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Abstract Resistance against aminoglycosides is widespread in bacteria. This study aimed to identify genes that are important for growth of E. coli during aminoglycoside exposure, since such genes may be targeted to re-sensitize resistant E. coli to treatment. We constructed three transposon mutant libraries each containing > 230.000 mutants in E. coli MG1655 strains harboring streptomycin (aph(3″)-Ib/aph(6)-Id), gentamicin (aac(3)-IV), or neomycin (aph(3″)-Ia) resistance gene(s). Transposon Directed Insertion-site Sequencing (TraDIS), a combination of transposon mutagenesis and high-throughput sequencing, identified 56 genes which were deemed important for growth during streptomycin, 39 during gentamicin and 32 during neomycin exposure. Most of these fitness-genes were membrane-located (n = 55) and involved in either cell division, ATP-synthesis or stress response in the streptomycin and gentamicin exposed libraries, and enterobacterial common antigen biosynthesis or magnesium sensing/transport in the neomycin exposed library. For validation, eight selected fitness-genes/gene-clusters were deleted (minCDE , hflCK , clsA and cpxR associated with streptomycin and gentamicin resistance, and phoPQ, wecA , lpp and pal associated with neomycin resistance), and all mutants were shown to be growth attenuated upon exposure to the corresponding antibiotics. In summary, we identified genes that are advantageous in aminoglycoside-resistant E. coli during antibiotic stress. In addition, we increased the understanding of how aminoglycoside-resistant E. coli respond to antibiotic exposure.

Escherichia coli carrying IncK-blaCMY-2 plasmids mediating resistance to extended-spectrum cephalosporins (ESC) has been frequently described in food-producing animals and in humans. This study aimed to characterize IncK-blaCMY-2-positive ESC-resistant E. coli isolates from poultry production systems in Denmark, Finland, and Germany, as well as from Danish human blood infections, and further compare their plasmids. Whole-genome sequencing (Illumina) of all isolates (n = 46) confirmed the presence of the blaCMY-2 gene. Minimum inhibitory concentration (MIC) testing revealed a resistant phenotype to cefotaxime as well as resistance to ≥3 antibiotic classes. Conjugative transfer of the blaCMY-2 gene confirmed the resistance being on mobile plasmids. Pangenome analysis showed only one-third of the genes being in the core with the remainder being in the large accessory gene pool. Single nucleotide polymorphism (SNP) analysis on sequence type (ST) 429 and 1286 isolates showed between 0–60 and 13–90 SNP differences, respectively, indicating vertical transmission of closely related clones in the poultry production, including among Danish, Finnish, and German ST429 isolates. A comparison of 22 ST429 isolates from this study with 80 ST429 isolates in Enterobase revealed the widespread geographical occurrence of related isolates associated with poultry production. Long-read sequencing of a representative subset of isolates (n = 28) allowed further characterization and comparison of the IncK-blaCMY-2 plasmids with publicly available plasmid sequences. This analysis revealed the presence of highly similar plasmids in ESC-resistant E. coli from Denmark, Finland, and Germany pointing to the existence of common sources. Moreover, the analysis presented evidence of global plasmid transmission and evolution. Lastly, our results indicate that IncK-blaCMY-2 plasmids and their carriers had been circulating in the Danish production chain with an associated risk of spreading to humans, as exemplified by the similarity of the clinical ST429 isolate to poultry isolates. Its persistence may be driven by co-selection since most IncK-blaCMY-2 plasmids harbor resistance factors to drugs used in veterinary medicine.

Abstract Background: Rucaparib is a poly(ADP-ribose) polymerase inhibitor approved in Europe as maintenance therapy for recurrent platinum-sensitive (Pt-S) ovarian cancer (OC). The Rucaparib Access Programme (RAP) was designed to provide early access to rucaparib for the above-mentioned indication, as well as for patients with BRCA-mutated Pt-S or platinum-resistant (Pt-R) OC and no therapeutic alternatives. Methods: In this observational, retrospective study we analysed the efficacy and safety of rucaparib within the RAP in Spain. Hospitals associated with the Spanish Ovarian Cancer Research Group (GEICO) recruited patients with high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer treated with rucaparib 600 mg twice daily as maintenance or treatment (Pt-S/Pt-R) in the RAP. Baseline characteristics, efficacy, and safety data were collected. Results: Between July 2020 and February 2021, 51 patients treated in 22 hospitals in the RAP were included in the study. Eighteen patients with a median of 3 (range, 1–6) prior treatment lines received rucaparib as maintenance; median progression-free survival (PFS) for this group was 9.1 months (95% confidence interval [CI], 4.2–11.6 months). Among 33 patients (median 5 [range, 1–9] prior treatment lines) who received rucaparib as treatment, 7 and 26 patients had Pt-S and Pt-R disease, respectively. Median PFS was 10.6 months (95% CI, 2.5 months-not reached) in the Pt-S group and 2.2 months (95% CI, 1.1–3.2 months) in the Pt-R group. Grade ≥ 3 treatment-emergent adverse events were reported in 39% of all patients, the most common being anaemia (12% and 15% in the maintenance and treatment groups, respectively). At data cut-off, 5 patients remained on treatment. Conclusion Efficacy results in these heavily pre-treated patients were similar to those from previous trials. The safety profile of rucaparib in real life was predictable and manageable.

Abstract This work investigates non-contact reflectance spectral imaging techniques, i.e. microscopic Fourier transform infrared (FTIR) imaging, macroscopic visible-near infrared (VNIR), and shortwave infrared (SWIR) spectral imaging, for the identification of bacteria on stainless steel. Spectral images of two Gram-positive (GP) bacteria (Bacillus subtilis (BS) and Lactobacillus plantarum (LP)), and three Gram-negative (GN) bacteria (Escherichia coli (EC), Cronobacter sakazakii (CS), and Pseudomonas fluorescens (PF)), were collected from dried suspensions of bacterial cells dropped onto stainless steel surfaces. Through the use of multiple independent biological replicates for model validation and testing, FTIR reflectance spectral imaging was found to provide excellent GP/GN classification accuracy (> 96%), while the fused VNIR-SWIR data yielded classification accuracy exceeding 80% when applied to the independent test sets. However, classification within gram type was far less reliable, with lower accuracies for classification within the GP (

