Obesity is the result of genetics which predisposes an individual to obesity and environmental factors, resulting in excessive weight gain. A well-established linear relationship exists between hypertension and obesity. The combined burden of hypertension and obesity poses significant health and economic challenges. Many environmental factors and genetic traits interact to contribute to obesity-linked hypertension. These include excess sodium re-absorption or secretion by the kidneys, a hypertensive shift of renal-pressure and activation of the sympathetic nervous system. Most individuals suffering from hypertension need drugs in order to treat their raised blood pressure, and while a number of antihypertensive therapeutic agents are currently available, 50% of cases remain uncontrolled. In order to develop new and effective therapeutic agents combating obesity-induced hypertension, a thorough understanding of the molecular events leading to adipogenesis is critical. With the advent of whole genome and exome sequencing techniques, new genes and variants which can be used as markers for obesity and hypertension are being identified. This review examines the role played by alternative splicing (AS) as a contributing factor to the metabolic regulation of obesity-induced hypertension. Splicing mutations constitute at least 14% of the disease-causing mutations, thus implicating polymorphisms that effect splicing as indicators of disease susceptibility. The unique transcripts resulting from the alternate splicing of mRNA encoding proteins that play a key role in contributing to obesity would be vital to gain a proper understanding of the genetic causes of obesity. A greater knowledge of the genetic basis for obesity-linked hypertension will assist in the development of appropriate diagnostic tests as well as the identification of new personalized therapeutic targets against obesity-induced hypertension. Obesity is a health problem, affecting individuals across the globe, and is on the increase in both emerging countries and the western world, regardless of gender race, ethnicity or socio-economic status. Recent estimates place the number of overweight adults at 1.9 billion worldwide, with 650 million being obese.10The relationship between obesity and hypertension is well documented and has been observed in multiple studies and populations, in both sexes.17The roots of obesity-linked hypertension can be traced to early childhood, 8–11 years, with the full effects being observed in adulthood.22fat mass and obesity-associated (FTO) gene also known as alpha-ketoglutarate-dependent dioxygenase. FTO is an RNA demethylase that regulates metabolic rate and body fat accumulation by controlling adipocyte differentiation into brown or white fat cells. These SNPs are strongly associated with BMI.Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) in the first intron of the This implies that these genetic variants that contribute to the development of obesity can be identified and used to customize treatments to suit the patient, in order to prevent or delay the development of obesity-associated hypertension. Model organisms can provide useful information regarding the role played by genetic polymorphisms during developmental stages and how the influence of the polymorphisms during development can contribute to obesity and hypertension; these results can then be used in humans to help prevent age-specific onset of human obesity-related hypertension.The mechanisms that lead to obesity-induced hypertension are yet to be fully understood; however, it is likely that distinct hypertensive phenotypes with distinct genetic determinants are related to disease progression. There are several abnormalities in the central and peripheral vasculature that contribute to the development of high blood pressure in severely obese individuals. These include an increase in the activity of the sympathetic nervous system, insulin resistance, activation of the RAAS, and altered vascular function.An increase in the activity of the sympathetic nervous system (SNS) is a common feature of obesity, which is present in both animals and humans, and it plays an important role in hypertension progression.332-receptor stimulator, resulting in reduced SNS activity. This is similar to α- and β-adrenergic blockers. When either clonidine or α- and β-adrenergic blockers are given to dogs fed a high fat diet, they helped to control the increased blood pressure normally observed in these dogs.Signal Transducer and Activator of Transcription 3 (STAT3). STAT3 acts downstream by downregulating the expression of endocabbanoids, which stimulate hunger. Disrupted STAT3 signaling leads to resistance to the anti-hunger effect of leptin, resulting in the consumption of excess food and possibly weight gain.39It remains uncertain as to what causes the activation of SNS in obesity as there may be several factors involved. One such factor seems to be increased adrenergic activity.Obesity is defined by a state of impaired glucose tolerance, high levels of circulating insulin, and reduced sensitivity to the metabolic actions of insulin.41Obesity-induced hypertension is a major factor contributing to cardiovascular disease, Another factor contributing to both cardiovascular disease and hypertension due to obesity is the RAAS.The RAAS plays an important role in the development and maintenance of high blood pressure associated with obesity.26Targeting the RAAS in hypertensive obese patients can result in a decrease in hypertension as well as in the incidences of associated metabolic disorders such as type 2 diabetes. Drugs that can be used to target the RAAS include ACEs or angiotensin II receptor blockers.56There is increasing evidence concerning the important role played by vascular endothelial dysfunction in the pathogenesis of hypertension. Obesity is a known contributing factor to endothelial dysfunction. Insulin signaling pathways stimulate inflammation and lead to aberrant excessive and uncontrolled endothelial growth resulting in endothelial dysfunction and hypertension . EndotheMAPK pathways also regulate the production of adhesion molecules by the endothelial cells. These adhesion molecules include vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin and promote the adhesion of monocytes to the vascular wall.59The kidneys and adrenal glands play an important central role in the regulation of blood pressure. Obese individuals and subjects with metabolic syndrome tend to be relatively salt sensitive.62Sodium retention is also associated with plasma aldosterone levels. Aldosterone is a steroid hormone produced by the adrenal gland. It is the main mineralocorticoid hormone in the body and is essential for the control of sodium retention in the kidney. Aldosterone levels in African American individuals are associated with hypertension. These hypertensive individuals also display high plasma aldosterone concentrations. The effect of obesity on aldosterone levels is partly due to the ability of adipokines to stimulate aldosterone production.44High concentrations of cortisone may also bind to mineralocorticoid receptors leading to an increase in arterial pressure in a similar way, but to a lesser degree, than aldosterone.49The development of obesity and hypertension in individuals involves behavioral, environmental, and genetic factors. Evidence of a genetic component is strongly implicated as playing a significant role in the risk of becoming obese.68fat mass and obesity-associated gene (FTO).FTO and GNDPA 2 genes are associated with obesity in the Chinese population. Multiple other genes have variants that may predispose individuals to obesity.Genome-wide association studies (GWASs) are able to detect if gene variants are associated with certain traits such as body mass index and obesity, with large GWAS meta-analysis revealing a number of genes that may influence an individual’s susceptibility to obesity.The splicing of precursor messenger RNA (pre-mRNA) occurs post-translationally and results in different isoforms of a protein, encoded by a single gene, being expressed. Alternative splicing (AS) allows for the generation of a greater diversity of proteins without increasing the number of genes. Alternative splicing generates these different isoforms by incorporating and excluding different combinations of exons, resulting in different mature mRNA molecules. The resulting protein isoforms may have related, distinct, or even opposing biological properties.78RNA-binding factor A (Rbm4a) gene is located on chromosome 11 at position q13.2. This gene has multiple functions in cellular processes such as the regulation of both AS and translation. RBM4α modulates 5-splice site and exon selection, thereby regulating AS. It is responsible for regulating the splicing of genes such as L-type Cav 1,2. The different isoforms of which play different roles in developmentally regulating blood pressure in the heart.Polypyrimidine tract-binding protein 1 (PTBP1) by activating exon skipping during muscle cell differentiation. PTBP1 is a splicing factor and it controls the splicing of alpha tropomyosin in muscle cells. RBM4A antagonizes the splicing modulation role of PTBP1.79The pyruvate kinase, contributing to changes in mitochondrial energy production in differentiating cells.Period circadian 1 protein (PER1) mRNA. PER1 plays an important role in controlling circadian rhythm and cell cycle progression in cells. Under stress conditions, it facilitates the initiation of the translation of specific target mRNAs by binding to ribosome entry sequences on these mRNAs. This results in the recruitment of the translation initiation factor EIF4A1 and the initiation of Internal Ribosome Entry Site IRES-dependent translation.Knockdown studies in mice showed that RBM4A is associated with proper pancreas development and therefore the expression of insulin. RBM4A also controls insulin receptor splicing switches.FTO was the first gene identified to contribute to non-syndromic human obesity.FTO is associated with abnormal adipose tissues and body mass. Characteristics that suggest a role for FTO in adipogenesis and energy homeostasis.FTO locus was found to be associated with BP. Individuals homozygous for the rs9930333 SNP in the FTO locus had a greater BMI by 1.8 kg/m2 and a higher blood pressure, 3.6 mm Hg higher than normal.23The FTO protein is a member of the AlkB family of non-heme Fe (II)/dioxygenases. FTO gene consists of nine exons . Only isoforms encoded by cFTO1 and cFTO4 retain demethylase activity. In the chicken, the expression of these various isoforms is associated with glucose metabolism, body weight, fatness, and body composition. Feeding chickens a high-glucose or high fat diet results in higher expression levels of cFTO1 in all tissues except in the pituitary.87The full-length ne exons  with theAnother possible link between obesity and the development of hypertension is hyperleptinemia. Leptin is a hormone produced by adipose tissue in direct proportion to adipose tissue mass.88leptin are known to be associated with an increase in body mass Index (BMI) and obesity.leptin gene are also associated with obesity. One such polymorphism known as C532T involves a base pair change at the 3ʹ-UTR and is associated with increased blood pressure.94Variations in the 5ʹ-region of TCF7L2 increase the chances of the individual developing type 2 diabetes.TCF7L2 gene leads to impaired TCF7L2 function. Different splice variants and expression patterns are found in different tissues.TCF7L2 consists of 18 exons and is characterized by alternative splicing of 3ʹ end exons 12, 13, 13a, and 13b. These alternate exons contain novel stop codons, leading to truncated isoforms because of the termination of transcription. The full exon 14 is required to code for the c terminus protein-binding domain (CTBP), which inhibits the Wnt signaling pathway. Exon 13 encodes