Lanice conchilega can have important consequences for the functioning of marine sediments. It is considered both an allogenic and an autogenic ecosystem engineer, affecting spatial and temporal biogeochemical gradients  and physical properties (grain size) of marine sediments, which could affect functional properties of sediment-inhabiting microbial communities. Here we investigated whether density-dependent effects of L. conchilega affected horizontal  and vertical  patterns in the distribution, diversity and composition of the typical nosZ gene in the active denitrifying organisms. This gene plays a major role in N2O reduction in coastal ecosystems as the last step completing the denitrification pathway. We showed that both vertical and horizontal composition and richness of nosZ gene were indeed significantly affected when large densities of the bio-irrigator were present. This could be directly related to allogenic ecosystem engineering effects on the environment, reflected in increased oxygen penetration depth and oxygen concentrations in the upper cm of the sediment in high densities of L. conchilega. A higher diversity (Shannon diversity and inverse Simpson) of nosZ observed in patches with high L. conchilega densities  at deeper sediment layers could suggest a downward transport of NO3− to deeper layers resulting from bio-irrigation as well. Hence, our results show the effect of L. conchilega bio-irrigation activity on denitrifying organisms in L. conchilega reefs.The presence of large densities of the piston-pumping polychaete  All seda), % clay and silt content and median grain size (MGS). Around 4 g of sediment (wet weight) was subsampled using sterilised tools for microbial analyses and stored in sterile 15-mL falcon tubes. Microbial and chl-a samples were immediately frozen at -80 °C, whereas the samples for grain size were dried at 60 °C before analysis.The sediment was subsampled by taking 3 to 5 mL using a cut-off syringe for the analysis of labile organic matter  analysis according to  content, CV, max OPD and OX. All assumptions [Multiple linear regressions were performed to identify the variables contributing significantly to the observed variations in the diversity indices of umptions  were cheumptions .L. conchilega) and depths . chl-a concentration was significantly affected by depth . Highest chl-a concentrations were observed at the top layer (0–0.5 cm)  followed by the second layer which was significantly higher than the deepest layer (2.5–3 cm) , were not significantly influenced by interactive and single effects of treatments  L. conchilega treatments . There were no significant differences between both L. conchilega treatments. SIMPER analysis showed that the difference in oxygen profiles between the control treatment and high L. conchilega treatment was attributed to 1–4 mm sediment depth whereas this difference was between 1–3.25 mm for the control and low L. conchilega treatments.Oxygen depth profiles  were sigL. conchilega (93–235 μmol L-1) and in the low L. conchilega treatment (60–203 μmol L-1). The lowest values were observed in the control treatment (80–157 μmol L-1) (Oxygen concentrations over time (over a period of 30–35 min) measured at 1 cm distance from the tube and at 1.5 mm sediment depth remained high in the high mol L-1) . Howevermol L-1) .L. conchilega density treatment  compared to the low  = 0.008) and control  = 0.001) treatments  and pair-wise tests showed a significantly higher max OPD in the high eatments ; Fig 2.PERMDISP analyses showed some heterogeneity of multivariate dispersion  in the analysis of maximum OPD but this was not the case for the analysis of the entire oxygen depth profiles (p > 0.05).nosZ reads were assigned to 502 OTUs. The current sampling effort was sufficient in most samples to reflect the variation of the observed OTUs . Of these abundant OTUs, the two dominant OTUs 1 and 2 combined constituted 50–77% of the community where OTU 1 alone accounted for 44–71% of the relative abundance in different samples .Numbers of reads and total numbers of OTUs per sample as well as the numbers of abundant OTUs (>1% relative abundance) in treatment-depth combinations are provided in nosZ sequences from Pseudomonas stutzeri constituting 76% of the community. The other abundant OTUs were less than 95% similar to protein sequences available in sequence databases. In addition, a blast search was performed of all 502 nosZ OTUs against NCBI’s non-redundant protein database (nr) with an e-value threshold of 1E-20 and taking into account only the first blast hit for each OTU. These results showed that 380 OTUs  were most closely related to Pseudomonas stutzeri.The results of the protein blast indicate that among the abundant OTUs, OTUs 1, 2, 4 and 10 were most closely (>97% AA identity) related to nosZ sequences retrieved from our samples showed considerable phylogenetic diversity (L. conchilega) contained sequences from across the phylogenetic tree . However, no difference in nosZ gene composition was observed between the low and high L. conchilega treatments, and also between low L. conchilega and control treatments (p > 0.05).Pairwise tests  indicatenosZ at the top layer (0–0.5 cm) was significantly different from the other layers  and only abundant OTUs  indicated a dispersion effect as well. Visualization by PCoA plot  showed tL. conchilega density and the two other treatments, reaching the lowest similarity (<50%) at the deepest sediment layer (2.5–3 cm). However, 231 out of 247 (93.5%) and 234 out of 247 (94.7%) of OTUs in Hd4 were shared with Cd4 and Ld4, respectively (L. conchilega treatment (The Venn diagram and Jaccard similarity index  indicateectively . Among dreatment .L. conchilega treatment at highest depth ((MC) > 0.05). In deeper sediment layers (2.5–3 cm), lower values were detected in the high L. conchilega treatment  = 0.015 and p(MC) = 0.016 compared with the control and low L. conchilega treatments). In addition, richness was significantly lower in the high L. conchilega compared to the control at the depth 0.5–1 cm  = 0.026).PERMANOVA did not show any significant effect on Shannon diversity and inverse Simpson for the factors “depth”, “treatment” or the interaction  although higher values were observed in the high st depth ; Fig 7. st depth . Pairwisst depth ; Fig 7 dL. conchilega treatment with increasing depth ((MC) = 0.022; We generally found a decreasing trend in OTU richness in the high ng depth . These cp > 0.05). We found significant negative relation  between max OPD and OTU richness; and a positive relation between this variable and Shannon diversity  (−3) and % mud content (β = -0.06). Apart from max OPD, chl-a concentration contributed significantly  to the model of richness.Maximum oxygen penetration depth  was an important variable incorporated in all diversity models. However, it was not significant in the model for the inverse Simpson index ( < 0.01) . The aveL. conchilega aggregations at the intertidal zone by focusing on expression of the gene encoding nitrous-oxide reductase (nosZ), the enzyme catalysing the final step of denitrification. We investigated typical nosZ genes, which play the major role in N2O reduction in coastal ecosystems [In the current study, we