Targeting Acetylcholinesterase: Identification of Chemical Leads by High Throughput Screening, Structure Determination and Molecular Modeling

Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e.g. in Alzheimer's disease), but may also act as dangerous toxins (e.g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685•mAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization, crystallography and computational chemistry provide a route to novel AChE inhibitors and reactivators

Food safety in informal public markets in Kenya: perceptions of stakeholders in the food chain

Introduction: Informal markets play a crucial role in providing fresh and nutritious foods for people in low and middle-income countries. However, the safety of food sold in these markets remains a major concern, contributing to a high burden of foodborne illnesses. Methods: This study was designed to analyze stakeholder perceptions of food safety in selected informal public markets in Kenya. Eight focus group discussions and 15 key informant interviews were conducted. In addition, two workshops were held to validate the findings. Results and discussion: We found that consumers and vendors confounded food quality, especially freshness, with safety, and paid more attention to appearance and physical qualities than food safety. Information (such as branding and expiry dates) and heuristic cues (such as the flow of buyers and the presence of flies) were used to assess food safety. Meat was perceived as the food most likely to cause disease. Chemical hazards in fresh produce and aflatoxins in maize were mentioned as priority hazards by both vendors and consumers. Stakeholders more knowledgeable about food safety considered microbial hazards as a top priority. Although diarrheal diseases were reported to be prevalent in the communities, these were often thought to be the result of agri-chemical residues in fresh produce or the consumption of high-fat foods, and not commonly linked to bacteria in food. Gaps identified during the interviews included poor infrastructure, inadequate food safety knowledge, insufficient or prohibitive policies, insufficient political will, and poor food safety-related practices. There were few mentions of lack of motivation or incentives for behavior change, or insufficient consumer demand for food safety. To fill the gaps, several opportunities were discussed, including contextualizing policies and regulations, investing in infrastructure, capacity building, and training, and promoting involvement and collaboration among various stakeholders. Conclusion: This study has highlighted gaps and misperceptions that need to be addressed through proper knowledge and awareness to effectively combat foodborne disease challenges. Behavioral change approaches to improve food safety are recommended

MILK Symposium review: Microbiological quality and safety of milk from farm to milk collection centers in Rwanda

The aim of this study was to generate knowledge on the most important milk quality and safety attributes, including somatic cell count (SCC), total bacterial count (TBC), Escherichia coli, Salmonella, and Brucella spp. antibodies and antibiotic residues in milk in the chain from farm to milk collection center (MCC) in Rwanda. In addition, we investigated farm and management factors associated with high TBC, SCC, and Salmonella counts. Raw milk was sampled at the farm and MCC levels. Milk samples were taken from dairy farms linked to 2 selected MCC in each of the 4 provinces in Rwanda. In total, 406 bulk milk samples from 406 farms and 32 bulk milk samples from 8 MCC were collected and analyzed. Farm milk average SCC varied between 180 × 103 and 920 × 103 cells/mL, whereas average SCC in milk samples at MCC varied between 170 × 103 and 1,700 × 103 cells/mL. The mean milk TBC of different farms per MCC varied between 1.1 × 106 and 1.6 × 107 cfu/mL, whereas in milk samples from different MCC, the mean TBC ranged between 5.3 × 105 and 2.4 × 108 cfu/mL. The high TBC in milk from MCC suggests proliferation or recontamination of milk by bacteria during transportation. Escherichia coli was detected in 35 of 385 farm milk samples and ranged between 5 cfu/mL and 1.1 × 104 cfu/mL, whereas in milk samples from the MCC, it was detected in 20 out 32 samples varying between 5 cfu/mL and 2.9 × 103 cfu/mL. Overall farm prevalence of Salmonella in milk samples was 14%, but no milk samples from MCC were positive for Salmonella. Five out of 22 bulk milk samples from different MCC were positive for Brucella spp. antibodies, but no Brucella antibodies were detected in milk samples from farms. The prevalence of antibiotic residues as detected by the Delvotest SP NT (DSM, Delft, the Netherlands) was low: 1.3% in farm milk samples and undetected in MCC milk samples. Lack of a separate milking area was associated with high TBC, whereas offering of supplemental feeds, keeping data of past diseases, and an unhygienic milking area were associated with high SCC. Lack of teat washing before milking was the only factor associated with Salmonella contamination of milk at the farm level. This study indicated high TBC and SCC of milk samples at the farm and MCC levels, which indicates both microbial contamination of milk and poor udder health in dairy cows. Presence of E. coli, Salmonella, and Brucella antibodies in milk was common, but finding antibiotic residues in milk was uncommon