a c-Clamp (CRARF) domain, which activates the Wnt signaling pathway. Since the Wnt signaling pathway is important in insulin signaling, it is also involved in the development of type 2 diabetes.100Transcription factor 7-like 2 (TCF7L2) is a transcription factor that contains a high mobility group (HMG) domain. These domains are involved with DNA binding and protein–protein interactions. TCF7L2 plays a role in the Wnt signaling pathway and is associated with blood glucose homeostasis. Mutations in The insulin receptor is activated through the binding of insulin, IGF-I and IGF-II. It is a tyrosine kinase receptor and exists in two isoforms in humans. The smaller isoform, IR-A, is normally expressed in the developing fetus. It lacks exon 11, which codes for 12 amino acids at the c terminus. The larger IRB isoform is normally expressed in adults. The isoforms have different binding affinities for (IGF)-2 and proinsulin, with an increase in the expression of IR-A being associated with diabetes and hyperinsulinemia.105LMNA are associated with lipodystrophy, hyperinsulinemia, dyslipidemia, diabetes, and hypertension.105,Prelamin (LMNA) mRNA contains 12 exons  enzyme that functions to convert phosphatidate to diacylglycerol and is therefore required for triacylglycerol synthesis. Lipin 1 also acts as an inducible transcriptional coactivator and in combination with the peroxisome proliferator-activated receptor α (PPARα) and PPARγ coactivator 1α (PGC-1α), it serves to regulate fatty acid metabolism. Lipin-1 is therefore required for adipocyte differentiation and lipid metabolism and influences fat mass and energy balance in adipose tissue and skeletal muscle.102Currently, there are no specific guidelines to treat hypertension associated with obesity-related hypertension and no adequate large-scale drug trial have been performed specifically targeting this problem. However, the vast majority of patients with hypertension are also overweight or obese. Any treatment to lower blood pressure should not contribute to weight gain. Choices of an appropriate treatment should be based on the mechanisms of obesity-induced hypertension, as well as clinical experience with these patients. Other important factors to consider would be patient comorbidities, side effects, and the frequency and cost of treatment.113Antagonizing the RAAS is a common drug target for lowering blood pressure. Targeting this system has the added advantage that the RAAS contributes to obesity. These drugs have an advantage over other commonly used drugs used to treat hypertension such as thiazide diuretics and β‐blockers, as the use of any of these classes of drugs can result in insulin resistance.114Metabolically neutral agents such as angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), direct renin inhibitors, and mineralocorticoid receptor antagonists are the first-line choice of drugs for obesity-related hypertension.123These are mainly complex mixtures of multiple components, each with its own chemical and pharmacological features. The combination of multiple phytochemicals may have synergistic activity that increases their bioavailability and their action on multiple molecular targets, which outweighs that of any single constituent.Proper regulation of alternative splicing relies on splicing machinery that can accurately and precisely identify the splice sites for intron removal and exon joining. Even a small error, such as single nucleotide addition or deletion, will shift the reading frame, leading to alteration of protein function. The splicing machinery responsible for achieving this accuracy is the spliceosome. The spliceosome consists of 5 snRNAs packaged with approximately 80 proteins.130Missense mutations have been predicted to cause diseases by contributing to 1.6% of all splicing errors and approximately 7% of the exonic variants may alter splicing which includes cryptic splicing.132Antisense oligonucleotides can act as splicing switches by binding to enhancer or silencing sequences within the target mRNA and influence the binding of splicing factors to these sequences.These splice switching oligonucleotides (SSO) have been used in trials to treat multiple ailments and seem to have few side effects for the patient.133Alternative splicing of the mRNA coding for genes that play a role in metabolism gives rise to protein isoforms that play important and sometimes conflicting roles in metabolic pathways. These metabolic pathways can influence an individual’s weight gain. Alternatively, these genes can control the development of adipocytes, resulting in obesity. Obesity is a major contributor to the development of hypertension. Regulation of alternative splicing or abnormal mRNA splicing can therefore be a major contributing factor to the development of obesity and consequently obesity-linked hypertension. The full effect of SNPs in obesity-linked loci is unknown, but SNPs may alter the splicing of genes associated with obesity and obesity-linked hypertension. This will then result in changes in the composition of the proteasome. One of the more important pieces of data that is missing from our knowledge regarding the role of alternative splicing in obesity-linked hypertension is the contribution of environmental factors to changes in the population of splice variants. Further characterization of splicing variants may allow these splice variants to be used as diagnostic or prognostic markers for metabolic and hypertensive disorders. They may also serve as lead targets for the development of drugs. The current strategies of using small molecules or antisense oligonucleotides to regulate and correct aberrant splicing are both promising avenues of investigation. One of the major sources for small molecules that may act on splicing is plants and plant-based compounds.


Introduction. On 23 October 2015, six related cases with gastroenteritis called the Netherlands Food and Consumer Product Safety Authority. They suspected filet américain, a raw beef spread, to be the source of infection. Leftovers and stool samples tested positive for Salmonella Typhimurium. Multiple locus variable-number of tandem repeat analysis (MLVA) revealed a MLVA pattern (02-23-08-08-212), which had not been detected in the Netherlands before. Concomitantly, an increase of this MLVA type was observed in the national Salmonella surveillance, amounting to 46 cases between 26 October and 9 December.Methods. To investigate whether filet américain or an alternative (related) source could  be linked to surveillance-reported cases, cases (n=38) were invited to complete a questionnaire and upstream source tracing to map the food supply chain was initiated.Results. Rapid interdisciplinary action resulted in identification of a contaminated 46-ton batch of beef distributed via a Dutch deboning plant as the likely source of infection. In total, 24/29 respondents (83%) could be linked to the incriminated batch of beef products (predominantly filet américain and minced beef).Discussion. Repeated identification of raw meat products as a source of infection emphasizes the importance of awareness of the risk of infection when handling or consuming these products. Improved measures and procedures on product labelling, pre-treatment or product testing should be considered. Enteritidis) – imported during a large avian influenza virus outbreak – and salmon (S. Thompson)4,S. Typhimurium with a novel phage type (23 cases), but microbiological analysis of incriminated meat products could not confirm the associationIn 2003 and 2012, the two largest recent national On 9 November 2015, an outbreak of Salmonella Typhimurium with a unique multiple locus variable-number of tandem repeat analysis (MLVA) pattern (02-23-08-08-212) was identified.Salmonella contamination to prevent further cases and new outbreaks.An outbreak investigation was started with the following aims (i) to find out whether cases of the local cluster and cases identified through the national surveillance shared a common or related source of infection and (ii) to trace the origin of the Case definitionSalmonella Typhimurium with MLVA pattern 02-23-08-08-212 had not been previously detected in the Netherlands, a case was defined as a person with laboratory-confirmed diagnosis for the outbreak Salmonella type. The sixth case within the local cluster was not laboratory-confirmed, but included as probable case due to the strong epidemiologic link with the other cases in the cluster.As Microbiological investigationSalmonella based on the presence of the invA gene, followed by transferring positive samples to the second enrichment (MSRV). Different from the method in the amendment, selective plating media BGA and MLCB, instead of XLD, were used for the isolation of Salmonella. This method, including the screenings PCR, has been validated for horizontal use in food matrices.Strains isolated from human samples and from food products were detected and isolated by medical microbiological laboratories and the Netherlands Food and Consumer Product Safety Authority , respectively. The method used for the food samples is based on the NEN-EN-ISO 6579/AI, which is an amendment to ISO 6579 published in 2007. Additionally to this method, the first enrichments (BPW) were screened for the presence of Salmonella standardsS. Typhimurium were also typed by means of MLVA, which can discriminate between S. Typhimurium strainsAt the RIVM, strains were serotyped based on O- and H-group antigens according to the World Health Organization (WHO) Collaborating Centre for Reference and Research on DNA testing of meatSalmonella source attribution model, as described earlierS. Typhimurium with this MLVA type. To investigate whether filet américain might have been contaminated with pork, we tested the leftovers for the presence of pig DNA using real-time PCR with the Biorad CFX 96 system.A Epidemiological investigationThe surveillance-reported cases were invited to complete a questionnaire, either by phone or by mail conducted by the regional Public Health Services. As the index cluster provided a strong lead towards the most likely source of infection, we used a questionnaire tailored to consumption of various meat products [beef (raw ingredient of filet américain) and pork (indicated by source attribution model)] and venues where meat products were purchased (such as butchers or supermarkets). The questionnaire comprised 12 pre-listed supermarket chains. All questions focused on the 7 days prior to the onset of symptoms. Other items of the questionnaire included sections on demography, clinical symptoms, date of onset and duration of gastrointestinal illness, hospitalization and travel history. Data from questionnaires was entered into Microsoft Access  and analyzed using descriptive epidemiology. The analyses were conducted in SAS for Windows  and Microsoft Excel.Ethical considerationsMedical Research Ethics Committee review and approval were not required as these kind of investigations are part of the routine public health response to an outbreak of salmonellosis. Cases were not asked for explicit informed consent, but indicated their willingness in participation in the interview. A case register including personal identifiers was used during the outbreak, which was needed for outbreak response and action. This register was only accessible by the outbreak management team who also performed all analyses.Trace-backS. Typhimurium isolated from filet américain consumed by the local cluster, the NVWA launched an investigation to trace-back the origin of the contamination and surveyed whether contaminated meat products were still on the market. The investigation aimed to map the food supply chain from retail/catering premises to processors/slaughterhouse, to assess production hygiene, ‘Hazard Analysis and Critical Control Points’ (HACCP) and traceability.During outbreaks, the NVWA is responsible for food trace