investigated the denitrifying community in osystems . The denosystems .nirS, nirK and nosZ) in marine sediments [nosZ community (502 OTUs) in the L. conchilega reef (area of L. conchilega aggregations plus zones without L. conchilega as a control). This is what can be expected in intertidal sediments where regular oxygen oscillations support a high genetic potential for denitrification [In comparison with previous studies on functional genes for denitrification . This is in agreement with previous studies in shelf sediments [The highest phylogenetic diversity was found in the Alphaproteobacteria and a high percentage of the sequences were closely related to ediments ,85 and sediments ,86,87. InosZ at both horizontal (m) and vertical (cm) scales between high L. conchilega and control treatments, and between top oxic (0–0.5 cm) and deeper layers. These differences in composition were mainly due to changes in the relative contributions of abundant OTUs to the community. The same pattern was also observed in the nosZ community structure in the high L. conchilega treatment at a depth of 2.5–3 cm   . This su October . Howeveroperties . TherefoLanice conchilega bio-irrigates its tube through piston-pumping activity for 1.5 min generally every 4 min, thereby transporting about 3 mmol O2 m-2 d-1 into the sediment [Lanice conchilega introduces oxygen-rich water in layers where oxygen is absent [L. conchilega tubes [L. conchilega bio-irrigation on the OPD . At this depth, a significant difference between the high and low L. conchilega treatments was also observed. In a vertical sediment depth profile, nitrate coexists with oxygen in the top layer. In the deeper layers where oxygen is low or absent, the nitrate concentration is an important factor determining similarity or difference in anaerobic denitrifying communities [nosZ between treatments at depth may thus be explained by the different availability of NO3− between treatments in the low oxygen or anaerobic deeper layers. The higher availability of NO3− in the high L. conchilega treatment can be by directly pumping NO3− [2 to deeper layers [L. conchilega and control treatments. The most prominent differences between the high L. conchilega and the other two treatments were at the deepest sediment layer (2.5–3 cm). These differences are not due to adaptation of certain OTUs specific to the high L. conchilega treatment: 93.5% and 94.7% of OTUs in Hd4 were shared with Cd4 and Ld4, respectively  of munities ,90. As amunities  while demunities ,91,92. Ting NO3−  and/or br layers  increasir layers  and alsoectively . InsteadLanice conchilega ventilates its tube in each pumping activity by a volume of water equivalent to 2.4 cm tube lengths [3− and little or no oxygen in the surrounding sediment) was met at 2.5–3 cm sediment. In the upper layers, high richness values resulted from the presence of a larger number of non-abundant and rare OTUs mainly belonging to the Alphaproteobacteria. This highly diverse pool may provide a background reservoir for suboptimal denitrification conditions increasing niche creation [nosZ community in the top oxic layer in all treatments  by denification .nosZ in the L. conchilega treatments compared with the control  affect composition, structure and diversity indices of the active nosZ community both vertically and horizontally in the sediment. These patterns can be linked with heterogeneity in geochemical environments in the sediment that are affected by allogenic ecosystem engineering effects of L. conchilega. The piston-pumping activity of the polychaete transports oxygen rich waters to the deeper layers of the sediment. This could also provide higher availability of NO3− at depth by increasing the surface for coupled nitrification-denitrification in the sediment along the tube.This study improves our understanding of the effects of bio-irrigation on the L. conchilega is present throughout the North Sea, often in dense aggregations, our results provide evidence for the implementation of this polychaete in marine Ecosystem-Based Management. This study illustrates that a future loss of macrobenthic diversity, and especially ecosystem engineers, will have important repercussions for benthic ecosystem functioning.Since S1 FignosZ sequences) as function of the number of sequences screened in samples with >4000 reads (A) and ≤4000 reads (B). “H” indicates high L. conchilega treatment. “L”: low L. conchilega treatment, “C”: control treatment, “d1”: 0–0.5 cm depth, “d2”: 0.5–1 cm, “d3”: 1–1.5 cm, “d4”: 2.5–3 cm.Plotting the number of observed OTUs (unique AA (TIF)Click here for additional data file.S2 Fig10% of OTUs (50 OTUs out of 502) making up around 84% of the total reads are shown. Abundant OTUs (>1% relative abundance in at least one treatment–depth combination) are indicated in red. The heat map (on the right) illustrates the average relative abundance of each OTU per sample. The dominant OTU 1 is shown in red. Numbers at nodes are bootstrap values . The scale bar represents 10% sequence divergence (10 mutations per 100 sequence positions).(TIF)Click here for additional data file.S3 FigTo construct a phylogeny with reference sequences, protein BLAST searches of 25 OTU representatives selected randomly across the phylogenetic tree in (TIF)Click here for additional data file.S4 FigL. conchilega treatment, Low: low L. conchilega treatment, Control) and four depth layers .A: per treatment; B: per depth. Each point represents a sample. Three treatments (High: high (TIF)Click here for additional data file.S1 TableAdaptor, Multiple Identifier (MID), pad, linker and primer sequences for the forward and reverse data sets are given.(DOCX)Click here for additional data file.S2 TableL. conchilega densities and control. Analyses were based on Euclidean distance similarity matrix. Where the treatment effect was significant, pairwise tests were performed within treatments. P-values were obtained by permutation and drawn from Monte-Carlo samplings (p(MC)) if the number of unique permutations was less than 100 [Treatments: high and low than 100 . Max OPD(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableL. conchilega densities and control; Depths: 0–0.5, 0.5–1, 1–1.5 and 2.5–3 cm.Analyses were carried out based on Generalized UniFrac distances (α = 0.5) on the data sets using all OTUs or only abundant OTUs with relative abundance >1%. Treatments: high and low (DOCX)Click here for additional data file.S5 TableL. conchilega densities and control; depths: 0–0.5, 0.5–1, 1–1.5 and 2.5–3 cm. Diversity indices  were calculated from the average values obtained from 1000 sub-samples of the data matrix to the minimum number of reads (1022). Univariate analyses of diversity indices were based on Euclidean distance similarity matrix. P-values obtained by permutation.Treatments: high and low (DOCX)Click here for additional data file.S6 TableL. conchilega treatment, Low: low L. conchilega treatment, Control) and four depths . Richness was calculated from the average values obtained from 1000 sub-samples of the data matrix to the minimum number of reads (1022). Analyses were based on Euclidean distance similarity matrix. P-values obtained by permutation.Three treatments (High: high (DOCX)Click here for additional data file.