Escherichia coli is the main cause of urinary tract infections (UTI). While genomic comparison of specific clones recovered from animals, and human extraintestinal infections show high identity, studies demonstrating the uropathogenicity are lacking. In this study, comparative genomics combined with bladder-cell and biofilm formation assays, were performed for 31 E. coli of different origins: 7 from meat (poultry, beef, and pork); 2 from avian-farm environment; 12 from human uncomplicated UTI, uUTI; and 10 from human complicated UTI, cUTI. These isolates were selected based on their genetic uropathogenic (UPEC) status and phylogenetic background. In silico analysis revealed similar virulence-gene profiles, with flagella, type 1 and curli fimbriae, outer-membrane proteins (agn43, ompT, iha), and iron-uptake (iutA, entA, and fyuA) associated-traits as the most prevalent (>65%). In bladder-cell assays, moderate to strong values of association (83%, 60%, 77.8%) and invasion (0%, 70%, 55.5%) were exhibited by uUTI, cUTI, and animal-derived isolates, respectively. Of interest, uUTI isolates exhibited a significantly lower invasive capacity than cUTI isolates (p

The increasing prevalence of antimicrobial resistance and the limited availability of new antimicrobial agents have created an urgent need for new approaches to combat these issues. One such approach involves reevaluating the use of old antibiotics to ensure their appropriate usage and maximize their effectiveness, as older antibiotics could help alleviate the burden on newer agents. An example of such an antibiotic is chloramphenicol (CHL), which is rarely used due to its hematological toxicity. In the current study, we employed a previously published transposon mutant library in MG1655/pTF2::blaCTX-M-1, containing over 315,000 unique transposon insertions, to identify the genetic factors that play an important role during growth in the presence of CHL. The list of conditionally essential genes, collectively referred to as the secondary resistome (SR), included 67 genes. To validate our findings, we conducted gene knockout experiments on six genes: arcA, hfq, acrZ, cls, mdfA, and nlpI. Deleting these genes resulted in increased susceptibility to CHL as demonstrated by MIC estimations and growth experiments, suggesting that targeting the products encoded from these genes may reduce the dose of CHL needed for treatment and hence reduce the toxicity associated with CHL treatment. Thus, the gene products are indicated as targets for antibiotic adjuvants to favor the use of CHL in modern medicine.

Cell adhesion proteins have been described at sites away from the cell surface, including in the nucleus. Here, the authors report the scale of nuclear localisation of adhesion proteins, establishing a nucleo-adhesome and showing that nuclear adhesion proteins can cooperate to control transcription.

Objective: To analyze the change in tooth color produced by two hydraulic and one resin-based sealers by means of spectrophotometry for 3 years. Methods: Forty maxillary anterior teeth were selected (n = 10 per group). Root canals were prepared by rotary instrumentation and irrigation was performed with NaOCl, which was also used in the final irrigation, followed by saline and activated with Endoactivator. Root canals were then filled using single cone technique. Negative control (NC): gutta-percha filling; Positive control: gutta-percha and AH Plus; experimental groups: gutta-percha and Bioroot RCS/TotalFill BC Sealer. Gutta-percha was cut 2 mm below the cementoenamel junction, the pulp chamber was sealed with flowable composite. The teeth were kept in PBS. Color was measured in the cervical and incisal halves before root canal treatment (RCT), one and six months after RCT, and after one, two and three years, with the Vita EasyShade spectrophotometer; positioned using an individualized splint. ΔEab and ΔE00 were calculated. Two-way ANOVA repeated measures test followed by Bonferroni post-test were performed to analyze the ΔL, Δa*, Δb*, ΔEab, and ΔE00, considering treatment groups and evaluation periods. Statistical significance was set at p NC > TotalFill > Bioroot. ΔE00 values were: 1.38 ± 0.61 NC, 2.37 ± 0.70 AH Plus, 1.86 ± 0.60 BioRoot and 1.53 ± 0.85 TotalFill. In the cervical half, the ΔEab values, showed the same descending order, except for Bioroot and TotalFill which alternated the order. The ΔE00 values were 1.86 ± 0.61 NC, 3.01 ± 0.70 AH Plus,1.89 ± 0.58 Bioroot and 1.65 ± 0.41 TotalFill, with no significant differences between groups and times in both locations. Conclusions: All groups presented some degree of discoloration. Lightness and b* component were the most influential. Calcium silicate-based cements (Bioroot RCS and TotalFill BC Sealer) produced acceptable levels of discoloration at the end of follow-up.

Salmonella enterica serovar Derby (S. Derby) ranks fifth among nontyphoidal Salmonella serovars causing human infections in the European Union. S. Derby isolates (36) collected between 2006 and 2018 in a Spanish region (Asturias) from human clinical samples (20) as well as from pig carcasses, pork- or pork and beef-derived products, or wild boar (16) were phenotypically characterized with regard to resistance, and 22 (12 derived from humans and 10 from food-related samples) were also subjected to whole genome sequence analysis. The sequenced isolates belonged to ST40, a common S. Derby sequence type, and were positive for SPI-23, a Salmonella pathogenicity island involved in adherence and invasion of the porcine jejune enterocytes. Isolates were either susceptible (30.6%), or resistant to one or more of the 19 antibiotics tested for (69.4%). Resistances to tetracycline [tet(A), tet(B) and tet(C)], streptomycin (aadA2), sulfonamides (sul1), nalidixic acid [gyrA (Asp87 to Asn)] and ampicillin (blaTEM-1-like) were detected, with frequencies ranging from 8.3% to 66.7%, and were higher in clinical than in food-borne isolates. The fosA7.3 gene was present in all sequenced isolates. The most common phenotype was that conferred by the tet(A), aadA2 and sul1 genes, located within identical or closely related variants of Salmonella Genomic Island 1 (SGI1), where mercury resistance genes were also present. Diverse IncI1-I(α) plasmids belonging to distinct STs provided antibiotic [blaTEM-1, tet(A) and/or tet(B)] and heavy metal resistance genes (copper and silver), while small pSC101-like plasmids carried tet(C). Regardless of their location, most resistance genes were associated with genetic elements involved in DNA mobility, including a class one integron, multiple insertion sequences and several intact or truncated transposons. By phylogenetic analysis, the isolates were distributed into two distinct clades, both including food-borne and clinical isolates. One of these clades included all SGI1-like positive isolates, which were found in both kinds of samples throughout the entire period of study. Although the frequency of S. Derby in Asturias was very low (0.5% and 3.1% of the total clinical and food isolates of S. enterica recovered along the period of study), it still represents a burden to human health linked to transmission across the food chain. The information generated in the present study can support further epidemiological surveillance aimed to control this zoonotic pathogen.