back investigations according to Directive 2003/99/ECStart of the outbreakOn 23 October 2015 , a citizen living in the Southern part of the Netherlands, called the NVWA to report that in total six persons fell ill with gastrointestinal illness after having consumed filet américain, a bread spread consisting of finely chopped raw beef mixed with a herb sauce. A stool sample from one case of the cluster tested positive for Salmonella spp. On 29 October, stool samples from four of the five remaing cases tested positive for S. Typhimurium. On the same day, the NVWA detected S. Typhimurium in the filet américain leftovers, informed the regional Public Health Service ‘Hart voor Brabant’ who subsequently reported this local cluster to the RIVM.On 9 November, typing results showed that the Salmonella isolates from the local cluster and the food leftovers had a unique MLVA-pattern (02-23-08-08-212). Furthermore, 26 additional cases were identified in that week. To check whether other European countries experienced a concurrent identical outbreak or had had an outbreak that could be linked to this one, an urgent inquiry was placed on the Epidemic Intelligence Information System for Food- and Waterborne Diseases and Zoonoses (EPIS-FWD) operated by the European Centre for Disease Prevention and Control (ECDC). This yielded no clues for solving the outbreak.Between 26 October and 9 December 2015, 45 outbreak cases were laboratory-confirmed (including five from the index cluster of six cases). For 34 cases, the date of onset of illness was known ; the majority  developed symptoms between 14 October and 21 October, whereas one case fell ill on 27 October. One case (4%) reported having been abroad for one day. The cases identified through the national surveillance (n=40) were distributed across the country. The majority of the laboratory-confirmed cases were female  and ages ranged from 2 to 79 years (median 21). Of these, 15 cases were 9 years of age or younger (33%), six cases were aged 10-17 years (13%), 16 were aged 18-49 years (36%) and eight cases were 50 years or older (18%).Information from questionnaireThe six cluster cases were not interviewed, as the only common source they have shared eating was filet américain and already a microbiological link with the filet américain was found for this cluster. All other cases typed until 23 November (n=38) were invited to fill in the questionnaire. Among those, 29 responded (response rate 76%). All 29 cases reported gastro-intestinal symptoms, eight (28%) were hospitalized. Diarrhoea lasted between 3 and 21 days (median: 9 days), based on information provided by 21 cases (72%).Consumption of filet américain was most often reported (76%), followed by minced beef (55%). In total 26 cases (90%) ate one or both products in the week before illness. In a continuous survey ongoing since 2008Trace-back investigations of food productsThe local cluster cases reported having purchased filet américain at supermarket chain D. Supermarket chain D produced filet américain locally by mixing prepared ground beef and a ready-made herb sauce. Inspection of the involved local production site did not reveal any breach of hygiene standards. Trace-back of the contaminated batch led to meat producer 1 . P1 reported delivering 70% of its total meat supply to supermarket chain D. Raw meat linked to the contaminated filet américain was distributed to 300 branches of this chain. All raw material for filet américain at supermarket chain D originated from P1. These tracing efforts could link another 15 cases to supermarket chain D.The raw material distributed by producer P1 was traced back to a Dutch deboning plant, which had processed ~58 tons of meat parts on 8 October 2015, originating from six slaughterhouses from four different European countries . In total, the deboning plant delivered ~46 tons of meat for human consumption to 55 meat-processing plants; of these, 32 were located in eight other EU Member States (~15 tons). Using information from forward and backward tracing, together with information from the questionnaires, we could establish links between the Dutch deboning plant and suppliers of three other supermarket chains, a diner and delicacy shop to which nine additional cases could be linked.Salmonella to an acceptable level taking into account consumers’ consumption habitsThe NVWA – via juridical ordinance – commissioned the plant on 1 December 2015 to inform all customers that received parts of the contaminated batch. Dutch customers of the deboning plant were legally required to inform the NVWA how much of the contaminated meat they had processed and distributed. As the contaminated 46-ton batch of beef was mixed with meat from other parties at various customers, the contaminated batch amounted to 400 tons in total. Of those 400 tons, around 65 tons (16%) were classified as risk products defined as “ready to eat” products or products very likely to be consumed with a mild treatment, insufficient to eliminate or reduce the risk of infection by Microbiological testing of food productsSalmonella spp. No samples were taken at the deboning plant as the particular batch was already distributed.Besides the positive leftovers, samples of filet américain and separate samples of sauce and raw meat meant to be used for filet américain were taken at producer P1. All samples were from other batches than the incriminated batch, and tested negative for Although the source attribution model indicated that the outbreak MLVA type was more likely to be linked with pork/pigs (58%), than with beef/cattle (3%), no pig DNA was detected in the leftover filet américain.S. Typhimurium with MLVA type 02-23-08-08-212 linked to raw and undercooked beef products. Based on microbiological and epidemiological evidence, beef sold as filet américain and minced beef was the source of infection. Timely involvement of the different authorities and exchange of information regarding epidemiological, laboratory and trace-back results led to the identification of a 46-ton batch of beef. The batch was distributed via a Dutch deboning plant. The plant received its meat from slaughterhouses outside the Netherlands, and thus outside Dutch authority, and with Salmonella not being a food safety criteria on farm level, further trace back was not performed. As parts of this batch could be traced forward to eight other EU countries, an alert in the Rapid Alert System for Food and Feed (RASFF) was placed. Risk products that were still on the market were withdrawn to protect consumers.Here we describe the investigation of a national outbreak of S. Typhimurium infectionS. Typhimurium outbreaks7,17,18,S. Typhimurium was linked to an outbreak in Denmark, which preceded the outbreak in the Netherlands‘Exposure to raw beef’ and ‘consumption of undercooked meat’ are known risk factors for ,Although trace-back revealed a batch of 46 tons as contaminated, only 46 cases were reported. The identified batch is the production of one day in the deboning plant. It is therefore likely that the batch will not be contaminated completely or evenly due to a point source contamination of one or more carcasses. Secondly, the number of cases will be an underestimation as most people with gastroenteritis do not visit a physician or are not tested. It was estimated earlier that around 1 in 20 Salmonella patients will be laboratory-confirmed25S. Typhimurium with identical MLVA type. Raw material used for this product was subsequently linked to the 46-ton batch distributed via the deboning plant .In outbreak situations where the source of infection has a short shelf life, microbiological detection of the pathogen in the food source is often not possible as the product is no longer on the market when trace-back efforts commence. In the current outbreak, the alert by the local cluster allowed timely microbiological testing of leftovers of filet américain, which expedited the process of identifying the underlying source. Parts of the contaminated batch of filet américain were still on the market (expiry date products producer P1: 17 October 2015) and could be recalled. Besides early identification of an outbreak, collaboration between regional and national authorities, and public health and food authorities is imperative to find the source. Regular contact and exchanging of gathered information proved to be crucial. Trace-back efforts could link 24 of 29 interviewed national surveillance-reported cases to the contaminated batch of beef. Although leftovers cannot be regarded as formal evidence for the source of infection, the microbiological results provided a strong clue for the trace-back, which was later confirmed by the positive results of a survey done independent from the outbreak investigations. An animal welfare organization performed microbiological tests in mini hamburgers included in a package containing a selection of different types of meat for raclette grilling. These mini hamburgers tested positive for The moment the national outbreak was detected, the local cluster could be linked to filet américain. Furthermore, the MLVA pattern had not been seen earlier in the Netherlands. Therefore, we refrained from conducting a classical case-control study and decided to tailor the questionnaire to meat products for two purposes. First, to confirm beef products as source of the outbreak and secondly, to gather information on type of beef products, purchase dates and purchase sites to feed the trace-back. This contributed to a timelier start of the trace-back investigation and a likely quicker withdrawal of incriminated meat. To check whether filet américain or other beef products indeed were eaten more often than expected, we used the national controlsurveySalmonella entering the food chain. However, it has to be acknowledged that trace-back operations remain cumbersome and time-consuming. During the trace-back operation, the complexity of the food supply chain and diverse stakeholders involved became apparent, once again. All parties involved were inspected by different divisions within the same competent authority and a good mutual communication is essential. Figure 3 gives a peek view of all parties involved in a one-day production of the slaughterhouse. Risks related to contamination at this stage of the supply chain should not be underestimated and can lead to great health risk as well as economic consequences due to recall and destruction of incriminated batches, as shown in this case.Trace-back investigation should be undertaken wherever possible to identify the initial source of contamination as this can lead to better source attribution and provide further evidence for policy changes at primary production and processing to reduce the risk of S. Typhimurium infection , possibly relating to a higher susceptibility of this age group. As the very young, elderly, pregnant and the immunocompromised are known to be at greatest risk of severe disease and death in relation to food- and waterborne infectionsOne third of cases involved in the current outbreak was 9 years or younger with the youngest case being two years old. Kivi et al.In general, to ensure food safety, batches of products that are intended to be eaten without sufficient heat treatment, should be submitted to risk based HACCP procedures in order to verify compliance with regulations. If such standards are not met – or as a general part of the production process - pre-treating risk products prior to sale could be considered. Pre-treatment methods, such as high-pressure processing that applies pressure to food products and thereby inactivates microorganism without alteration of taste or texture of the food product, constitute promising methods for that purposeS. Typhimurium outbreak. This outbreak yet again emphasizes the importance of awareness among consumers of the risk of infection when consuming or handling raw meat products. To increase transparency for consumers, we recommend adding warning labels to risk food products.In conclusion, this outbreak investigation highlighted the importance of close collaboration between regional Public Health Services, the NVWA, the RIVM and diagnostic laboratories. Through a combined microbiological, epidemiological and source tracing approach, raw and undercooked beef products could be identified as the source of the The authors have declared that no competing interests exist.Access to data are restricted to protect the confidentiality of individuals and premises involved in this outbreak. Researchers interested in accessing an anonymised minimal data set should write to the National Institute for Public Health and the Environment (details below), who will assess the request. Requests for data should be addressed to: Department Epidemiology and Surveillance of Gastroenteritis and Zoonoses (pb 75), Centre for Infectious Disease Control (CIb), National Institute for Public Health and the Environment (RIVM), Pbox 1, 3720 BA Bilthoven, the Netherlands (eelco.franz@rivm.nl).Ingrid Friesema: ingrid.friesema@rivm.nl