Antimicrobial resistance (AMR) has reached an end point, prompting a worldwide scare as no new antibiotics are in the pipeline, particularly for treatment of Gram-negative bacteria. To prevent further development and spread of AMR and to inform empirical treatment guidelines, surveillance of AMR is necessary.We aim to provide a framework for a stepwise approach toward implementation of laboratory-based surveillance for AMR in African countries.Building up a surveillance system is a robust process that begins with a gap analysis in each participating country. This framework provides practical guidance on how to set up surveillance, identify responsibilities and set timelines in sustainable manner for African countries. It addresses sampling strategies, human resources, procurement and maintenance issues for AMR testing at routine clinical and national reference and public health laboratories involved in AMR surveillance. Key issues such as laboratory capacity building, training and continuous education, quality and diagnostic stewardship are discussed in detail.There are several priorities for AMR surveillance that need to be addressed in a comprehensive manner at regional and national levels, whilst keeping in line with current and proposed initiatives for laboratory capacity building, in order for African countries to achieve goals for combatting the real and current threat of AMR. The impact that AMR and its current pace of expansion will have on economic and health outcomes cannot be underestimated.3 AMR is now considered a threat to global health security and, as such, is a key element of the Global Health Security Agenda (GHSA).4 The control of AMR requires a holistic approach involving human, animal and environmental domains. The strategies necessary to deal with the AMR threat developed by the World Health Organization (WHO) include: (1) to create awareness of AMR; (2) to introduce antimicrobial stewardship and appropriate use of antibiotics; (3) to restrict and regulate overuse of antibiotics in human and animals; (4) to improve infection prevention and control measures; and (5) to enhance surveillance of AMR.5 For some countries, AMR is already a high priority, whereas other countries are still in the initial stages of recognising it as a threat.6 However, we have now reached the point at which all countries, including low- and middle-income countries (LMIC), are required to develop strategies for containment of AMR as part of the WHO’s Global Action Plan (GAP) for AMR.5 This plan contains a number of features aimed at avoiding the emergence of new resistance and preventing transmission of existing resistance  to bacterial pathogens is a global threat that ignores borders between countries. The emergence of multidrug-resistant bacteria means that very few effective antibiotics are available for the empirical or targeted treatment of many infectious diseases,sistance .78 That study also underlined the flaws in currently-available data and the challenges faced in LMICs when implementing AMR surveillance, including a lack of allocated human and economic resources.8Most information on AMR prevalence comes from high-income countries, and very little is known about AMR in African countries. A systematic review of AMR in sub-Saharan Africa by Leopold et al. revealed a high prevalence of AMR to commonly-used antibiotics in clinical bacterial isolates.9 The WHO has sent an open call to all countries to enroll in the Global AMR Surveillance System and to participate in a structured surveillance programme.6 LMICs are encouraged to use every opportunity to apply for transfer of resources and funds in order to comply with the GAP. There are clear and sometimes large differences between countries in the African region as to what extent AMR surveillance is currently taking place. These differences are dependent on many factors, including socio-economic factors; however, surveillance of AMR is most likely to be successfully implemented when approached using a stepwise process and when tailored to the country’s level of preparedness.10 Here we address the essential components of an AMR surveillance implementation plan.As one of its multifaceted responses to combat AMR, the first step proposed in the WHO GAP is to perform a baseline assessment of the AMR prevalence in all countries.5 which is a multi-sectorial and multi-disciplinary way to optimise use of antimicrobials among both humans and animals. Monitoring and evaluation of all tasks should be implemented with clear indicators and targets within the AMR strategic framework. Finally, the benefits of AMR surveillance for clinically-relevant reporting should be made apparent  that can inform the surveillance programme, which may be steered by a central body . In addiFor a national laboratory-based surveillance programme to be successful, government commitment to support the surveillance programme for AMR at the country level is essential . Such coEstablishment of a national laboratory-based surveillance system at the country level, as established by each country’s National Department or Ministry of Health, also includes the establishment of a coordinating centre with the responsibility to systematically collect, analyse and share data at the national and international level. Governments may decide to set up their coordinating centres differently, but all will need expertise in epidemiology, clinical microbiology, infectious diseases, veterinary medicine, data management and governance. Such expertise may be built and subsequently sustained through continuous education programmes that take place locally or within the region. For example, South Africa, Kenya, Ghana, Uganda, Nigeria and Burkino Faso all provide a Field Epidemiology Training Programme.Establishment of at least one reference laboratory that can undertake infectious disease diagnostics and antimicrobial susceptibility testing, and that has competence in phenotypic and genotypic confirmation of the presence of resistance genes, is required. Initially, in the absence of capacity to perform advanced phenotypical and molecular testing in the national reference laboratory, a clinical hospital bacteriology laboratory with demonstrated capacity to perform these functions, could be selected as a reference laboratory. In addition, instead of performing genotyping at the national level, molecular typing and gene characterisation could take place in another reference laboratory in the African region. National reference laboratories perform their role in close collaboration with provincial and district clinical laboratories, which generally receive samples directly from patients. Together, these laboratories form the national surveillance network. Countries should also consider including private laboratories in their network. Although specifics vary by country, such laboratories may receive large numbers of samples from a substantial proportion of the population. Strategies on how to involve and include private laboratories in national AMR surveillance networks must be developed to achieve this inclusion.11 The organisation of regional AMR surveillance programmes creates opportunities for concentrating specific expertise in dedicated national laboratories, thus potentially reducing costs and increasing efficiency. In addition, such regional programmes may provide support for solving logistical issues, such as those described below.Setting up a regional network for AMR surveillance in Africa to provide locally relevant AMR data to serve as a bench mark may be the eventual goal. Whilst several regional networks exist, the Central Asia and Eastern European Surveillance of Antimicrobial Resistance network is one model for the African region. This network is a joint initiative of the WHO Regional Office for Europe, the European Society of Clinical Microbiology and Infectious Diseases, and the Dutch National Institute for Public Health and the Environment. The Central Asia and Eastern European Surveillance of Antimicrobial Resistance network includes all countries in the WHO European Region that are not part of the European Antimicrobial Resistance Surveillance Network, which is coordinated by the European Centre for Disease Prevention and Control in the European Union.Laboratories that are participating in AMR surveillance as sentinel or reference sites should comply with baseline requirements as indicated under this heading. The quality of laboratory-based AMR surveillance data depends to a large extent on the microbiology laboratory. Firstly, the quality of a laboratory system should be assessed in order to identify gaps and areas for improvements. In order for participating laboratories to meet minimal quality standards requirement as defined by the network , establishment of a laboratory quality system is essential. However, the logistical difficulties of joining external quality assessment programmes and receiving quality control materials present significant challenges to countries in sub-Saharan Africa, because of customs and other policies for shipment of samples and microorganisms between different countries.Laboratory assessments should include pre-analytical, analytical and post analytical steps for reporting of antimicrobial susceptibility testing (AST) and its limitations. For the pre-analytical phase, diagnostic stewardship is critically important. Diagnostic stewardship is the process that guides and improves the use of microbiological tests to ensure they are timely, appropriate and accurate. Reports sent to clinicians should include a feedback form to help