Due to the rapid spread of CTX-M type ESBLs, the rate of resistance to third-generation cephalosporin has increased among Gram-negative bacteria, especially in Escherichia coli, and there is a need to find ways to re-sensitize ESBL E. coli to cephalosporin treatment. A previous study showed that genes involved in protein synthesis were significantly up-regulated in the presence of subinhibitory concentration of cefotaxime (CTX) in a CTX-M-1-producing E. coli. In this study, the interaction between CTX and gentamicin (GEN), targeting protein synthesis, was evaluated in MG1655/pTF2, and the MIC of CTX was strongly reduced (128-fold) in the presence of this combnation therapy. Since the underlying mechanism behind this synergy is not known, we constructed a saturated transposon mutant library in MG1655/pTF2::blaCTX-M-1 containing 315,925 unique transposon insertions to measure mutant depletion upon exposure to CTX, GEN, and combination treatment of CTX and GEN by Transposon Directed Insertion-site Sequencing (TraDIS). We identified 57 genes that were depleted (log2FC ≤ −2 and with q.value ≤ 0.01) during exposure to CTX, 18 for GEN, and 31 for combination treatment of CTX and GEN. For validation, we deleted eight genes that were either uniquely identified in combination treatment, overlapped with monotherapy of GEN, or were shared between combination treatment and monotherapy with CTX and GEN. Of these genes, we found that the inactivation of dnaK, mnmA, rsgA, and ybeD increased the efficacy of both CTX and GEN treatment, the inactivation of cpxR and yafN increased the efficacy of only CTX, and the inactivation of mnmA, rsgA, and ybeD resulted in increased synergy between CTX and GEN. Thus, the study points to putative targets for helper drugs that can restore susceptibility to these important drugs, and it indicates that genes involved in protein synthesis are essential for the synergy between these two drugs. In summary, the study identified mutants that sensitize ESBL-producing E. coli to CTX and a combination of CTX and GEN, and it increased our understanding of the mechanism behind synergy between β-lactam and aminoglycoside drugs. This forms a framework for developing new strategies to combat infections caused by resistant bacteria.

Abstract Background Possible cases of SARS-CoV-2 infection were diagnosed in primary care in Madrid, some of these cases had pneumonia. Most of the SARS-CoV-2 pneumonia published data came from hospitalised patients. This study set out to describe clinical characteristics of patients with SARS-CoV-2 pneumonia diagnosed in primary care across age groups and type of pneumonia. Methods Observational retrospective study obtaining clinical data from the electronic health records of patients who were followed-up by SARS-CoV-2 possible infection in a primary care practice in Madrid. All the cases were collected by in-person or remote consultation during the 10th March to the 7th of April. Exposure: Diagnosis of SARS-CoV-2 pneumonia by chest X-ray ordered by the GP. Main outcomes and measures: Symptoms of SARS-CoV-2 pneumonia, physical examination and diagnostic tests as a blood test, nasopharyngeal swab results for RT-PCR (Reverse transcriptase-polymerase chain reaction) and chest X-ray results. Results The overall SARS-CoV-2 pneumonias collected were 172 (female 87 [50.6%], mean age 60.5 years standard deviation [SD] 17.0). Comorbidities were body mass index ≥ 25 kg/m2 (90 [52.3%]), hypertension (83 [48.3%]), dyslipidaemia (68 [39.5%]) and diabetes (33 [19.2%]). The sample was stratified by age groups (

Objective: Zinc oxide is used to prevent post-weaning diarrhoea in pigs as an alternative to antimicrobial growth promoters. This study aims to determine if the use of zinc oxide selects for extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and affects the expression of blaCTX-M-1 in E. coli. Methods: Using an in vitro faecal micro-cosmos model, the selective properties of zinc were investigated using an E. coli strain with blaCTX-M-1 encoded by a natural IncI1 resistance plasmid (MG1655/pTF2) and another strain where the same gene was located on the chromosome (MG1655::blaCTX-M-1). The micro-cosmos was seeded with faecal material containing an increasing concentration of zinc (0–8 mM). Outcome measurements consisted of colony-forming units (CFU) of the inoculated ESBL E. coli and naturally occurring coliforms as determined by plate counting on MacConkey with and without 5 mg/L cefotaxime as well as total viable bacteria determined on Luria agar without cefotaxime. Expression of blaCTX-M-1 under the experimental zinc concentrations was determined by quantitative polymerase chain reaction. Results: The proportion of MG1655/pTF2 of the total viable bacteria was significantly higher at high zinc concentrations (6 and 8 mM) compared with low concentrations (0–4 mM). The messenger RNA (mRNA) levels of blaCTX-M-1 in the two ESBL strains increased at increasing zinc concentrations and varied with the growth phase. Conclusion: The growth of the inoculated CTX-M-1-encoding E. coli MG1655 strains and naturally occurring coliforms was impacted differently when exposed to zinc oxide. The blaCTX-M-1 mRNA expression levels seemed to increase with increasing zinc concentrations, but varied with growth phase, but not gene location.

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infection (UTI), a widespread infectious disease of great impact on human health. This is further emphasized by the rapidly increase in antimicrobial resistance in UPEC, which compromises UTI treatment. UPEC biology is highly complex since uropathogens must adopt extracellular and intracellular lifestyles and adapt to different niches in the host. In this context, the implementation of forefront ‘omics’ technologies has provided substantial insight into the understanding of UPEC pathogenesis, which has opened the doors for new therapeutics and prophylactics discovery programs. Thus, ‘omics’ technologies applied to studies of UPEC during UTI, or in models of UTI, have revealed extensive lists of factors that are important for the ability of UPEC to cause disease. The multitude of large ‘omics’ datasets that have been generated calls for scrutinized analysis of specific factors that may be of interest for further development of novel treatment strategies. In this review, we describe main UPEC determinants involved in UTI as estimated by ‘omics’ studies, and we compare prediction of factors across the different ‘omics’ technologies, with a focus on those that have been confirmed to be relevant under UTI-related conditions. We also discuss current challenges and future perspectives regarding analysis of data to provide an overview and better understanding of UPEC mechanisms involved in pathogenesis which should assist in the selection of target sites for future prophylaxis and treatment.