Intracellular gene delivery is normally required to study gene functions. A versatile platform able to perform both chemical transfection and viral transduction to achieve efficient gene modification in most cell types is needed. Here we demonstrated that high throughput chemical transfection, virus packaging, and transduction can be conducted efficiently on our previously developed superhydrophobic microwell array chip (SMAR-chip). A total of 169 chemical transfections were successfully performed on the chip in physically separated microwells through a few simple steps, contributing to the convenience of DNA delivery and media change on the SMAR-chip. Efficiencies comparable to the traditional transfection in multi-well plates (~65%) were achieved while the manual operations were largely reduced. Two transfection procedures, the dry method amenable for the long term storage of the transfection material and the wet method for higher efficiencies were developed. Multiple transfections in a scheduled manner were performed to further increase the transfection efficiencies or deliver multiple genes at different time points. In addition, high throughput virus packaging integrated with target cell transduction were also proved which resulted in a transgene expression efficiency of >70% in NIH 3T3 cells. In summary, the SMAR-chip based high throughput gene delivery is efficient and versatile, which can be used for large scale genetic modifications in a variety of cell types. In the post-genome era, with the continuous development of genomics, the study of gene function has become a hotspot in life science research. The modification of gene expressions by introducing genetic materials into cells is a common strategy of studying gene function . UsuallyChemical transfection and viral transduction are two of the most widely used methods for introducing genetic materials into cells. Chemical transfection is easier to conduct, however it obtains high efficiencies only in a few fast dividing cell lines, such as HEK293 cells . On the Reverse transfection ,7,8 has Microfluidic devices for gene delivery have been numerously reported, which offer the advantages of high throughput, low reagent consumption, and independently controlled liquid conditions, however most of the studies focused on chemical transfection ,14 or elIn our previous study, we developed the superhydrophobic microwell array chip (SMAR-chip) for investigating the microenvironmental factors that influence stem cell behavior . Here, wThe chip fabrication was done as previously described . Briefly2 and passaged every 3–5 days for fewer than 10 passages before being abandoned.The cells  were cultured in high glucose DMEM, supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin . All the cells were cultured in a 37 °C incubator with saturate humidity and 5% COPrior to loading cells, the SMARchip was soaked in 75% ethanol for 15 min and exposed to UV light for 2 h. Microwells were first filled with liquid by dripping DMEM onto the chip with a 10-mL stripette, which was held ~20 cm above the chip to expel the air out of the microwells. Cells were then loaded into the microwells by submerging the entire chip into a cell suspension solution for 15 min to allow the cells to settle, followed by aspiration of the excess cell suspension. The chip was then submerged in the culture media overnight for cell attachment.6/well in six-well plates 1 day before the transfection. The next day, cells were transfected using Lipofectamine® 2000  according to the manufacturer’s instruction. The images of the transfected cells were acquired 48 h after the transfection.293T cells were seeded at a density of 1 × 10® 2000 (Invitrogen) for 10 min. Then, the medium was aspirated, and the chip was swept with a cell spreader to form a uniform droplet array. Plasmid solutions  encoding green  or red (constructed by replacing the GFP sequence in pll 3.7 with an RFP sequence) fluorescent protein were spotted on a glass slide in a desired format using a microarray spotter  that emitted a volume of 30 nL per spot. The glass slide was aligned and sandwiched onto the microwell array with the aid of a custom-made aligner. Then, 4–6 h after the addition of plasmids, the transfection was stopped by submerging the chip in normal culture medium. For multiple-round transfections, three rounds of transfections, each of which lasted 4 h, were consecutively performed without interruption. The images of the transfected cells were acquired 48 h after the last round. For sequential transfections, the two rounds of transfections were performed on day 1 and 2, respectively, with an 18-h interval in between.293T cells were seeded at a density of ~80 cells/well. After overnight culture, cells in the microwells reached 70–80% confluency. The normal culture medium was aspirated, and the chip was submerged in Opti-MEM with  encoding the red fluorescent protein for 6 h. Viruses were harvested 48 h after the end of the transfection. A total of 1 mL of the virus media supplemented with 8 mg/mL polybrene (Sigma Aldrich) was added to the NIH-3T3 cells (~50% confluent) seeded in the six-well plate. The virus containing media was replaced with fresh media after 6 h and the images of the transducted cells were acquired 48 h later.The virus packaging cells Plat-E were seeded in six-well plates at the density of 1 × 10The virus packaging cells Plat-E were seeded on the SMARchip at a density of ~80 cells/well and incubated overnight. The next day, cells were transfected with the retroviral vector pMX-red (Addgene) encoding the red fluorescent protein for 6 h, using the above-mentioned procedures. The chip was then submerged in normal cell culture media for 10 min to stop the transfection, followed by aspiration of the excess media to form droplets in the microwells. The chip was placed in a petri dish with sterile water to prevent evaporation of the droplets. Virus was produced and accumulated in the droplets for a period of 24 h before aligned and sandwiched onto the chip seeded with the target cells . Then, 6 h after the assembling, the chip with target cells was submerged in fresh culture media to complete the transduction. The images of the target cells were acquired 48 h later.cS is the fluorescent signal from the transfected/transducted cells, acS is the total signal from the thresholded cells, wS is the total signal from the microwell, wA is the total area of the microwell, and cA is the total area of the thresholded cells [Images of the cells were taken using an inverted microscope  equipped with a charge-coupled device (CCD) camera  using a 10× objective and suitable filters for the fluorescence wavelength. The microwell array was scanned with a motorized stage  using a 4× objective. The transfection and the transduction efficiencies were calculated as the number of cells expressing the fluorescent protein genes over the total number of cells in the microwells. The number of fluorescent positive cells was counted manually. All quantification was performed by three researchers blinded to the conditions. The results are reported as mean + standard deviation. The total fluorescent intensity was measured using ImageJ. The images were first thresholded to identify the transfected or infected cells. The fluorescent signal from these cells were calculated as: The SMAR-chip with a 13 × 13 microwell array was microfabricated which was composed of a PDMS substrate with the microwells  were spotted on a glass slide in a desired format using a microarray spotter  that jetted a volume of ~30 nL per spot. The glass slide was then upside-down aligned and sandwiched onto the microwell array with the aid of a custom-made aligner to deliver the DNA to the cells. After 4–6 h of incubation, the transfection was stopped by submerging the chip in normal culture medium. The DNA-Lipofectamine® 2000 mixtures should be delivered to the cells immediately after spotting to avoid evaporation.We developed two procedures (the dry method and the wet method) to conduct the high-throughput chemical transfection through a few simple steps as shown in ® 2000) was added to the Opti-MEM and delivered to the cells during the submerging step (step 2). The glass slide with the spotted plasmid solution can be dried and preserved for up to 15 months [While for the dry method, the transfection reagent  in published data [Although the chemical transfection is easy to conduct, its efficiency is low for most of the primary cells . Exploithed data ,24. As sThe SMAR-chip-based gene delivery is convenient and versatile which overcomes the limitations of the conventional multi-well plate-based techniques and the reverse transfection. Here, 169 transfections were done simultaneously in physically separated microwells on the SMAR-chip. The throughput can be further increased by fabricating larger chips with more microwells. The labor consuming well-by-well operations are avoided since media exchange in all the microwells is done by submerging the chip in fresh media for 10 min followed by aspirating the excess media. The genetic materials are delivered to the microwells by sandwiching the DNA array with the SMAR-chip. In the future, DNA can be added to the microwells through centrifuge using our previously reported nanoliter centrifugal liquid dispenser to further simplify the whole procedure . In cont® 2000) facilitates the formation of the DNA-liposome complex.In addition, the dry and the wet methods have distinct advantages, and both achieve efficiencies higher than 60%, similar to the conventional transfection in cell culture plates. The dry method is more convenient to the end users since the glass slides with spotted DNA arrays can be preserved for a long time, thus not requiring the availability of the microarray spotter. While, the wet method has higher transfection efficiency as the premixing of DNA with the transfection reagent (LipofectamineMore importantly, on chip high-throughput virus production combined with in situ target cell infection was achieved. As a proof of concept, transduction of the NIH 3T3 cells results in expression of the transgene in about 76% of the target cells, much higher than the previously reported 22% for chemical transfections ,24. ThesIn summary, the SMAR-chip can be used as an efficient platform for high throughput gene delivery through chemical transfection or virus transduction. In the future, a suitable method can be chosen according to the cell type and the experimental design. In addition, the SMAR-chip technology can be used in other high-throughput cell microarray-based assays, such as drug screening and the analysis of factors that influence cell behavior, owning to the convenience of reagent delivery and the isolated media conditions in individual microwells.