on-going improvements. To apply required standards for AST, laboratories must have the required infrastructure, equipment, supplies and staffing in place. Laboratories should be able to identify gaps and recommend improvements. For example, some laboratories may have all the equipment needed but no supply of reagents or other consumables needed for basic microscopy, culture and AST implementation. This, in turn, affects diagnostic stewardship as clinicians may lose confidence in laboratory reporting when timely, appropriate and accurate results cannot be achieved. A country’s national AMR surveillance plan and the regional AMR surveillance network may provide for policies and procedures that make timely ordering and receipt of consumables and other supplies feasible and affordable. ASLM could play an active role in such advocacy activities. Laboratory experts should be involved in ordering supplies to avoid receiving supplies that are irrelevant, not useful or of poor quality.12 Other initiatives such as those from the RESAOLAB network13 have created their own open source LIS that includes an option to store and report AMR data.The use of a LIS is limited in most LMICs, which is unfortunate, as it improves AMR surveillance by reducing workload and errors, thus improving the overall quality of microbiology laboratories. Laboratories should use LIS to retrieve and share AST data with national and regional bodies . Whilst 14 or from EUCAST,15 the latter of which is freely available. For additional standardisation of data collection and reporting to a central data collection point, a regional network would require additional software that can be linked to local LISs, including newly-designed or WHONET, European Antimicrobial Resistance Surveillance Network, or Central Asia and Eastern European Surveillance of Antimicrobial Resistance network software. This software may be expensive to set up, and maintenance is an additional consideration. It is also important to consider that these software systems may not be sufficient for outbreak detection due to the lack of an alert system. In addition, such systems must be compatible with existing or future national LIS systems, in order to prevent time-consuming transfers of data, as is currently needed for WHONET. Thus, the available data collection systems for each country and the costing structure for implementation of region-wide AMR surveillance need further investigation.As mentioned, national and regional networks will require (inter)national standardisation of methods. For standardisation of bacteriology test methods, guidelines are available and are often used, e.g. those from the Clinical and Laboratory Standards InstituteHuman resource capacity may be the most challenging issue, as shortages of qualified and trained professionals can have a serious negative impact on the ability of laboratories to perform testing. Human resources are a weak spot in microbiology and have been ignored. Quality microbiology requires sufficient number of qualified technicians, as well as supervision by MD or MSc level clinical microbiologists who are capable of reporting results to clinicians. Finally, veterinarians should be involved in the process of developing surveillance systems to strengthen diagnostic stewardship in animal health, provide assistance about status of AMR in animal health, and implement preventative measures in animal health, if needed at a later stage. For capacity building that includes education, training and refresher courses and other forms of networking, the WHO and ASLM should be excellent sources of support.The following components should be considered when designing an AMR surveillance plan .16 An analysis of existing surveillance systems should be undertaken before development of a new system is considered.A gap analysis that includes all levels of the health system must be performed in order to implement or further expand existing AMR surveillance activities. For example, it was recently reported that many countries still lack reference to laboratories in their health policies and plans and do not consider laboratories at all in their health budgets.17 as well as by ASLM as part of the GHSA.18 These tools are targeted at different levels of the health system. The WHO Stepwise Laboratory Improvement Process Towards Accreditation tool is a generic quality system assessment tool for laboratories and is not designed to look specifically at AMR surveillance capacity.19 The audit checklist tools developed by ASLM are established to assess laboratory capacity at national level and include a bacterial AMR testing module. Whichever tool is used, it should be applied for the purpose for which it was designed.There are currently several tools available to assess laboratory capacity and quality that could be used as a part of a gap analysis. These include the Stepwise Laboratory Improvement Process Towards Accreditation tools developed by WHO,6 should be established.In order to ensure AMR surveillance of relevant pathogens, it is important to establish case definitions. For example, standard definitions should be established for patients with symptoms of syndromes that have high clinical importance, such as central nervous system infections and septicaemia. The selection of specimens should be directed according to local, national, and regional relevance, after considering site of infection, such as meningitis or bacteraemia. Isolates from cerebrospinal fluid and blood cultures, with particular pathogens of focus, including both Gram-negative and Gram-positive bacterial organisms, should be included. In addition, exclusion criteria, such as de-duplication based on one specimen per patient in 21 days or based on Global AMR Surveillance System criteria,Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Streptococcus pneumoniae, Salmonella spp., Shigella spp. and Neisseria gonorrhoea. Each country should prioritise which drug/bacteria combination to test and report according to local relevance.In addition, there are number of priority organisms and antibiotics recommended by WHO that should be considered in a stepwise process. These include There are several types of surveillance and current recommendations are to perform laboratory-based surveillance. For each drug/bacteria combination, the approach toward laboratory-based surveillance should be decided on . WhichevFor technical support and strategy advice, an AMR reference laboratory for African region should be established. The role of an African region AMR reference laboratory would be multi-faceted. Firstly, the regional reference laboratory should provide training of laboratory personnel from network member countries to help build capacity for development of surveillance systems in the trainees’ home countries. Secondly, the regional reference laboratory should take the lead in introducing and providing training on new techniques. Thirdly, it should provide confirmation of difficult AST results and offer advice when necessary. Finally, it should manage other laboratory networks in the region. A public/private partnership should be sought as the funding source.20Considering the growing impact of AMR worldwide and the lack of quality surveillance systems in the African region, an enormous effort must be undertaken through international and multi-sectorial collaboration. A recent report analysing the AMR surveillance capacity of the East Africa Public Health Laboratory Network by the Center for Disease Dynamics, Economics & Policy, commissioned by the World Bank, summarised the following key issues for laboratories to carry out AMR surveillance:Lack of demand of bacteriology diagnostics from clinicians, related to length of time to get results (at least two days); lack of trust in results; lack of laboratory capacity for blood cultures, which are needed for many of the most serious, life-threatening infections.Low priority given to bacteriology diagnostic supplies by hospitals and other decision makers who control purchasing.The multi-component nature of bacterial culture and antimicrobial susceptibility testing, which makes it especially vulnerable to weak supply chains and frequent stock outs; results cannot be obtained if essential components are unavailable when testing is needed.Lack of recognition that microbiology requires dedicated, trained personnel, leading some facilities and/or ministries of health to rotate staff in and out of microbiology.Few options for automated testing relative to haematology, chemical pathology or HIV testing, which may be more satisfying to staff, leading to low morale.Requirement for patients to pay out of pocket for diagnostic tests in many countries.22 Whichever the situation, solving these issues will require collaborative efforts of multiple stakeholders. ASLM could play a key role in facilitating these efforts due to its central position between ministries of health, policy makers, laboratory professionals and, increasingly, clinicians and professional societies.Whilst some of these may be generic to LMICs, others may be more specific to certain countries or settings.In conclusion several key steps should be implemented for an AMR surveillance programme such as: assessment of laboratory capacity to perform surveillance for AMR; identification of gaps; adoption of the Global AMR Surveillance System manual or other guidelines; designation of national reference laboratories for AMR and organisation of advisory committees; implementation of laboratory-based AMR surveillance; and reporting to the Global AMR Surveillance System.