The objective of this study is to compare portable visible spectral imaging (443–726 nm) and conventional RGB imaging for detecting products stored beyond the recommended “use-by” date and predicting the number of days poultry products have been stored. Packages of chicken thighs with skin on were stored at 4 °C and imaged daily in pack through plastic lidding film using spectral and RGB imaging over 10 days. K-nearest neighbour (KNN) models were built to detect poultry stored beyond its recommended “use-by” date and partial least squares regression (PLSR) models were built to predict the storage day of samples. Model overfitting in the spectral PLSR model was prevented using a geostatistical approach to estimate the number of latent variables (LV). All models were built at the object level by using mean spectra and colour values per image. The KNN model built using spectral images (acc. = 93 %, sen. = 75 %, spec. = 100 %) was more suitable than the model built using RGB images (acc. = 80 %, sen. = 42 %, spec. = 96 %) for detecting poultry stored beyond its “use-by” date. The PLSR model built using spectral images (R2 = 0.78 RMSEC = 0.92, RMSEV = 1.11, RMSEP = 1.34 day) was more suitable than the model built using RGB images (R2 = 0.60, RMSEC = 1.66, RMSEV = 1.67, RMSEP = 1.92 day) for predicting storage day of poultry products.

Metastatic melanoma is a highly immunogenic tumor with very poor survival rates due to immune system escape-mechanisms. Immune checkpoint inhibitors (ICIs) targeting the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and the programmed death-1 (PD1) receptors, are being used to impede immune evasion. This immunotherapy entails an increment in the overall survival rates. However, melanoma cells respond with evasive molecular mechanisms. ERK cascade inhibitors are also used in metastatic melanoma treatment, with the RAF activity blockade being the main therapeutic approach for such purpose, and in combination with MEK inhibitors improves many parameters of clinical efficacy. Despite their efficacy in inhibiting ERK signaling, the rewiring of the melanoma cell-signaling results in disease relapse, constituting the reinstatement of ERK activation, which is a common cause of some resistance mechanisms. Recent studies revealed that the combination of RAS–ERK pathway inhibitors and ICI therapy present promising advantages for metastatic melanoma treatment. Here, we present a recompilation of the combined therapies clinically evaluated in patients.

Within the process of progressive digitization of materials and tools for teaching and distance learning of a subject of introduction to Microeconomics (quarterly, in year three of the Degree in Social Work), taught by the authors at the National University of Distance Education (UNED), a virtual assistant in the form of chatbot, or conversational robot, called EconBot, has been designed and made available to students from 2017. This paper presents the reasons that led to its adoption, the process of its development, differentiating two phases, its characteristics and functions, the assessment of its usefulness and the role of teachers in the implementation of this type of technological innovation.

Complex gill disorder (CGD) is an important condition in Atlantic salmon aquaculture, but the roles of the putative aetiological agents in the pathogenesis are uncertain. A longitudinal study was undertaken on two salmon farms in Scotland to determine the variations in loads of CGD-associated pathogens (Desmozoon lepeophtherii, Candidatus Branchiomonas cysticola, salmon gill pox virus (SGPV) and Neoparamoeba perurans) estimated by quantitative PCR. In freshwater, Ca. B. cysticola and SGPV were detected in both populations, but all four pathogens were detected on both farms during the marine stage. Candidatus B. cysticola and D. lepeophtherii were detected frequently, with SGPV detected sporadically. In the marine phase, increased N. perurans loads associated significantly (p < 0.05) with increases in semi-quantitative histological gill-score (HGS). Increased Ca. B. cysticola load associated significantly (p < 0.05) with increased HGS when only Farm B was analysed. Higher loads of D. lepeophtherii were associated significantly (p < 0.05) with increased HGS on Farm B despite the absence of D. lepeophtherii-type microvesicles. Variations in SGPV were not associated significantly (p > 0.05) with changes in HSG. This study also showed that water temperature (season) and certain management factors were associated with higher HGS. This increase in histological gill lesions will have a deleterious impact on fish health and welfare, and production performance.

Abstract Background Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T). Results Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed. Conclusions Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

Deep learning (DL) has recently achieved considerable successes in a wide range of applications, such as speech recognition, machine translation and visual recognition. This tutorial provides guidelines and useful strategies to apply DL techniques to address pixel-wise classification of spectral images. A one-dimensional convolutional neural network (1-D CNN) is used to extract features from the spectral domain, which are subsequently used for classification. In contrast to conventional classification methods for spectral images that examine primarily the spectral context, a three-dimensional (3-D) CNN is applied to simultaneously extract spatial and spectral features to enhance classification accuracy. This tutorial paper explains, in a stepwise manner, how to develop 1-D CNN and 3-D CNN models to discriminate spectral imaging data in a food authenticity context. The example image data provided consists of three varieties of puffed cereals imaged in the NIR range (943–1643 nm). The tutorial is presented in the MATLAB environment and scripts and dataset used are provided. Starting from spectral image pre-processing (background removal and spectral pre-treatment), the typical steps encountered in development of CNN models are presented. The example dataset provided demonstrates that deep learning approaches can increase classification accuracy compared to conventional approaches, increasing the accuracy of the model tested on an independent image from 92.33 % using partial least squares-discriminant analysis to 99.4 % using 3-CNN model at pixel level. The paper concludes with a discussion on the challenges and suggestions in the application of DL techniques for spectral image classification.

Uropathogenic Escherichia coli (UPEC) are the most common cause of urinary tract infection (UTI). UPEC normally reside in the intestine, and during establishment of UTI, they undergo metabolic adaptations, first to urine and then upon tissue invasion to the bladder cell interior. To understand these adaptations, we used quantitative proteomic profiling to characterize protein expression of the UPEC strain UTI89 growing in human urine and when inside J82 bladder cells. In order to facilitate detection of UPEC proteins over the excess amount of eukaryotic proteins in bladder cells, we developed a method where proteins from UTI89 grown in MOPS and urine was spiked-in to enhance detection of bacterial proteins. More than 2000 E. coli proteins were detected. During growth in urine, proteins associated with iron acquisition and several amino acid uptake and biosynthesis systems, most prominently arginine metabolism, were significantly upregulated. During growth in J82 cells, proteins related to iron uptake and arginine metabolisms were likewise upregulated together with proteins involved in sulfur compound turnover. Ribosomal proteins were downregulated relative to growth in MOPS in this environment. There was no direct correlation between upregulated proteins and proteins reported to be essential for infections, showing that upregulation during growth does not signify that the proteins are essential for growth under a condition.