This paper presents Neutron Capture Enhanced Particle Therapy (NCEPT), a method for enhancing the radiation dose delivered to a tumour relative to surrounding healthy tissues during proton and carbon ion therapy by capturing thermal neutrons produced inside the treatment volume during irradiation. NCEPT utilises extant and in-development boron-10 and gadolinium-157-based drugs from the related field of neutron capture therapy. Using Monte Carlo simulations, we demonstrate that a typical proton or carbon ion therapy treatment plan generates an approximately uniform thermal neutron field within the target volume, centred around the beam path. The tissue concentrations of neutron capture agents required to obtain an arbitrary 10% increase in biological effective dose are estimated for realistic treatment plans, and compared to concentrations previously reported in the literature. We conclude that the proposed method is theoretically feasible, and can provide a worthwhile improvement in the dose delivered to the tumour relative to healthy tissue with readily achievable concentrations of neutron capture enhancement drugs. The objective is to minimise normal tissue complication probability (NTCP) - which includes the probability of developing treatment-induced cancers - by maximising the conformity of the delivered dose to the target volume5. Technological advancements in radiotherapy such as intensity modulated radiotherapy, image-guided radiotherapy and particle therapies have enabled more accurate and selective targeting of the tumour6. Many of these treatments can also be enhanced by the use of tumour-specific radiosensitisers, which increase the contrast between the biological dose delivered to the tumour and healthy tissues7.The principal aim of all forms of radiation therapy is to deliver the maximum therapeutic radiation dose to the target, while sparing surrounding healthy tissue. One of the greatest challenges of radiotherapy is to minimise its latent effects - including the risk of secondary cancer, which can occur anywhere from five years to many decades post-treatment9.Particle therapy is a form of radiotherapy in which a beam of highly energetic protons or heavy ions (most commonly carbon) is used to deliver a therapeutic radiation dose to a treatment region. Monoenergetic beams of protons and heavy ions exhibit a very well-defined Bragg peak, depositing most of their kinetic energy within a narrow energy-dependent depth range. By tuning the beam energy, this property enables the delivery of a highly conformal radiation dose distribution to the target. Particle therapy is therefore ideal for treatment of deep tissues, since it results in a lower dose to healthy tissues in front of or behind the tumour compared to photon and electron beams11. The thermal neutrons irradiate both target and non-target tissues indiscriminately, and they deposit a fraction of the beam’s kinetic energy outside of the target volume9. Such interactions are typically regarded as a nuisance when they occur outside of the treatment region, since their existence undermines one of the main advantages of particle therapy - the large peak-to-entrance-plateau dose ratio. It is also important to emphasise that these thermal neutrons are generated internally - in the vicinity of the target area inside the patient. This is quite distinct from the neutrons which result from contamination of the beam due to collisions between the beam and beam modifying devices.During particle therapy, most of the primary particles in the beam deposit their kinetic energy through multiple electromagnetic interactions with the target. However, a fraction of these particles will undergo nonelastic collisions with nuclei in the target. This results in the production of a range of nuclear fragments at the target site, including short-range, high-LET charged particles, and a mixture of fast and thermal neutrons. Due to scattering within the target tissue, the thermal neutrons are emitted nearly isotropically from the point of collision, and they deposit their energy in the region surrounding the path of the incident ion beam via a succession of elastic and inelastic collisions12), which can be greatly increased by the introduction of agents containing isotopes such as 10B or 157Gd with very high neutron cross sections . While nonelastic thermal neutron interactions with water primarily result in hydrogen capture of the neutron and the release of a high-energy gamma photon, nonelastic thermal neutron interactions with 10B and 157Gd result in the production of energetic charged particles with high relative biological effectiveness - the basic operating principle of neutron capture therapy (NCT)16. Our central hypothesis is that if a sufficient thermal neutron fluence is generated during heavy ion therapy, it can be exploited therapeutically via the administration of a suitable non-toxic neutron capture agent containing 10B or 157Gd, preferentially absorbed by the tumour at an elevated concentration compared to the surrounding normal tissue as in conventional NCT. This combined therapeutic modality is denoted neutron capture enhanced particle therapy (NCEPT). To validate our hypothesis, and demonstrate the feasibility of NCEPT, it is necessary to determine the concentrations of neutron capture agents which would be required to achieve a therapeutically significant boost to tumour dose during a typical proton or carbon ion treatment plan.Light water, the principal constituent of human tissue, has a moderate thermal neutron cross-section (0.335 barnsin vitro vs. in vivo); it is also specific to each specific neutron capture agent. For this work, only values reported for in vivo studies are relevant. In BNCT literature this compound-specific RBE factor is commonly referred to as compound biological effectiveness (CBE), however most researchers working with gadolinium simply refer to it as RBE. For consistency we refer to it henceforth as CBE for both boron and gadolinium.In conventional neutron capture therapy, the biological dose due to the presence of the capture agent depends on the physical dose , together with the relative biological effectiveness (RBE) of the secondary particles as determined by the specific neutron capture agent (NCA). The latter factor varies significantly between different cell types and context , CBE values of 3.6–3.8 and 0.9–1.3 have been reported for brain tumour cells and normal tissues, respectively, with tumour to healthy tissue concentration ratios between 5:1 and 8:123. An alternative capture agent, borocaptate sodium (BSH), has shown potential for NCT applications; the reported range of CBE is between 1.2 and 2.3 in brain tumours and 0.37 to 0.5 in normal tissues, although the uptake concentration ratio tends to be much lower than for BPA (1.2–3.5 in the brain)25. The specific values differ for other target tissues, with higher values of CBE reported for liver tumours for both agents 26.For the most widely used 157Gd neutron capture reaction follows a somewhat different path, and results in the production of an excited 158Gd nucleus and a high-energy gamma ray:32. A yield of 5 Auger electrons, 1.8 γ photons and 0.69 IC electrons and 1.0 recoil nucleus has been estimated for the thermal neutron capture reaction34.The et al. conducted a review of dosimetry of Auger electrons at DNA, subcellular and organ level, and recommended a CBE of 10 when the emitter is electrostatically bound to the DNA of the nucleus35. Fairlie performed a review of the radiation-weighted factors and RBEs of Auger-emitting isotopes, and recommended the adoption of CBEs of 5 and 20 for emitters of multiple Auger electrons incorporated within cells and DNA, respectively36. Cerullo et al. estimated the lineal energy distribution of Auger electrons within a 6 nm diameter cylinder (representing a DNA fragment) using the PENELOPE Monte Carlo simulation code, and obtained CBEs of 12.6, 6 and 1.5 where electrons are localised inside, on the surface and outside of the cylinder, respectively31.A number of studies have utilised Monte Carlo simulations to quantify the macroscopic, microscopic and nanoscopic components of the dose delivered by Auger-emitting isotopes in order to estimate their corresponding CBE. Humm 157Gd is of great interest for neutron capture therapy due to its extremely high thermal neutron cross-section - the highest of any stable isotope. While free Gd3+ ion is highly toxic to organisms both in vitro and in vivo, chelated Gd3+ compounds can be used safely due to their physiological stability37. Very high cellular concentrations of gadolinium can be achieved in vitro without significant cytotoxicity 39. While gadolinium contrast agents such as Gd-DOTA and Gd-DTPA are approved for use in humans diagnostically, neither accumulates to significant concentration within the cell nucleus40. Amongst the experimental gadolinium compounds, motexafin-gadolinium (MGd) been proposed as a potential candidate for GdNCT37. It is a tumour-specific radiosensitiser, and its combined use with whole-brain radiation therapy has reached Phase III clinical trials41. With a 70:1 tumour to healthy tissue uptake ratio, prolonged retention of gadolinium in vitro (up to 2 months) and 90% uptake in glioblastoma cell nuclei, it is a promising candidate for use in NCT44. Recent efforts towards the development of DNA and mitochondria-targeting gadolinium agents has resulted in a number of promising agents. Morrison et al. have reported on the development of a tumour-cell selective mitochondrial agent designed for NCT applications, with cellular concentrations of up to 3000 ppm46.10B capture agents with external neutron beams is already an established radiotherapy modality, with traditional reactor-based neutron sources now being supplanted by more compact accelerator-based beam facilities. A number of accelerator-based epithermal neutron beam are in various stages of development and commissioning in hospitals in Japan, Finland, Russia, Taiwan, Argentina, Italy and the UK56. Two 10B delivery agents, L 10B-BPA) and sodium mercaptoundecahydro-closo-dodecaborate  have been used clinically to treat patients suffering from glioblastoma multiforme and