A multi-country One Health foodborne outbreak simulation exercise: cross-sectoral cooperation, data sharing and communication

IntroductionThe awareness of scientists and policy makers regarding the requirement for an integrated One Health (OH) approach in responding to zoonoses has increased in recent years. However, there remains an overall inertia in relation to the implementation of practical cross-sector collaborations. Foodborne outbreaks of zoonotic diseases continue to affect the European population despite stringent regulations, evidencing the requirement for better ‘prevent, detect and response’ strategies. Response exercises play an essential role in the improvement of crisis management plans, providing the opportunity to test practical intervention methodologies in a controlled environment.MethodsThe One Health European Joint Programme simulation exercise (OHEJP SimEx) aimed at practicing the OH capacity and interoperability across public health, animal health and food safety sectors in a challenging outbreak scenario. The OHEJP SimEx was delivered through a sequence of scripts covering the different stages of a Salmonella outbreak investigation at a national level, involving both the human food chain and the raw pet feed industry.ResultsA total of 255 participants from 11 European countries (Belgium, Denmark, Estonia, Finland, France, Italy, Norway, Poland, Portugal, Sweden, the Netherlands) took part in national level two-day exercises during 2022. National evaluations identified common recommendations to countries aiming to improve their OH structure to establish formal communication channels between sectors, implement a common data sharing platform, harmonize laboratory procedures, and reinforce inter-laboratory networks within countries. The large proportion of participants (94%) indicated significant interest in pursuing a OH approach and desire to work more closely with other sectors.DiscussionThe OHEJP SimEx outcomes will assist policy makers in implementing a harmonized approach to cross-sector health-related topics, by highlighting the benefits of cooperation, identifying gaps in the current strategies and suggesting actions required to better address foodborne outbreaks. Furthermore, we summarize recommendations for future OH simulation exercises, which are essential to continually test, challenge and improve national OH strategies

Longitudinal study of Staphylococcus aureus genotypes isolated from bovine clinical mastitis

Bovine clinical mastitis is an important problem for the dairy industry, and Staphylococcus aureus is a common mastitis-causing pathogen in many countries. Detailed knowledge on genetic variation of Staph. aureus strains within the bovine population, including changes over time, can be useful for mastitis control programs, because severity of disease and effects on milk production are at least partly strain-associated. Therefore, the major aim of this study was to compare sequence types of Staph. aureus isolated from cases of bovine clinical mastitis from 2002 to 2003 with sequence types of a more recent set of isolates collected from 2013 to 2018, using core genome multi-locus sequence typing (cgMLST). We also wanted to compare antibiotic resistance genes of isolates from the 2 sets, to identify changes that may have occurred over time in the Staph. aureus population. A total of 157 isolates of Staph. aureus, almost equally distributed between the 2 time periods, were subjected to high-throughput sequencing and cgMLST. The results showed that the most prevalent sequence types found among the 2002 to 2003 isolates belonged to the clonal complexes CC97, CC133, and CC151, and that those complexes still dominated among the isolates from 2013 to 2018. However, a population shift from CC133 to CC97 and CC151 over time was observed. Likewise, no important differences in prevalence of antibiotic resistance genes were found between the 2 sets of isolates. As expected, genes belonging to the major facilitator superfamily of transporter proteins, and multidrug and toxic compound extrusion transporters, were very common. Moreover, several genes and mutations conferring resistance to fosfomycin were present, but not in CC97 isolates. The beta-lactamase gene blaZ was found in only 3 out of 81 isolates from 2002 to 2003 and 1 out of 76 isolates in 2013 to 2018. In conclusion, the results indicate that mastitis-associated Staph. aureus strains circulating among dairy cows in Sweden exhibit a remarkable genotypic persistence over a time frame of close to 15 yr