Las técnicas de reproducción asistida (TRA ) pueden estar asociadas a complicaciones obstétricas y perinatales, incluido un mayor riesgo de malformaciones congénitas y de patología adquirida. Es un deber de los profesionales sanitarios informar a quienes desean someterse a estas técnicas, sobre los riesgos para la salud de los hijos y las madres. A parte de los riesgos biológicos comentados, las TRA plantean también múltiples problemas bioéticos, que se han de abordar partiendo del valor fundamental de la vida humana. El objetivo principal de este estudio retrospectivo es el análisis bioético y biomédico del impacto de las TRA en la salud de los niños y sus madres, comparando el riesgo de patología entre los nacidos por TRA y por concepción natural.

Salmonella Gallinarum only infects avian species, where it causes a severe systemic infection in birds of all ages. It is generally accepted that interaction with phagocytic cells plays an important role in the development of systemic, host-specific Salmonella infections. The current study detailed the interaction of S. Gallinarum with macrophages derived from chicken (HD11) and cattle (Bomac) compared to interaction of the broad host range serovar, Salmonella Typhimurium and the cattle adapted serovar Salmonella Dublin. Results showed a weaker invading ability of S. Gallinarum in both kinds of macrophages, regardless whether the bacteria were opsonized or not before infections. However, opsonization of S. Gallinarum by chicken serum increased its intracellular survival rate in chicken macrophages. No significant induction of nitrogen oxide was observed in the infected HD11 cells within the first 6 h, and levels of reactive oxygen species (ROS) were similar among the three serovars. S. Gallinarum infection was associated with low cell deaths in both chicken and cattle macrophages, whereas S. Dublin only induced a comparable high level of cell death in chicken macrophages, but not in macrophages of its preferred host species (Bomac) compared to host generalist S. Typhimurium. S. Gallinarum-infected HD11 macrophages exhibited low induction of pro-inflammation genes [interleukin (IL)1β, CXCLi1, and CXCLi2] compared to the two other serovars, and contrary to the other serovars, it did not induce significant downregulation of Toll-like receptor (TLR)2, TLR4, and TLR5. In in vivo infection of 1-week-old chicken, a significant upregulation of the TLR4 and TLR5 genes in the spleen was observed in S. Gallinarum-infected chickens, but not in S. Typhimurium-infected chicken at 5 days post-infections. Taken together, results show that S. Gallinarum infection of macrophages was characterized by low uptake and low cytotoxicity, possibly allowing long-term persistence in the intracellular environment, and it caused a low induction of pro-inflammatory responses.

This work investigates the application of reflectance Fourier transform infrared (FTIR) microscopic imaging for rapid, and non-invasive detection and classification between Bacillus subtilis and Escherichia coli cell suspensions dried onto metallic substrates (stainless steel (STS) and aluminium (Al) slides) in the optical density (OD) concentration range of 0.001 to 10. Results showed that reflectance FTIR of samples with OD lower than 0.1 did not present an acceptable spectral signal to enable classification. Two modelling strategies were devised to evaluate model performance, transferability and consistency among concentration levels. Modelling strategy 1 involves training the model with half of the sample set, consisting of all concentrations, and applying it to the remaining half. Using this approach, for the STS substrate, the best model was achieved using support vector machine (SVM) classification, providing an accuracy of 96% and Matthews correlation coefficient (MCC) of 0.93 for the independent test set. For the Al substrate, the best SVM model produced an accuracy and MCC of 91% and 0.82, respectively. Furthermore, the aforementioned best model built from one substrate was transferred to predict the bacterial samples deposited on the other substrate. Results revealed an acceptable predictive ability when transferring the STS model to samples on Al (accuracy = 82%). However, the Al model could not be adapted to bacterial samples deposited on STS (accuracy = 57%). For modelling strategy 2, models were developed using one concentration level and tested on the other concentrations for each substrate. Results proved that models built from samples with moderate (1 OD) concentration can be adapted to other concentrations with good model generalization. Prediction maps revealed the heterogeneous distribution of biomolecules due to the coffee ring effect. This work demonstrated the feasibility of applying FTIR to characterise spectroscopic fingerprints of dry bacterial cells on substrates of relevance for food processing.

Summary: Modern omics technologies allow us to obtain global information on different types of biological networks. However, integrating these different types of analyses into a coherent framework for a comprehensive biological interpretation remains challenging. Here, we present a conceptual framework that integrates protein interaction, phosphoproteomics, and transcriptomics data. Applying this method to analyze HRAS signaling from different subcellular compartments shows that spatially defined networks contribute specific functions to HRAS signaling. Changes in HRAS protein interactions at different sites lead to different kinase activation patterns that differentially regulate gene transcription. HRAS-mediated signaling is the strongest from the cell membrane, but it regulates the largest number of genes from the endoplasmic reticulum. The integrated networks provide a topologically and functionally resolved view of HRAS signaling. They reveal distinct HRAS functions including the control of cell migration from the endoplasmic reticulum and TP53-dependent cell survival when signaling from the Golgi apparatus. : Santra et al. develop MiNETi (Mixed Network Integration) to integrate multi-omics data. Applying MiNETi to analyze the interactome, phosphoproteome, and transcriptome regulated by HRAS signaling from different subcellular compartments shows that HRAS controls phosphorylation-dependent signaling mainly from the cell membrane but regulates a large number of genes from endomembranes. Keywords: RAS, subcellular compartmentalization, signal transduction data integration, network biology, proteomics, transcriptomics, cell migration, TP53, apoptosis

Applications of hyperspectral imaging (HSI) to the quantitative and qualitative measurement of samples have grown widely in recent years, due mainly to the improved performance and lower cost of imaging spectroscopy instrumentation. Data sampling is a crucial yet often overlooked step in hyperspectral image analysis, which impacts the subsequent results and their interpretation. In the selection of pixel spectra for the calibration of classification models, the spatial information in HSI data can be exploited. In this paper, a variety of sampling strategies for selection of pixel spectra are presented, exemplified through five case studies. The strategies are compared in terms of the proportion of global variability captured, practicality and predictive model performance. The use of variographic analysis as a guide to the spatial segmentation prior to sampling leads to the selection of representative subsets while reducing the variation in model performance parameters over repeated random selection.