malignant melanoma, with Phase II and III clinical trials for the treatment of glioblastoma (GBM), head and neck tumours and liver metastases underway in Japan, Finland, Sweden, Japan, Taiwan, and the United States65. However, treatment of tissues deeper than approximately 3 cm is challenging with external epithermal neutron beams, due to the very high neutron fluence at the surface which is required to achieve a therapeutic effect at the target - a consequence of the neutron-moderating effect of the water in human tissue67.Neutron capture radiotherapy based on 10B and 157Gd-based NCAs required to achieve an arbitrary 10% boost to the tumour biological effective dose are calculated, and compared to tissue concentrations in different tissues previously reported in the literature, with the aim of demonstrating the feasibility of NCEPT.In this paper, the quantity and spatial distribution of the thermal neutron fluence generated by typical therapeutic scanned-beam proton and carbon ion irradiations of homogeneous PMMA targets has been estimated via Monte Carlo simulations. The tissue concentrations of several Details of the Monte Carlo simulations, including the primary beam energy and geometry, phantoms and physical models, as well as the method used in the quantification of the neutron capture dose enhancement are presented in Section 2. Neutron fluence results and the estimated neutron capture dose enhancement for boron and gadolinium is presented in Section 3, with the implications for enhanced biological effective dose discussed in Section 4. Conclusions and proposed future work are presented in Section 5.The method described here aims to establish whether NCEPT, when used in conjunction with a typical treatment plan, offers a significant therapeutic benefit compared to proton or heavy ion therapy alone. For the purpose of this study, significance is arbitrarily defined as an average 10% increase in photon-equivalent dose within the tumour resulting from the administration of a non-toxic bolus of neutron-capture agent; however, the method can be used with any desired dose increase factor. To answer this question, it is necessary to determine the concentration of neutron capture agent which is required in order to provide a 10% increase in effective photon-equivalent dose, for a simple simulated therapeutic proton/heavy ion treatment plan, and compare this with concentrations reported in the literature.12C beams, is conducted with four different energies in a homogeneous PMMA target. Dose and thermal neutron fluence distributions are recorded for each simulation; corresponding distributions at energies in between these are also estimated by interpolating between the distributions obtained at these energies.The first step is to evaluate the thermal neutron fluence (defined as neutrons with kinetic energy below 0.4 eV) resulting from pencil-beam irradiation of a point within a target volume. A set of simulations of this pencil beam, for both proton and A simple treatment plan is then implemented, in which the pencil beam is stepped across an array of points inside a treatment volume at a series different energies. The primary particle fluence at each energy is then weighted such that a specified, approximately flat biological effective dose (BED) is delivered to a defined treatment volume by the ion beam. Two 50 mm cubic volumes are evaluated, once centred at a depth of 125 mm and a second centred at a depth of 165 mm. Thermal neutron fluence distributions throughout the entire phantom are then estimated based on the primary particle fluence weights for each treatment volume.10B and 157Gd-based NCAs required to achieve a 10% increase in biological effective dose is then determined for each treatment volume. The resulting NCA concentrations in normal tissue  are calculated using the reported tumour to normal tissue ratios for these NCAs. Maximum additional dose delivered to normal tissue is then determined as a percentage increase in dose to normal tissue in excess of the dose delivered by the primary beam.Based on the thermal neutron fluence estimates obtained through this process, the concentrations of a number of 69. Electromagnetic interactions were modelled using the standard Geant4 physics option 3 model (G4EmStandardPhysics_option3), while the hadronic physics models used in the simulations are listed in Table All Monte Carlo simulations were performed using the Geant4 toolkit version 10.2.p03)0.2.p0369M = 50 runs of M simulations. The inter-run standard deviation should approach zero as k until the ratio of inter-run standard deviation to mean was less than an arbitrary threshold of 5%. This analysis showed that N = 5 × 107 incident protons and N = 5 × 106 12C ions is sufficient to obtain a satisfactory estimate of thermal neutron fluence (99% probability of the estimated fluence being within ±5% of the true fluence).A simple variance analysis method was used to determine the minimum number of primary particles to use in the simulations. A series of test simulations were conducted, each with 12C ions with a rotationally symmetric 5 mm FWHM Gaussian beam profile were directed perpendicularly towards the surface of a homogeneous PMMA phantom. Four reference primary beam energies were chosen for the 12C beam, resulting in Bragg peak depths in PMMA of between 45 mm and 191 mm. Beam energies were then calculated for the proton beam such that the Bragg peaks were located at approximately the same depths. The full set of beam energies for each primary particle type and the corresponding locations of Bragg peaks in each phantom are listed in Table 70. PMMA is chosen as the target material as it has a similar electron density and hence depth-dose profile to human tissue, and yields a similar range of fragmentation products despite its differing elemental composition71; moreover, it will be a convenient target for future experimental work.The Geant4 simulation and analysis configuration is shown in Fig. 12C beams, for energies in steps of 1 MeV/u in the range 73–182 MeV/u for protons and 150–350 MeV/u for 12C. While the method is only an approximation, its accuracy can be improved if desired by performing simulations at additional energies in the range of interest.Pencil beam physical dose and thermal neutron fluence distributions were obtained for each beam type and energy, as listed in Table 12C it was assumed to be 2.5, 3.0 and 3.3 for the pencil beams corresponding to the proximal, centre and the distal edges of the spread-out Bragg peak, 1.5 in the entrance plateau and buildup region , and a linear interpolation between these values in the intermediate regions74. The biological dose distributions were then used to develop a simple treatment plan for two target volumes for each beam type. These three-dimensional dose distributions for the centred pencil beam at the k th energy  plane of the treatment volume corresponding to the Bragg peak depth at each energy, through R row and C column positions  for each of the k energies:D, then the objective is to achieve the most uniform approximation of this dose possible within the treatment volume by determining the number of primary particles Nk must be positive. The total number of primary particles required at each energy can then be multiplied by the corresponding map of neutron production per primary particle for each energy, to yield a map of total neutron fluence ϕ throughout the phantom (both inside and outside of the treatment volume):75:−14 for 10B and 9.27 × 10−15 for Gd77), NNCA is the concentration of neutron capture agent in parts per million, and the the compound biological effectiveness CBE = 3.8 for 10B-BPA and ≈40 for the DOTA 157-Gadolinium triphenylphosphonium salt complex 76. For this study, the target dose was set to D = 1 GyE, R = C = 11, and steps between rows and columns were set to 5 mm (i.e. the same as the FWHM of the beam) for a 50 mm square treatment plane at each energy. A range of energies were selected to extend the spread out Bragg peak (SOBP) between depths of 100 mm and 150 mm for the first treatment volume and 140 mm to 190 mm for the second; energies were incremented in steps of 1 MeV/u. Therefore, each treatment volume is a 50 mm cubic volume, with 1 GyE of dose delivered by the ion beam whilst the remainder of the phantom is regarded as normal tissue.As this study is concerned with determining the feasibility of NCEPT rather than evaluating a specific treatment plan, a set of simple treatment plans were developed for the PMMA target in to estimate the order of neutron capture agent concentration that would be required to achieve a 10% increase in photon-equivalent biological dose. For each energy, the BED and thermal neutron fluence maps  are stepped across the transverse  is shown in Fig. The per-primary-particle thermal neutron distributions corresponding to each of the energies in the treatment plan were scaled by the number of primary particles determined for each plan and summed for all energies required to form the spread out Bragg peak. An example of the resulting distribution of thermal neutron fluence  is shown in Fig. The maximum, mean and minimum thermal neutron fluences obtained within the treatment volumes are listed in Table 10B and 157Gd required to achieve a 10% increase in biological effective dose are listed in Tables The tumour concentrations of Normal tissue NCA concentrations are calculated on basis of the tumour concentrations required for a 10% increase in biological effective dose and the tumour:normal tissue concentration ratios reported in the literature. The maximum additional dose to normal tissue is then determined using this estimate of normal tissue NCA concentration together with normal-tissue CBEs reported in the literature and the maximum neutron fluence estimated in the normal tissue volume. The maximum percentage increases in dose to normal tissue resulting from a 10% increase in biological effective dose in both treatment volumes, are listed for all treatment volumes and all evaluated NCAs in Table et al.26. On the other hand, BSH concentrations have been reported in the literature which would realise a dose boost of close to 10% - for example, Suzuki et al.78 