateral sclerosis patients: a multicenter Italian survey

Background: Law 219/2017 was approved in Italy in December 2017, after a years-long debate on the autonomy of healthcare choices. This Law, for the first time in Italian legislation, guarantees the patient's right to request for withdrawal of life-sustaining treatments, including mechanical ventilation (MV). Objective: To investigate the current status of MV withdrawal in amyotrophic lateral sclerosis (ALS) patients in Italy and to assess the impact of Law 219/2017 on this practice. Methods: We conducted a Web-based survey, addressed to Italian neurologists with expertise in ALS care, and members of the Motor Neuron Disease Study Group of the Italian Society of Neurology. Results: Out of 40 ALS Italian centers, 34 (85.0%) responded to the survey. Law 219/2017 was followed by an increasing trend in MV withdrawals, and a significant increase of neurologists involved in this procedure (p 0.004). However, variations across Italian ALS centers were observed, regarding the inconsistent involvement of community health services and palliative care (PC) services, and the intervention and composition of the multidisciplinary team. Conclusions: Law 219/2017 has had a positive impact on the practice of MV withdrawal in ALS patients in Italy. The recent growing public attention on end-of-life care choices, along with the cultural and social changes in Italy, requires further regulatory frameworks that strengthen tools for self-determination, increased investment of resources in community and PC health services, and practical recommendations and guidelines for health workers involved

Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation.Objective: To identify the genetic variants associated with juvenile ALS.Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism.Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members.Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway.Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.</p