The treatment of diarrhea in the postweaning period is a common reason for the use of antimicrobials in pig production, and Escherichia coli is the single most important causative agent for this condition. Colistin has recently been classified as a critically important antimicrobial for human health, as it is a last-resort drug against certain multi-drug-resistant Gram-negative bacteria. Therefore, the use of colistin has been significantly reduced in some countries, including Denmark. Despite this, the drug is still commonly used to treat diarrhea in pigs in many countries, and there is a need to understand the risks associated with this practice. We performed a prospective cohort study to investigate the effect of colistin treatment on the changes in the average minimum inhibitory concentration (MIC) in commensal E. coli in a pig herd where no colistin-resistant bacteria were detectable before treatment. One group of pigs was batch treated with colistin after the clinical observation of diarrhea, one group was batch treated with colistin approximately 10 days before the expected onset of diarrhea, and a control group was not treated with colistin but provided with nonantimicrobial antidiarrheal feed supplement. Treatment with colistin in the dose and time combinations used did not result in a significant increase in the average colistin MIC values in E. coli. Moreover, no E. coli strains showed a MIC above the breakpoint of >2 mg/L against colistin. Co-selection of resistance to other antimicrobials was not observed.

Amino acid hydroxylation is a post-translational modification that regulates intra- and inter-molecular protein-protein interactions. The modifications are regulated by a family of 2-oxoglutarate- (2OG) dependent enzymes and, although the biochemistry is well understood, until now only a few substrates have been described for these enzymes. Using quantitative interaction proteomics, we screened for substrates of the proline hydroxylase PHD3 and the asparagine hydroxylase FIH, which regulate the HIF-mediated hypoxic response. We were able to identify hundreds of potential substrates. Enrichment analysis revealed that the potential substrates of both hydroxylases cluster in the same pathways but frequently modify different nodes of signaling networks. We confirm that two proteins identified in our screen, MAPK6 (Erk3) and RIPK4, are indeed hydroxylated in a FIH- or PHD3-dependent mechanism. We further determined that FIH-dependent hydroxylation regulates RIPK4-dependent Wnt signaling, and that PHD3-dependent hydroxylation of MAPK6 protects the protein from proteasomal degradation.

La neurofibromatosis tipo 1 (NF-1) o enfermedad e Von Recklinhousen es una enfermedad multisistémica de herencia autosómica dominante que afecta principalmente a la piel y al sistema nervioso. El diagnóstico es clínico y puede confirmarse por estudio genético, pero técnicamente es complejo y no predice la aparición de complicaciones, por lo que no está indicado realizarlo de forma rutinaria. La neurofibromatosis tipo 1 se asocia a distintas enfermedades endocrinológicas, siendo una de ellas el feocromocitoma. Los feocromocitomas compuestos son tumores raros que también se han relacionado con este síndrome. Asocian feocromocitomas con tumores con un mismo origen embrionario, siendo los que asocian ganglioneuromas los más frecuentes. La prevalencia de feocromocitoma y ganglioneuroma puede estar aumentada en pacientes con NF-1 y esta asociación se ha relacionado con tumores más agresivos, por lo que en el presente artículo se insiste en la importancia de la evaluación de estos pacientes para evitar posibles complicaciones relacionadas con el tumor si no se hace un diagnóstico precoz. Presentamos el caso de un feocromocitoma compuesto con ganglioneuroma en una paciente asintomática afecta de NF-1 y se revisan los aspectos más relevantes de este tipo de tumores Abstract Neurofibromatosis type 1 (NF-1) or Von Recklinhousen´s disease is a multisystem disease of autosomal dominant inheritance that primarily affects the skin and nervous system. Diagnosis is clinical and can be confirmed by genetic testing, but technically complex and does not predict the occurrence of complications, so it is not indicated routinely perform it. Neurofibromatosis type 1 is associated with various endocrine diseases, one of which pheochromocytoma. The compounds pheochromocytomas are rare tumors have also been associated with this syndrome. Pheochromocytomas associated with tumors with the same embryonic origin, associating ganglioneuromas being the most frequent. The prevalence of pheochromocytoma and ganglioneuroma may be increased in patients with NF-1 and this association has been associated with more aggressive tumors, so in this article stresses the importance of evaluating these patients to avoid complications related to the tumor early diagnosis is if it does. We report the case of a pheochromocytoma compound in an asymptomatic patient ganglioneuroma affect NF-1 and the most relevant aspects of this tumor are reviewed.

The resistance plasmid pUO-StVR2, derived from virulence plasmid pSLT, is widespread in clinical isolates of Salmonella enterica serovar Typhimurium recovered in Spain and other European countries. pUO-StVR2 carries several genes encoding a FetMP-Fls system, which could be involved in iron uptake. We therefore analyzed S. Typhimurium LSP 146/02, a clinical strain selected as representative of the isolates carrying the plasmid, and an otherwise isogenic mutant lacking four genes (fetMP-flsDA) of the fetMP-fls region. Growth curves and determination of the intracellular iron content under iron-restricted conditions demonstrated that deletion of these genes impairs iron acquisition. Thus, under these conditions, the mutant grew significantly worse than the wild-type strain, its iron content was significantly lower, and it was outcompeted by the wild-type strain in competition assays. Importantly, the strain lacking the fetMP-flsDA genes was less invasive in cultured epithelial HeLa cells and replicated poorly upon infection of RAW264.7 macrophages. The genes were introduced into S. Typhimurium ATCC 14028, which lacks the FetMP-Fls system, and this resulted in increased growth under iron limitation as well as an increased ability to multiply inside macrophages. These findings indicate that the FetMP-Fls iron acquisition system exceeds the benefits conferred by the other high-affinity iron uptake systems carried by ATCC 14028 and LSP 146/02. We proposed that effective iron acquisition by this system in conjunction with antimicrobial resistance encoded from the same plasmid have greatly contributed to the epidemic success of S. Typhimurium isolates harboring pUO-StVR2.