reported up to 200 and 234 ppm for BSH plus two different embolising agents, which would offer dose boosts of the order of 6.4–7.5% in the liver.Several conclusions may be drawn by examining the tumour concentrations of each NCA listed in Tables 10B-BPA concentrations required to achieve a 10% increase in biological effective dose during proton therapy in the brain would need to be around three times greater than the highest concentrations reported in the literature to date, while the concentration needed for carbon ion therapy is even greater. Conversely, with the highest BPA concentration reported in the literature of 125 ppm, the increase in dose is approximately 3.2–3.6% for proton therapy, and about half that for carbon. These results do not rule out the use of boron neutron capture agents for NCEPT treatment in the brain, but demonstrate the need for further development of boron-based NCAs.The situation is somewhat less positive for the brain; 79. While there appears to be very little research into BNCT specifically applied to the pancreas , it would appear to be a good candidate for NCEPT.Tantalisingly, there are reports in the literature of strong uptake of BPA in the pancreas, an organ in which cancer is notoriously difficult to treat10B-based NCAs are still in development80. BSH has been somewhat disappointing as an NCA in BNCT, chiefly due to its inability to directly penetrate the cell membrane. However, several BSH-derived compounds have been proposed that combine up to 8 instances of the BSH compound with peptide chains, which are able to penetrate the membrane and deliver high concentrations of boron within the cell. Boron concentrations in excess of 5000 ppm have been reported for these compounds81. Other promising recent studies have investigated the use of using boron nitride nanotubes as NCAs in BNCT, which can also potentially deliver very high 10B concentrations to the tumour82.Several promising new 157Gd, the situation is more complex. The values are highly dependent on how the 157Gd atoms are distributed; when they are either electrostatically attached to DNA or concentrated in the cell nucleus, the required concentrations are well within the ranges reported in the literature; this remains true even when the gadolinium is present in the cytoplasm or outside of the cell membrane. Several of the gadolinium compounds now in development appear to have many very promising properties for highly selective tumour uptake, and in particular high uptake in the nucleus and mitochondria, where they are most effective for neutron capture therapy. Significantly, many of the recently developed gadolinium-based compounds appear to offer very high tumour:normal tissue concentration ratios.For 26) and gadolinium (up to 3000 ppm in vitro45) it is clear that for certain existing combinations of NCA and target tissues, achieving an increase in biologically effective dose of at least 10% - or, equivalently, reducing the primary radiation dose and hence reducing the radiation dose to normal tissue - is feasible.By comparing the required tumour concentrations obtained in this study to values previously published for both boron  or magnetic resonance imaging (MRI) to improve the efficacy of neutron capture therapies90. 18F-BPA, a radio-labelled analogue of 10B-BPA has been successfully employed to determine the macrodistribution of boron in the tumour and estimate the tumour to normal tissue uptake ratio prior to treatment for treatment planning purposes95. A similar approach to quantify the biodistribution of the NCAs in target volume is necessary to optimise the treatment delivery in NCEPT.Intra-tumour and inter-patient tumour heterogeneity resulting in variable biodistribution and pharmacokinetics is a major problem in cancer treatments which rely on the targeted delivery of drugs to the malignancy, such as chemotherapy, therapeutic nuclear medicine and BNCTWith the additional dose provided through neutron capture, achievement of a prescribed uniform (or non-uniform) total radiation dose (ion plus neutron capture) throughout the tumour volume will require modifications to the treatment planning process. An initial plan can be constructed which only considers the primary ion dose. From this, an estimate of the thermal neutron field can be computed. Finally, either by assuming a flat distribution of NCA in the tumour, or by measuring it voxel-by-voxel using the theranostic approach mentioned previously, the estimated additional dose can be computed, after which the primary dose can be iteratively modulated until the total dose converges on the desired target dose prescription.98. From a practical perspective, this makes addition of a boron-bearing drug infusion step to the treatment process a minimal additional burden on the patient, as it may only need to be performed once or twice.One possible criticism of the NCEPT approach is the need to fractionate the delivery of the therapeutic dose, which would either necessitate the use of a NCA with a long residence time or require repeated infusion of the NCA. However, the most recent literature recommends delivery of heavy ion radiotherapy via hypofractionation (1–2 fractions only)99. NCEPT has the potential to offer a new source of thermal neutrons at any proton or heavy ion treatment facility, conveniently situated at the point of treatment inside the patient’s own body. With the prospect of further progress in the development of new NCAs, with greater tumour specificity and potentially very high achievable tumour concentrations, and possibly in combination with ultrasonic or other uptake enhancement methods, it may be possible to achieve even greater dose enhancement in the future.As a final observation on the practicality of NCEPT: the main impediment to widespread adoption of neutron capture therapy is the limited availability of suitable epithermal neutron sources rather than the availability of appropriate NCAsOur results demonstrate that the thermal neutron fluence distribution resulting from proton and carbon ion therapy mostly originates in the vicinity of the Bragg peak , with the neutron fluence falling with increased distance from the Bragg peak in all directions. The fluence distribution resulting from a realistic treatment plan is sufficient to enable a significant increase of the order of 10% with realistic NCA concentrations of the order of magnitude previously reported in the literature. The resulting dose increase in normal tissues is quite modest, and is unlikely to cause additional harm to the patient. To our knowledge, this is the first time this concept has been proposed.


Bordetella bronchiseptica rotor flagellum is highly PMF dependent and that flagellar motility represents an important factor involved in colonization, we monitored the swimming and swarming motilities of bacteria and showed a strong inhibition in the presence of the lead selected compound. Taken together, our results suggest that this class of molecules are able to increase treatment efficacy and decrease drug consumption.The spreading of multidrug-resistant bacteria and the lack of novel antibiotic molecules leave clinicians and veterinarians with very limited options to treat bacterial infections, especially those caused by Gram-negative pathogens. To reduce the selection of antibiotic resistance mechanisms and their transfer to human pathogens, veterinary pharmaceutical companies have dramatically decreased the number of antibiotics used. Among all the investigated alternate solutions, chemosensitizers, which decrease the amount of the used drugs, appear to be one of the most promising strategies. In this study, we reported that polyamino-isoprenyl derivatives can potentiate florfenicol activity against veterinary sensitive reference strains as well as clinical isolates. These molecules induce inner membrane depolarization and subsequently inhibit efflux pumps by collapsing the proton-motive force (PMF). Considering that  The bac3(3)], hydroxyethyl piperazineethanesulfonic acid (HEPES), ethylene diamine tetraacetic acid (EDTA), and benzalkonium chloride were purchased from Sigma-Aldrich . The polyamino-isoprenyl derivatives chemolibrary was described previously . The antibiotic imipenem was purchased from Sequoia Research Products . All the chemicals polymyxin B, polymyxin B nonapeptide (PMBN), phenylalanine-arginine beta- naphthylamide (PAβN), carbonylcyanide m-chlorophenylhy- drazone (CCCP), cetyl trimethylammonium bromide (CTAB), dimethyl sulfoxide (DMSO), 3,3′-dipropylthiacarbocyanine iodide  supplemented with agar 0.3% (w/v) . Swarmin1 was added where indicated and immediately observed. Acquisition was performed in resonant mode with the LEICA SP5 CLS by using a laser HeNe at 633 nm and a transmission PMT. The scans were recorded at a 512 × 512 resolution size and time laps over 200 frames. Their subsequent analysis was realized with the free software ImageJ using the MtrackJ plugging permitting to determine the average speed in micrometers per second of each bacterium followed. The results reported correspond to mean values obtained from 15 tracking measurements for each condition analyzed.A drop of each bacterial culture in exponential growth was deposited on a microscope slide and Cpd1 and florfenicol combination against the reference strain CIP 55.110 was determined by the time-kill assay. An overnight culture was diluted to 50-fold into 5 ml of Columbia agar and grown until the cell suspension reached the mid-logarithmic phase. The inoculum was then adjusted to 107 CFU/ml in Columbia broth supplemented with either no addition, sub-inhibitory concentration of florfenicol (MIC/4), sub-inhibitory concentration of Cpd1 (7.5 μM), or sub-inhibitory concentration of both. Each tube containing 1 ml was incubated at 35°C, and the bacterial count was determined after several times of incubation: 0, 1, 3, 6, 9, and 12 h by spreading the appropriate dilutions on Columbia agar plates. The plates were incubated overnight at 35°C before colony counting. The curves obtained are the result of two independent experiments.The effectiveness of CpdLibraries were prepared with NebNext kit  after Covaris ME220 Covaris Focused-ultrasonicator  shearing, according