A nanofabrication method for the production of flexible core-shell structured silk elastin nanofibers is presented, based on an all-aqueous coaxial electrospinning process. In this process, silk fibroin (SF) and silk-elastin-like protein polymer (SELP), both in aqueous solution, with high and low viscosity, respectively, were used as the inner (core) and outer (shell) layers of the nanofibers. The electrospinnable SF core solution served as a spinning aid for the nonelectrospinnable SELP shell solution. Uniform nanofibers with average diameter from 301 ± 108 nm to 408 ± 150 nm were obtained through adjusting the processing parameters. The core-shell structures of the nanofibers were confirmed by fluorescence and electron microscopy. In order to modulate the mechanical properties and provide stability in water, the as-spun SF-SELP nanofiber mats were treated with methanol vapor to induce β-sheet physical crosslinks. FTIR confirmed the conversion of the secondary structure from a random coil to β-sheets after the methanol treatment. Tensile tests of SF-SELP core-shell structured nanofibers showed good flexibility with elongation at break of 5.20% ± 0.57%, compared with SF nanofibers with an elongation at break of 1.38% ± 0.22%. The SF-SELP core-shell structured nanofibers should provide useful options to explore in the field of biomaterials due to the improved flexibility of the fibrous mats and the presence of a dynamic SELP layer on the outer surface. Bombyx mori cocoons, is a protein polymer which has been extensively studied and used for biomaterial applications [Electrospinning is a relatively simple and effective method for the production of uniform, continuous fibers with diameters ranging from the nanoscale to the microscale. These micro/nanofibers in the form of nonwoven mats have been used widely in various biotechnologies, such as wound healing, tissue engineering, and drug delivery ,2. Silk ications ,4,5,6. Sications  or from ications . HoweverSilk-elastin-like protein polymers (SELPs) are genetically-engineered block copolymers consisting of silk-like (GAGAGS) and elastin-like (GVGVP) repeating units in defined ratios. SELPs are stimuli sensitive materials  ,10,11 anIn order to gain the benefits of both SF and SELPs, here we report a method to generate SF-SELP core-shell nanofibers from aqueous solutions by coaxial eletrospinning. Theoretical and experimental studies on coaxial electrospinning suggest that this research area has significantly advanced nanofabrication techniques and has potential for biomedical applications, such as for tissue engineering scaffolds, drug release systems, wound dressings, and related topics ,25,26,27In the present work, the objective was to produce SF-SELP core-shell structured nanofibers by using the core solution as a spinning aid to electrospin nonelectrospinnable shell solutions. This method would serve as a roadmap for the fabrication of functional nanofibers using nonelectrospinnable materials, such as genetically designed functional peptides. To demonstrate this concept, SF was chosen as a core material because of its mechanical strength and good electrospinnability, while SELP was used as the shell due to its elasticity and stimuli sensitive features; towards responsive and dynamic shell systems on the fibers. In order to determine the most suitable concentration of the core and shell solutions, the rheological behavior of the SF and SELP aqueous solutions with different concentrations were evaluated. In addition, the effects of coaxial electrospinning process parameters, such as the flow rate of the core and shell solutions, the applied voltage, and the resultant fiber diameters were investigated.E. coli strain BL21Star (DE3)  in a New Brunswick BioFlo 3000 bioreactor . The protein was purified by the inverse transition cycling (ITC) method, as described previously [The SELP, named S2E8Y, was genetically designed to have a silk to elastin block ratio of 1:4, and molecular weight of 53 kDa, where S is the silk block with sequence GAGAGS and E is the elastin block with sequence GYGVP. The expression plasmids were constructed using our previously established procedures  and expreviously ,10. The B. mori cocoons as described previously [2CO3 (0.02 M), rinsed and then dried at ambient conditions overnight. The dried degumming fibers were solubilized in a 9.3 M LiBr solution at 60 °C for 4 h, yielding a 20% (w/v) solution. Silk solutions were dialyzed against deionized water using dialysis tubing (MWCO 3.5 kDa) for 72 h to remove salts. SF solution was concentrated by airflow in Slide-a-Lyzer dialysis cassettes  at 10 °C to produce a 20 wt % SF solution, then the pH of the solution was adjusted to 6.0 by adding a 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES)-trishydroxymetnyl aminomethane (Tris) buffer solution into a 20 wt % solution in a volume ratio of 1:2. SF solution was concentrated again by airflow in a 50 mL beaker at 10 °C to 26–29 wt %. The concentration of the solution was monitored by weighing the remaining solid after drying.Silk fibroin (SF) solution was prepared from eviously . Briefly−1.Rheological analysis of SF and S2E8Y solutions was performed on an ARES-LS2 rheometer  using a 25 mm cone and plate geometry  at 25 °C. The viscosities of SF and S2E8Y aqueous solutions with different concentrations under shear were investigated through a logarithmic steady shear rate from 0.01 to 1000 sCoaxial electrospinning was performed using a vertical setup containing a variable high DC voltage power supply  and two individual syringe pumps (Thermo Scientific). The coaxial spinneret  was comprised of a 20G (inside diameter (i.d.) 0.584 mm, outside diameter (o.d.) 0.889 mm) inner needle concentrically mounted on a 12G  outer needle. SF and S2E8Y solutions were poured into 5 and 3 mL plastic syringes which were connected to the syringe pumps and the coaxial spinneret. The coaxial spinneret was connected to the high voltage power supply. The flow rates of the core (SF solution) and shell (S2E8Y solution) were controlled by two separate pumps. The electrospun fibers were collected on an aluminum foil wrapped 10 cm-diameter grounded metal plate. The applied voltage was varied in the range of 20–30 kV, and the distance between the tip and the collector was 10 or 12 cm, respectively. All the electrospinning processes were carried out at around 23 ± 3 °C and 45% ± 5% humidity.SF fiber mats were produced by the same vertical setup using a single needle 16G (i.d. 1.19 mm) to serve as a control sample. The process parameters were set as following: the concentration of SF was 29 wt %; the flow rate of solution was 0.8 mL/h; applied voltage was 25 kV; the distance between the tip and the collector was 12 cm; and electrospinning processes were carried out at around 23 ± 3 °C and 45% ± 5% humidity.Morphology of the electrospun SF-SELPs fiber mats was observed with a field emission scanning electron microscope (FE-SEM) . Samples were sputter-coated with gold for 60 s for improved conductivity. The SEM images were taken at 5 kV. The average diameter of the fibers in each specimen was generated from 100 randomly selected fibers on images and expressed as mean ± standard deviation (SD). All measurements and statistics were handled by Image J. software .To visualize the core and shell phases, 5 μg/mL rhodamine-B  and 40 μg/mL 70 kDa fluorescein isothiocyanate-dextran  were added to the 29 wt % SF solution and 12 wt % S2E8Y