El objetivo de este estudio ha consistido en seleccionar aquellas pruebas que mejor permitieran estudiar desde un punto de vista formal, el lenguaje del niño/a con hipoacusia. Aunque no hay un consenso general, consideramos que es fundamental analizar todos los aspectos del lenguaje, para tener un conocimiento más completo del desarrollo lingüístico, porque no es paralelo en todos sus ámbitos. Las pruebas escogidas han sido: el test PEABODY; el registro fonológico de Laura Bosch; EDAF; EMLE-TALE 2000; PLON-R y BLOC-SR. Esta evaluación se complementa con un cuestionario dirigido a quienes aplican los test y con la opinión de expertos logopedas en el terreno práctico. En términos generales todas las pruebas han resultado adecuadas en función de su finalidad. Las que mejor se corrigen son EDAF, PEABODY y BLOC-SR y la que peor, el registro fonológico de Laura Bosch. Para nosotros, el procedimiento de aplicación de PEABODY y PLON-R es ideal para niños con hipoacusia, presentando mayores dificultades el EDAF y BLOC-SR y siendo muy difíciles de aplicar, el registro fonológico de Laura Bosch y el EMLE. Consideramos que la selección realizada para evaluar el desarrollo del lenguaje en el niño/a con discapacidad auditiva es adecuada, siempre y cuando tengamos en cuenta la edad de desarrollo y las modificaciones específicas que debemos adoptar en esta población.

In the current study, we identified plasmids carrying antimicrobial resistance genes in draft whole genome sequences of 16 selected Salmonella enterica isolates representing six different serovars from humans in Ghana. The plasmids and the location of resistance genes in the genomes were predicted using a combination of PlasmidFinder, ResFinder, plasmidSPAdes and BLAST genomic analysis tools. Subsequently, S1-PFGE was employed for analysis of plasmid profiles. Whole genome sequencing confirmed the presence of antimicrobial resistance genes in Salmonella isolates showing multidrug resistance phenotypically. ESBL, either blaTEM52−B or blaCTX−M15 were present in two cephalosporin resistant isolates of S. Virchow and S. Poona, respectively. The systematic genome analysis revealed the presence of different plasmids in different serovars, with or without insertion of antimicrobial resistance genes. In S. Enteritidis, resistance genes were carried predominantly on plasmids of IncN type, in S. Typhimurium on plasmids of IncFII(S)/IncFIB(S)/IncQ1 type. In S. Virchow and in S. Poona, resistance genes were detected on plasmids of IncX1 and TrfA/IncHI2/IncHI2A type, respectively. The latter two plasmids were described for the first time in these serovars. The combination of genomic analytical tools allowed nearly full mapping of the resistance plasmids in all Salmonella strains analyzed. The results suggest that the improved analytical approach used in the current study may be used to identify plasmids that are specifically associated with resistance phenotypes in whole genome sequences. Such knowledge would allow the development of rapid multidrug resistance tracking tools in Salmonella populations using WGS.

This is the first report on the genetic diversity of commensal E. coli from pigs reared in an antibiotic free production system and belonging to different age groups. The study investigated the genetic diversity and relationship of 900 randomly collected commensal E. coli strains from non-antimicrobial treated pigs assigned to five different age groups in a Danish farm. Fifty-two unique REP profiles were detected suggesting a high degree of diversity. The number of strains per pig ranged from two to 13. The highest and the lowest degree of diversity were found in the early weaners group (Shannon diversity index, H' of 2.22) and piglets (H' of 1.46) respectively. The REP profiles, R1, R7 and R28, were the most frequently observed in all age groups. E. coli strains representing each REP profile and additional strains associated with the dominant profiles were subjected to PFGE and were assigned to 67 different genotypes. Whole genome sequence analysis of 52 isolates leading to unique REP profiles identified a high level of sequence variation. Six and six strains were assigned to sequence type ST10 and sequence type ST58, respectively. Virulence and antimicrobial resistance genes, as well as, genes associated with mobile genetic elements were commonly found among these commensal E. coli strains. Interestingly, strains yielding the three most common REP profiles clustered together in the SNPs phylogenetic tree, and such strains may represent the archetypal commensal E. coli in Danish pigs.

Sugar beet is the second biggest world contributor to sugar production and the only one grown in Europe. One of the main limitations for its competitiveness is the lack of effective tools for assessing sugar content in unprocessed sugar beet roots, especially in breeding programs. In this context, a dedicated near infrared (NIR) fiber-optic probe based approach is proposed. NIR technology is widely used for the estimation of sugar content in vegetable products, while optic fibers allow a wide choice of technical properties and configurations. The objective of this research was to study the best architecture through different technical choices for the estimation of sugar content in intact sugar beet roots. NIR spectral measurements were taken on unprocessed sugar beet samples using two types of geometries, single and multiple fiber-probes. Sugar content estimates were more accurate when using multiple fiber-probes (up to R2 = 0.93) due to a lesser disruption of light specular reflection. In turn, on this configuration, the best estimations were observed for the smallest distances between emitting and collecting fibers, reducing the proportion of multiply scattered light in the spectra. Error of prediction (RPD) values of 3.95, 3.27 and 3.09 were obtained for distances between emitting and collecting fibers of 0.6, 1.2 and 1.8 µm respectively. These high RPD values highlight the good predictions capacities of the multi-fiber probes. Finally, this study contributes to a better understanding of the effects of the technical properties of optical fiber-probes on the quality of spectral models. In addition, and beyond this specificity related to sugar beet, these findings could be extended to other turbid media for quantitative optical spectroscopy and eventually to validate considered fiber-optic probe design obtained in this experimental study.