to manufacturer protocol, and sequenced on a NextSeq550  with High Output 300 cycles V2 cartridge (ref. FC-404-2004). Antibiotic resistance profiles of the four strains were tested against CARD (22) and oneCodex, with confirmation of phenotypic resistance except for florfenicol.B. bronchiseptica CIP 55.110 to identify compounds able to decrease florfenicol MIC below 1 mg/L. To avoid compounds with intrinsic activity, a control experiment was performed in the absence of florfenicol. We then identified compounds that could only kill bacteria in the presence of florfenicol. Among the tested compounds, only Cpd1 led to an 85% inhibition of the bacterial growth under these experimental conditions.The MICs to florfenicol of 10 clinical strains and the reference one were determined, with values ranging from 2 to 32 mg/L showing different levels of resistance to this antibiotic . A polya1 on 10 animal isolates. This compound was able to restore the susceptibility of 8/10 isolates to florfenicol. Nevertheless, two of them were still resistant to florfenicol alone and in combination . On the other hand, we were unable to determine the FIC indexes for the geranyl ones (Cpd5–Cpd7) due to the high concentrations needed to inhibit the bacterial growth as mentioned by ND in To determine both the minimal efficient combinations of the designed compounds and florfenicol that are necessary to inhibit the bacterial growth, we performed a checkerboard assay on animal isolates, with the strain CIP 55.110 being used as control. The results are summarized in B. bronchiseptica, we measured the ability of the strain CIP55.110 to expel the 1,2′dNA, a fluorescent dye that is a substrate of efflux pumps  did not exhibit any permeabilization activity while farnesyl ones (Cpd1–Cpd4) increased the permeability of the outer membrane from 30 to 50%, considering that a maximum rate of permeation was obtained by using polymyxin B (100 μM) (4 (branched amine) presenting a similar activity with respect to the three other farnesyl compounds bearing a linear amine.The other parameter that could modulate the antibiotic intracellular concentration is the outer membrane permeability. Thus, we evaluated the permeabilization activity of the polyamino-isoprenyl derivatives by using a previously reported nitrocefin hydrolysis method (15). We took advantage of the isolate 77-A12 that produces a β-lactamase (not shown). Interestingly, geranyl compounds (Cpd(100 μM) . HoweverB. bronchiseptica. As shown in 1 and Cpd2 cause the most important depolarization at about 60% of the inner membrane. Surprisingly, derivatives Cpd6 and Cpd4 were also able to depolarize the inner membrane up to 40%. Nevertheless, we observe a slight effect for Cpd5 and Cp7, which depolarized the inner membrane up to 20 and 10%, respectively. Molecules responsible for the most important depolarization remained the farnesyl ones, while the geranyl compounds displayed a lower rate of depolarization. In addition, the presence of a branched amine on the farnesyl moiety greatly influenced the activity (compare Cpd1/Cpd4 and Cpd2/Cpd4) except for Cpd3, but one must consider that it is due to the presence of only three amino groups instead of Cpd4, as it is encountered for the other compounds. Thus, conversely to the results obtained from the outer membrane permeation assays, herein the activity depends on the isoprenyl moiety not only of the compound but also of the polyamine structure.In Gram-negative bacteria, the PMF drives many of the efflux pumps and inner membrane transporters, and our compounds are able to inhibit efflux of the fluorescent dye used . Consequ1, we were wondering if this compound could influence the flagella-driven motility that is highly dependent from the energy triggered by the PMF. We tested here the ability of Cpd1 to inhibit the swimming and swarming of two strains of B. bronchiseptica, the CIP 55.110, which is susceptible to florfenicol, and the SR11-14, which is resistant both to florfenicol and to the combination florfenicol/Cpd1. Thus, we measured the diameter of the motility halo after 24 h of incubation at 25°C to assess the functioning of the flagella.As the derivatives can largely impair the PMF and particularly Cpd1 and/or NaCl by considering that the flagella could use both the proton and the sodium gradient. We observed that Cpd1 inhibited the swimming motility at about 40% for both strains  parameters were evaluated using time-kill kinetics assays  had no effect on the cell growth such as the Cpded alone . The comfloR gene was identified in some isolates as responsible for this resistance  exhibited resistance to florfenicol . Such resistance is not common but has already been described and the sistance . To checfloR-derived one, we mapped our two libraries made from resistant strains against reference strain B. bronchiseptica 253, by using BWA MeM software (version 0.7.16a-r1181) with a minimum seed length of 28; all other options were kept to standard. Owing to the fact that this strain does not have resistance to florfenicol, we selected reads that did not map to this strain. We reconstructed de novo the two other strains CIP55-110 and 77H12 with SPAdes version 3.9.1 . Unfortunately, we did not find any match.In order to be able to highlight a new gene of resistance or a on 3.9.1  and we mon 3.9.1 . We recoEnterococcus faecium, have been detected co-localized on same contigs, which also exhibit plasmid associated features. Interestingly, OptrA was shown to afford cross-resistance to chloramphenicol and florfenicol in E. faecium . Interestingly, genes sharing homology with ABC transporters and OptrA , which h faecium  that can faecium .The international and European authorities requested to use with parsimony antibiotics that can favor emergence of resistance in human and veterinary medicine. For the first time, an alternate way is studied by using adjuvant molecules that enhance florfenicol antibiotic activity. This strategy allowed us to decrease by four the MIC of the majority of strains tested here with small amounts of polyamino-isoprenyl derivatives, previously known to potentiate antibiotics by acting on membrane functions . Thus, eToday, it is recognized that human health is directly related to animal health and the environment. The dramatic increase in antibiotic resistance and the scarcity of new drugs on the market in recent decades highlight the threat of moving to the past era of antibiotics, with a significant increase in infection mortality. In this context, taking into account the “One Health” problem becomes an urgent need. This must allow collaborative efforts between health actors, as well as breeders and those involved in environmental management.This includes the involvement of a research at the interface between chemistry and microbiology, to develop new infection control strategies, by developing molecules that target specific new bacterial processes and/or new molecules that block/inhibit resistance mechanisms already under way. In recent decades, the efflux mechanisms have become increasingly important for human and animal health, and we propose to block the mechanisms of resistance by efflux of Gram-negative bacteria.B. bronchiseptica, CIP 55.110 reference strain. Thus, Cpd1 was identified as an interesting chemosensitizer of B. bronchiseptica to florfenicol and evaluated against 10 animal isolates leading to the restoration of susceptibility for 8 of these 10 strains. Two isolates were resistant to the combination, and the mechanism of resistance involved remains unknown.According to our previous work , we eval1 remained the better-tested compound, needing the lowest concentration to potentiate florfenicol activity. It is also noteworthy that all farnesyl compounds showed better efficiency than geranyl ones. As polyamines are well-known agents to disrupt membranes , suggesting the presence of putative RND efflux pump coding sequences ]. Moreover, we observed a dose-response effect for each farnesyl compound as encountered in a previous work on Pseudomonas  and the reference strain CIP55.110 gave the same response to inhibition of motility after treatment with Cpd1. More interestingly, the study of the resistance mechanism of these strains by bioinformatic investigations showed the presence of a gene sharing homologies with the gene coding the ABC-F type ribosomal protection protein OptrA, known to confer florfenicol and chloramphenicol resistance in E. faecium and to use ATP as a source of energy (1 targets the PMF and correlates with the absence of efficacy toward the strains exhibiting this mechanism of resistance. In the strain SR 11-14, one might consider that OptrA and/or efflux pumps are overexpressed in this strain. We can hypothesize that florfenicol resistance could have been acquired through horizontal transfer from Enterococcus, and this hypothesis is strengthened by the fact that the contig containing resistance-associated genes showed high homology with plasmids found in such bacteria.The flagella that potentially contribute to virulence could be considered as a new target for the development of motility inhibitors to circumvent the spread of bacterial infections . The efff energy . This is1 combination reduces the antibiotic efficient concentration four times and thus could allow drastically reducing the amount of the antibiotic used. Because Cpd1 did not modify the bacteriostatic activity of florfenicol, but increased its efficiency, one may consider that Cpd1 allows an increase of the intracellular concentration of the antibiotic (Finally, to better characterize the antibiotic/derivative combination, we did time-kill assays at the concentrations that have shown a synergistic effect. This florfenicol/Cpdtibiotic . ExperimAll datasets generated for this study are included in the manuscript and/or the All authors conceived the approach and reviewed the manuscript. DB carried out all the experiments related to bacteria. JBr carried out the synthesis, purification, and analyses of the compounds. OG performed the DNA sequencing and the sequence analyses. DB, JBr, and JBo wrote the main manuscript text. DB and JBr prepared the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.