solution, respectively, and coaxially electrospun as described previously. The nanofibers were directly deposited on the glass coverslips and imaged under phase contrast and fluorescence modes using a BZ-X700 fluorescence microscope . Wide-green and wide-blue filter cubes were used to detect the rhodamine B-stained core and FITC-dextraned-stained shell phases, respectively [−1 over the wavenumber range from 600 to 4000 cm−1. The background spectra were collected under the same conditions and subtracted from the scan for each sample.FTIR analysis was performed using an FT/IR-6200 Spectrometer , equipped with a triglycine sulfate detector in attenuated total reflection (ATR) mode. The absorbance spectrum for each sample was obtained by averaging 64 scans with a resolution of 2 cmThe as-spun electrospun fiber mats were treated with methanol vapor in a vacuum desiccator for 72 h. During treatment, a Petri dish containing 200 mL MeOH was placed in the bottom chamber of a sealed desiccator, while the SF and SF-SELP fiber mats with aluminum foil were placed on a ceramic desiccator plate in the top chamber. Treated nanofiber mats were then peeled off the aluminum foil for FTIR and mechanical analysis.−1 with gauge length of 20 mm. The stress strain data were obtained automatically. Young’s modulus was calculated by the slope of each stress-strain curve in its elastic deformation region [Specimens were prepared for mechanical testing following previous methods ,31. Fiben region . Data waAn SDS-PAGE gel for the purified S2E8Y and regenerated SF is shown in 0), which suggested the formation of replacement interaction in a dynamic equilibrium [Rheological curves of SF and S2E8Y solutions with different concentrations are shown in ilibrium . As the ilibrium . CompareIn most coaxial electrospinning, the shell polymer solution has sufficient viscoelasticity and good electrospinnability, and the core solution can have either poor or good electrospinnability ,38. In tThe effect of SF concentrations on nanofiber morphology is shown in The effect of SELP concentration on the nanofiber morphology is shown in In summary, the SF concentration had a major impact on fiber morphology in the coaxial electrospining process. The SF solution with appropriate viscosity was able to serve as a spinning aid to successfully prepare nanofibers from low viscosity SELP solutions.In order to generate a good “compound Taylor cone” with a core-shell structure, controlled and balanced injection speeds of the inner and outer fluids are critical to keep the “compound Taylor cone” in dynamic stabilization . In the The influence of the core SF flow rate on the morphology and diameter of the fibers is shown on the top-line of The middle line of The last line of The results suggested that in this coaxial electrospinning process, the SF solution served as a spinning aid to successfully prepare nanofibers from nearly nonelectrospinnable SELP solution. The core SF solution carried the shell SELP solution through the formation of a stable Taylor cone and continuous jet ejection during the process. The optimum conditions to electrospin uniform SF-SELP nanofibers with different processes parameters are shown phase diagram in TEM imaging were used to confirm the formation of SF-SELP core-shell structured fibers . Sharp b−1 , shiructures ,47), resThe tensile stress-strain curves of SF fibers mats and SF-SELP core-shell fiber mats before and after methanol treatment are shown in The recombinant protein polymer SELP and SF aqueous solutions were used to produce SF-SELP core-shell structured nanofibers by coaxial electrospinning. SF solution with high viscosity and SELP solution with low viscosity were utilized as core and shell materials to cospin, respectively. The effects of processing parameters on morphology and diameters of electrospun SF-SELP nanofibers were studied and the SF core solution served as spinning aid to prepare uniform nanofibers with average diameter from 301 ± 108 nm to 408 ± 150 nm via adjusting process parameters. The core-shell structure of SF-SELP nanofibers was confirmed via fluorescence imaging and TEM. The as-spun SF-SELP nanofiber mats were treated with methanol vapor to make fiber mats insoluble in water based on conversion of the secondary structures from random coils to β-sheets. The mechanical testing of nanofiber mats showed that SF-SELP core-shell structured nanofibers had good flexibility with elongation at break of 5.20% ± 0.57% after methanol treatment, compared with SF nanofibers with elongation at break of 1.38% ± 0.22%. SF-SELP core-shell structured nanofibers should provide interesting utility in biomaterial systems and in the biomedical field such as for drug delivery, due to the functional layer on the surface of the SF fibers.


Adolescence is an age of heightened vulnerability to develop psychiatric disorders that involve alterations in prefrontal cortex circuitry and cognitive dysfunction. The maturation of prefrontal cortex function is linked to the establishment of dopamine connectivity in this region. Development of mesocortical dopamine is a gradual process that continues until early adulthood. Because of its extended maturational course, this system is particularly susceptible to environmental influences. Yet there is a significant gap in our knowledge about the cellular and molecular mechanisms underlying adolescent prefrontal cortex dopamine development and how they are influenced by experience.We examined the role of the Netrin-1 guidance cue receptor, DCC, and its microRNA repressor, miR-218, on adolescent mouse prefrontal cortex development. We used axon-initiated recombination and cell-specific knock-down techniques to characterize the spatiotemporal growth of mesocortical dopamine axons and the role that DCC and miR-218 play in this process. Next, we assessed whether stimulant drugs in adolescence alter miR-218/DCC signaling, thereby disrupting mesocortical dopamine axon growth. Finally, we determined whether altered dopamine axon growth influences prefrontal cortex development by quantifying pyramidal neuron morphology and cognitive performance in adulthood.Here we show, for the first time, that dopamine axons continue to grow from the nucleus accumbens to the prefrontal cortex during adolescence. We discovered that DCC receptors control the extent of this protracted growth by determining where and when dopamine axons recognize their innervation target. Exposure to stimulant drugs or to stress leads to disruption of DCC-dependent adolescent targeting events, causing dopamine axons that should innervate the nucleus accumbens, to grow ectopically to the prefrontal cortex. This effect profoundly changes prefrontal cortex structural and functional development, producing alterations in cognitive processes known to be impaired across psychiatric conditions, including schizophrenia. Importantly, miR-218 controls DCC receptor expression in dopamine neurons across postnatal development and acts as a molecular mediator of the effects of stimulant drugs on prefrontal cortex development.The prolonged growth of dopamine axons during adolescence represents an extraordinary period for experience to influence their growth and predispose to or protect against psychopathology. MicroRNA control of DCC receptor in dopamine neurons is a molecular link where genetic and environmental factors seem to interact in adolescence to influence the development and function of the prefrontal cortex.