Tillgänglighet och delaktighet i offentlig miljö för personer med fysisk funktionsnedsättning : En litteraturstudie

Riksdagen beslutade år 2000 om en nationell handlingsplan för att förbättra tillgängligheten för persoaner med funktionsnedsättning. Hindren skall vara åtgärdade före 2010. Syfte. Att studera hur tillgänglighet i offentlig miljö beskrivs i vetenskapliga artiklar och hur det påverkar delaktigheten i socila aktiviteter för personaer med fysisk funktionsnedsättning. Metod. Systematisk litteraturstudie av artiklar sökta i fyra olika databaser. Resultat. Elva artiklar publicerade 2002-2009 studerades och resultatet visar att tillgänglighet i offentlig miljö har stor betydelse för delaktighet i sociala aktiviteter men också graden av funktionsnedsättning, ålder samt upplevelse av hinder är faktorer som är avgörande. Konklusion. Resultatet styrker vikten av att följa den nationella handlingsplanen från 2000 för att öka möjligheten för personer med funktionsnedsättning att leva enligt sina egna önskemål och inte vara tvingade att acceptera hinder i omgivningen.The Swedish Parliament decided in 2000 on a national plan to improve the accessibility to people with disability. Environmental barriers should be removed prior to 2010. Purpose. To investigate how accessibility to public environments is described in scientific articles and how it influences participation in social activities for persons with physical functional limitations. Method. Review of scientific articles found in four different databases. Results. Eleven scientific articles published 2002-2009 were studied and the result showed that accessibility to public environments is found to be of great importance for participation in social activities. For persons with physical functional limitations, level of disability, age and experience of environmental barrioers are utmost importance. Conclusion. The result strengthen the necessity of following the national plan from 2000 and thereby increase the possibilities for persons with disability to live according to their own wishes and not be forced to accept environmental barriers

Staphylococcus aureus and Lipopolysaccharide Modulate Gene Expressions of Drug Transporters in Mouse Mammary Epithelial Cells Correlation to Inflammatory Biomarkers.

Inflammation in the mammary gland (mastitis) is the most common disease in dairy herds worldwide, often caused by the pathogens Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Little is known about the effects of mastitis on drug transporters and the impact on transporter-mediated excretion of drugs into milk. We used murine mammary epithelial HC11 cells, after lactogenic differentiation into a secreting phenotype, and studied gene expressions of ABC- and SLC- transporters after treatment of cells with S. aureus and lipopolysaccharide, an endotoxin secreted by E. coli. The studied transporters were Bcrp, Mdr1, Mrp1, Oatp1a5, Octn1 and Oct1. In addition, Csn2, the gene encoding β-casein, was analyzed. As biomarkers of the inflammatory response, gene expressions of the cytokines Il6 and Tnfα and the chemokine Cxcl2 were determined. Our results show that S. aureus and LPS treatment of cells, at non-cytotoxic concentrations, induced an up-regulation of Mdr1 and of the inflammatory biomarkers, except that Tnfα was not affected by lipopolysaccharide. By simple regression analysis we could demonstrate statistically significant positive correlations between each of the transporters with each of the inflammatory biomarkers in cells treated with S. aureus. The coefficients of determination (R2) were 0.7-0.9 for all but one correlation. After treatment of cells with lipopolysaccharide, statistically significant correlations were only found between Mdr1 and the two parameters Cxcl2 and Il6. The expression of Csn2 was up-regulated in cells treated with S. aureus, indicating that the secretory function of the cells was not impaired. The strong correlation in gene expressions between transporters and inflammatory biomarkers may suggest a co-regulation and that the transporters have a role in the transport of cytokines and chemokines. Our results demonstrate that transporters in mammary cells can be affected by infection, which may have an impact on transport of essential compounds and contaminants into milk