In this study, the effect of temperature  and roasting time  on the bioactive compound content  of cold-pressed oil from yellow-seeded rapeseed lines of different colors was investigated. Roasting increased the peroxide value in the seed oils compared to the oils from the control samples. However, roasting did not affect the acid values of the oils, which were 1.15–1.47 and 1.30–1.40 mg KOH/g, for line PN1 03/1i/14 (yellow seeds) and line PN1 563/1i/14 (brown seeds), respectively. In this study, the seeds of line PN1 03/1i/14 were characterized by different changes in canolol content during roasting than the seeds of PN1 563/1i/14. There was a 90-fold increase in canolol for the line PN1 03/1i/14 (768.26 µg/g) and a 46-fold increase for the line PN1 563/1i/14 (576.43 µg/g). Changes in tocopherol and PC-8 contents were also observed. There was an increase in the contents of γ-T and PC-8 in the oils obtained from the seeds roasted at 180 °C for 10 and 15 min. γ-T content increased by 17–18% after 15 min of roasting, whereas the PC-8 content increased twofold. Rapeseed oil is the third most commonly produced vegetable oil in the world, just behind palm and soy oil. . Black rversus linolenic (n-3) fatty acid and high contents of active biological compounds, such as tocopherols, plastochromanol-8, phytosterols and phenol compounds [in vitro conditions [d-glucopyranosyl sinapate; 1,6-di-O-sinapoylglucose; 1,2,6′-tri-O-sinapoylgentiobiose; sinapic acid methyl ester, 3-dihexoside-7-sinapoyl-hexoside kaempferol and 3-hexoside-7-sinapoyl hexoside kaempferol [In recent years, consumer interest in natural, ecological, and low-processed products is on the increase . Cold-pressed oils are considered ecologically friendly and natural by consumers . These oompounds –15. Rapeompounds . Phenol nditions .The mainnditions . Other dempferol –23. Duriempferol , 25. In empferol –30.However, these studies are concerned with black rapeseed. There is no data on the effect of the roasting process on yellow-seeded rapeseeds, which are characterized by thinner seed coats and lower fiber contents. The goal of this project was to assess the effect of roasting (various times and temperatures) on the contents of canolol, tocopherol, plastochromanol-8 in oils obtained from yellow-seeded rapeseeds. The effect of yellow-seeded rapeseed on the rate of decarboxylation of sinapinic acid was studied.n-Hexane (HPLC-grade), and 1.4-dioxane (HPLC-grade) were purchased from Merck . All other solvents and chemicals used in this study were of analytical grade.All tocopherol homologues (purity > 95% by HPLC) were obtained from Calbiochem-Merck Biosciences . Brassica napus × Brassica rapa crosses [Two yellow-seeded winter rapeseed lines were obtained from the Laboratory of Genetics and Quality Breeding of the IHAR-PIB Research Division in Poznań. The origin of the yellow-seeded lines lies in crosses of a spontaneous winter rapeseed mutant with lighter-colored seeds with spotted seed coats—a spring line obtained from earlier  crosses . Seed co crosses .The lineThe control consisted of oil from seeds that had not undergone roasted prior to pressing. The processes of roasting and cold pressing have been presented in an earlier study applied to black-seeded rapeseeds . For eacThe PV and AV of the cold-pressed rapeseed oils were determined using the standard methods from ISO 3960:2001   and ISO n-hexane, made up to 10 ml, and transferred to vials for analysis. Tocopherols were qualitatively and quantitatively identified using a Waters HPLC system  consisting of a pump (Waters 600), a fluorimetric detector (Waters 474), a photodiode array detector (Waters 2998 PDA), an autosampler (Waters 2707), a column oven (Waters Jetstream 2 Plus), and a LiChrosorb Si 60 column  from Merck . The mobile phase was a mixture of n-hexane with 1.4-dioxane (96:4 v/v). The flow rates were 1.0 ml/min (for tocopherols and PC-8) and 2.0 ml/min (for canolol). To detect the fluorescence of tocopherols and PC-8, the excitation wavelength was set at λ = 295 nm and the emission wavelength at λ = 330 nm. To detect the fluorescence of canolol, the excitation wavelength was set at λ = 280 nm and the emission wavelength at λ = 325 nm. Standards of α-, β-, γ- and δ-tocopherols (>95% of purity) were purchased from Merck . The plastochromanol-8 (PC8) contents were assayed and calculated following to Siger et al. [Rapeseed oil (200 mg) was dissolved in r et al. .post hoc analysis was performed using Tukey’s test. Intra-sample quantity variation of the oils was assayed using principal component analysis (PCA). The results of all tests were considered significant at p < 0.05. The statistical analysis was performed using Statistica 10.0 software .The experiment with the roasting rape seeds was performed twice. Results are presented as means ± standard deviations from three replicates for each bioactive compound analysis. The differences between the mean values were determined by analysis of variance (ANOVA). The The protein contained in the pomace left behind after pressing the rapeseed oil could receive better and broader use if the seeds had a lower fiber content, as fiber reduces the energy value of the pressed oil. The solution to this problem is to breed yellow-seeded varieties, which have thinner seed coats and lower concentrations of polyphenolic compounds, with higher contents of protein and fat. Such features are very desirable and reflect the effectively of breeding aimed at improved the quality characteristics of rape , 37.The seeds' fat content was determined as 46.7 ± 0.51% in the case of the line PN1 03/1i/14 and 46.6 ± 0.35% for the line PN1 563/1i/14. The seeds of line PN1 03/1i/14 were found to contain 20.8 ± 0.41% protein, and the seeds of line PN1 563/1i/14 had 21.4 ± 0.36% protein. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were also analyzed. In the line PN1 03/1i/14, the levels of NDF and ADF were lower than in the line PN1 563/1i/14 . The total glucosinolate content was 15.25 ± 0.17 M/g free fatty dry mater (ffdm) of the seeds  for the seeds of line PN1 03/1i/14 and 15.31 ± 0.15 M/g ffdm  for the seeds of line PN1 563/1i/14 (data not show).2/kg. The peroxide values for the cold-pressed oil from the unroasted seeds (the control sample) were 1.27 and 1.20 meq O2/kg, for the seeds of line PN1 03/1i/14 and seeds of line PN1 563/1i/14, respectively. Roasting the seeds resulted in an increase in the peroxide content of the oils obtained from both lines of yellow-seeded rapeseed. Roasting the seeds at 140 °C caused a statistically significant increase (p < 0.0001) in peroxide over that of the control sample. The peroxide levels increased to 2.31 meq O2/kg in the oil from the line PN1 03/1i/14 and to 1.83 meq O2/kg in that from the line PN1 563/1i/14. Increasing the roasting temperature to 160 °C caused further a statistically significant (p < 0.0001) increase in the peroxide levels of the oils. In the case of the seeds line PN1 03/1i/14, following 15 min of roasting at 160 °C, the peroxide content reached 3.09 meq O2/kg. For the seeds line PN1 563/1i/14, peroxide reached 2.55 meq O2/kg (Table p > 0.05) in peroxide content over that of the seeds roasted at 160 °C. It should be noted that the peroxide content of the oil from the line PN1 563/1i/14—which have a darker seed coat—is lower than that from the line PN1 03/1i/14, which have brighter seed coats. The free fatty acid contents show no substantial difference (p > 0.05) between roasted seeds and unroasted seeds  in the canolol content of the oil. The canolol content was influenced both by the increase in temperature and in the roasting time. In the case of the line PN1 03/1i/14, roasting at 140 °C led to an increase in canolol to 22.36 μg/g after 5 min of roasting, to 45.44 μg/g after 10 min of roasting, and to 132.28 μg/g after 15 min of roasting. Increasing the temperature to 160 °C caused a further increase in the canolol content of the oil. After 15 min of roasting, the canolol content increased to 258.93 μg/g (30 times that obtained from the control sample). In oil obtained from seeds roasted at 180 °C, the canolol contents after 5 and 10 min were 264.32 μg/g and 473.65 μg/g, respectively. The highest canolol levels of 768.26 μg/g were noted after roasting the seeds of the line PN1 03/1i/14 for 15 min; this is 90 times the value from the control sample. A smaller increase in canolol was noted in the oil pressed from the roasted seeds of line PN1 563/1i/14: a eightfold increase in canolol was seen for the oil from line PN1 563/1i/14 roasted at 140 °C for 15 min. After 5 min of roasting at 160 °C, the canolol content in the oil was 58.72 μg/g; after 10 min, 137.42 μg/g; and after 15 min, 222.65 μg/g . In the roasting at 140 °C for 5 or 10 min, the α-T and γ-T homologue contents were statistically significantly lower (p < 0.0001) than in the control sample. The content of γ-T in the oil from seeds (line PN1 03/1i/14) roasted at 140 °C for 15 min was statistically significantly higher (p < 0.0001) than in the case of the oils from the seeds roasted at the same temperature but for only 5 or 10 min (Table p < 0.0001) of these compounds, at 76.46 and 81.34 mg/100 g, respectively.The results are shown in Tables p > 0.05) from the control sample. However, in the other samples, an increase in α-T can be seen; this was not noted in the line PN1 03/1i/14-seed oils. In the case of the oils from the line PN1 563/1i/14 seeds that had been roasted at 180 °C for 10 and 15 min, a 12% increase in the α-T content was noted (Table p > 0.05) between the oils from seeds roasted at 140 °C and the control sample. Roasting at 160 °C led to a statistically significant difference (p < 0.0001) in the levels of this homologue, which exceeded 36 mg/100 g  in PC-8: 29% greater after 5 min roasting, 61% after 10 min roasting, and 66% after 15 min roasting. In the oils from the seeds roasted at 180 °C for 10 and 15 min, a 100% increase in PC-8 was seen, reaching 4.21 mg/100 g. No statistically significant difference (p > 0.05) was shown among the oils from the seeds roasted at 180 °C for 10 and 15 min. Gawrysiak-Witulska et al. [et al. [et al. [et al. [The two lines of yellow rapeseed studied here also differed in their PC-8 content. The oil from the seeds of line PN1 03/1i/14 contained between 2.53 and 5.63 mg/100 g of this compound, and that from seeds of line PN1 563/1i/14 had 1.98–4.21 mg/100 g , γ-T levels , total tocopherols  and PC-8 . The results recorded for line PN1 563/1i/14 show that canolol levels are correlated with α-T levels , γ-T levels , total tocopherols , and PC-8 . To show the differences between new breeding line yellow-seeded Brassica napus a principal component analysis was performed. Principal component analysis confirmed the variation in the tested oils obtained from two new breeding lines of yellow-seeded rapeseed, differing by seed-coat color: line PN1 03/1i/14 (yellow) and line PN1 563/1i/14 (brown)  to be obtained than in the case of darker-hulled rapeseed (line PN1 563/1i/14).


The lack of translatable in vitro blood-tumor barrier (BTB) models creates challenges in the development of drugs to treat tumors of the CNS and our understanding of how the vascular changes at the BBB in the presence of a tumor.In this study, we characterize a novel microfluidic model of the BTB (and BBB model as a reference) that incorporates flow and induces shear stress on endothelial cells. Cell lines utilized include human umbilical vein endothelial cells co-cultured with CTX-TNA2 rat astrocytes (BBB) or Met-1 metastatic murine breast cancer cells (BTB). Cells were capable of communicating across microfluidic compartments via a porous interface. We characterized the device by comparing permeability of three passive permeability markers and one marker subject to efflux.−3, n = 4) than the BBB model . Similar permeability increases were observed in the BTB model for molecules ranging from 600 Da to 60 kDa. The function of P-gp was intact in both models and consistent with recent published in vivo data. Specifically, the rate of permeability of Rhodamine 123 across the BBB model , increased 14-fold in the presence of the P-gp inhibitor verapamil  and eightfold with the addition of Cyclosporine A . Similar values were noted in the BTB model.The permeability of Sulforhodamine 101 was significantly (p < 0.05) higher in the BTB model (13.1 ± 1.3 × 10The dynamic microfluidic in vitro BTB model is a novel commercially available model that incorporates shear stress, and has permeability and efflux properties that are similar to in vivo data. The occurrence of brain metastases in breast cancer patients is approximately 10–16% . After the determination of a perfusion time where an adequate amount of fluorescent marker was allowed to pass into brain, while still remaining in the linear uptake zone, kin was determined [Unidirectional uptake transfer constants (ktermined .in) was determined with linear regression using best-fit values. One-way ANOVA analysis and unpaired t test with Welch’s correction, followed by an F test to compare variances were used for the comparison of the kin values between unrestricted diffusion, BBB, and BTB among each tracer and with Rho123 in absence and presence of inhibitors. For all data, errors are reported as standard error of the mean unless otherwise indicated. Differences were considered statistically significant at the p < 0.05 levels .The slope of the line   and >60 kDa .In initial kinetic experiments, we determined unrestricted diffusion rates of difference sized molecules by perfusing solutes through microfluidic chips without endothelial cells or astrocytes/cancer cells. To quantify tracer accumulation, regions of interest were selected to determine sum fluorescence intensity in the outer compartment ROI 136), central compartment (ROI 139), and background (ROI 165) over time (1D). ROI 165 was taken to ensure data received in the outer and central compartments were significant when compared to the background sum fluorescence. We observed  according to the equation found in our methods. Free Texas Red kin values  and BTB  were significantly different (p < 0.05) between each other. Texas Red 3 kDa values  and BTB  and Texas Red 70 kDa values  and BTB  were also significant (p < 0.05) when compared to the unrestricted diffusion kin, but significance was not observed between the BBB and BTB models of these dyes.In our next experiments, we qualitatively imaged Texas Red accumulation from 0 to 90 min in the BBB model Fig. a–d. Lineues Fig. a for thet p < 0.0 between t p < 0.0 between −3, n = 3), and a significant (p < 0.05) eight fold increase of Rho123 with Cyclosporine A  when compared to control Rho123  in the BBB model , and a twofold increase with Cyclosporine A  when compared to Rho123 control  , and Verapamil (50 mM)—concentrations that ensured maximal inhibition . We qualThe results of the studies presented herein suggest that a novel microfluidic chip in part mimics the in vivo BTB with regard to passive permeability and efflux . ImportaPredominant in vitro BBB models have some key similarities. First, there is a presence of some type of “barrier” cell in a luminal or outer compartment (representing the vascular lumen). These cells range from primary or immortalized brain endothelial cells , peripheral endothelial (HUVECs), or stem-cell derived cells. In this study HUVECs were chosen as they are commonly used by a number of labs, and using a co-culture of astrocytes or even astrocyte-conditioned media alone has been shown to induce BBB-like expression of tight junctions and barrier tightness in a variety of endothelial cells , 29, 35.The use of dyes has been a long-standing method to evaluate the integrity of the BBB and the breakdown of the BTB . Some ofin), from the outer compartment to the central compartment, of all three sized molecules were not significantly different from each other. This data is consistent with previous work showing that if the diameter of each molecule being tested is at least 12× less than the barrier defects, then diffusion will remain constant for all molecules [In measuring the unrestricted diffusion (the absence of cells) of molecules from the outer chamber to the center chamber, we observed that the diffusion rates  forced on the endothelial cells, which may contribute to the low passive permeability measurements which can be as low as ~74 Ω cm2 , compare2 [2 . While a2 [2 –31, this2 [2  in additThis novel and dynamic microfluidic in vitro BTB model mimics the in vivo barrier with regard to shear stress, permeability, and efflux. Permeability of large molecule dextrans, as well as small molecule dextrans and Rhodamine 123 (with and without inhibitors) were characteristic and relatable to what is seen in vivo. The shear stress from adding flow over HUVECs eliminates the unstirred water layer and allows for different tracers to be added and followed in real time from outer to central compartment. Based on these characteristics, this microfluidic chip shows potential for use in BBB and BTB research. Expanding on these data, future work should entail the use of different drugs, and the comparison of different endothelial cell models to in vivo data with regards to passive permeability and influx/efflux.