BACKGROUND:Antiangiogenic therapies for glioblastoma (GBM) such as bevacizumab (BVZ), have been unable to extend survival in large patient cohorts. However, a subset of patients having angiogenesis-dependent tumors might benefit from these therapies. Currently, there are no biomarkers allowing to discriminate responders from non-responders before the start of the therapy. METHODS:40 patients from the randomized GENOM009 study complied the inclusion criteria (quality of images, clinical data available). Of those, 23 patients received first line temozolomide (TMZ) for eight weeks and then concomitant radiotherapy and TMZ. 17 patients received BVZ+TMZ for seven weeks and then added radiotherapy to the treatment. Clinical variables were collected, tumors segmented and several geometrical measures computed including: Contrast enhancing (CE), necrotic, and total volumes; equivalent spherical CE width; several geometric measures of the CE 'rim' geometry and a set of image texture measures. The significance of the results was studied using Kaplan-Meier and Cox proportional hazards analysis. Correlations were assessed using Spearman correlation coefficients. RESULTS:Kaplan-Meier and Cox proportional hazards analysis showed that total, CE and inner volume (p = 0.019, HR = 4.258) and geometric heterogeneity of the CE areas (p = 0.011, HR = 3.931) were significant parameters identifying response to BVZ. The group of patients with either regular CE areas (small geometric heterogeneity, median difference survival 15.88 months, p = 0.011) or those with small necrotic volume (median survival difference 14.50 months, p = 0.047) benefited substantially from BVZ. CONCLUSION:Imaging biomarkers related to the irregularity of contrast enhancing areas and the necrotic volume were able to discriminate GBM patients with a substantial survival benefit from BVZ. A prospective study is needed to validate our results.

Introduction: Corticobasal syndrome (CBS) has a heterogeneous clinical presentation with no specific pathologic substratum. Its accurate diagnosis is a challenge for neurologists; in order to establish CBS definitively, postmortem confirmation is required. Some clinical and radiological features can help to distinguish it from other neurodegenerative conditions, such as Creutzfeldt-Jakob disease (CJD). Clinical Case: A 74-year-old woman presented with language impairment, difficulty in walking and poor attentiveness that had begun 10 days before. Other symptoms, such as asymmetrical extra-pyramidal dysfunction, limb dystonia and ‘alien limb’ phenomena, were established over the next 2 months, with rapid progression. Death occurred 3 months after symptom onset. Laboratory results were normal. Initially, imaging only showed restricted diffusion with bilateral parieto-occipital gyri involvement on DWI-MRI, with unspecific EEG changes. An autopsy was performed. Brain neuropathology confirmed sporadic CJD (sCJD). Conclusions: CBS is a heterogeneous clinical syndrome whose differential diagnosis is extensive. CJD can occasionally present with clinical characteristics resembling CBS. MRI detection of abnormalities in some sequences (FLAIR, DWI), as previously reported, has high diagnostic utility for sCJD diagnosis – especially in early stages – when other tests can still appear normal. Abnormalities on DWI sequencing may not correlate with neuropathological findings, suggesting a functional basis to explain the changes found.

The reform of the autonomous financing system in 2009, motivated by thestatutory reforms approved in recent years, particularly the reform of theCatalan Statute, was implemented as a response to the primary weaknessespresented by the previous system, specifically in relation to the problems offinancial inadequacy, deficiencies in the capping system, the insufficient taxdevolution and the weak fiscal co-responsibility. After analysing the backgroundinformation of the reform and the negotiating process, the articledescribes the new model and the different elements integrating it, and thenit identifies both the aspects of the reform that deserve a positive evaluationand the problems that it has not managed to solve, in terms of both the taxautonomy and the fiscal co-responsibility of the Communities, and the budgetcapping mechanisms, also indicating the possibilities of improvement inthe institutional articulation and system governance.

El cáncer de mama es un problema socio-sanitario de primer orden en España debido a su elevada incidencia. En las últimas tres décadas se han producido notables mejoras en la supervivencia debidas tanto a la introducción de las campañas de cribado mamográfico como a los nuevos tratamientos médicos. Pese a estos datos optimistas, no hay que olvidar que aún quedan pacientes que recaen tras el tratamiento del tumor mamario (cerca del 20%) y fallecen a causa de la enfermedad, por lo que es importante continuar con los esfuerzos de investigación en cáncer de mama hasta conseguir una tasa de recaída cero. En este artículo se revisan distintos aspectos de esta enfermedad como son sus síntomas y signos, el diagnóstico, la quimioprevención, así como el tratamiento del cáncer de mama localizado y metastásico.

In current industrial environments there is an increasing need for practical and inexpensive quality control systems to detect the foreign food materials in powder food processing lines. This demand is especially important for the detection of product adulteration with traces of highly allergenic products, such as peanuts and tree nuts. Manufacturing industries dealing with the processing of multiple powder food products present a substantial risk for the contamination of powder foods with traces of tree nuts and other adulterants, which might result in unintentional ingestion of nuts by the sensitised population. Hence, the need for an in-line system to detect nut traces at the early stages of food manufacturing is of crucial importance. In this present work, a feasibility study of a spectral index for revealing adulteration of tree nut and peanut traces in wheat flour samples with hyperspectral images is reported. The main nuts responsible for allergenic reactions considered in this work were peanut, hazelnut and walnut. Enhanced contrast between nuts and wheat flour was obtained after the application of the index. Furthermore, the segmentation of these images by selecting different thresholds for different nut and flour mixtures allowed the identification of nut traces in the samples. Pixels identified as nuts were counted and compared with the actual percentage of peanut adulteration. As a result, the multispectral system was able to detect and provide good visualisation of tree nut and peanut trace levels down to 0.01% by weight. In this context, multispectral imaging could operate in conjuction with chemical procedures, such as Real Time Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay to save time, money and skilled labour on product quality control. This approach could enable not only a few selected samples to be assessed but also to extensively incorporate quality control surveyance on product processing lines.

BRAF functions in the RAS-extracellular signal-regulated kinase (ERK) signaling cascade. Activation of this pathway is necessary to mediate the transforming potential of oncogenic BRAF, however, it may also cause a negative feedback that inhibits the epidermal growth factor receptor (EGFR). Mitogen-inducible gene-6 (MIG-6) is a potent inhibitor of the EGFR and has been demonstrated to function as a tumor suppressor. As MIG-6 can be induced via RAS-ERK signaling, we investigated its potential involvement in this negative regulatory loop. Focus formation assays were performed and demonstrated that MIG-6 significantly reduces malignant transformation induced by oncogenic BRAF. Although this genetic interaction was mirrored by a physical interaction between MIG-6 and BRAF, we did not observe a direct regulation of BRAF kinase activity by MIG-6. Interestingly, a selective chemical EGFR inhibitor suppressed transformation to a similar degree as MIG-6, whereas combining these approaches had no synergistic effect. By analyzing a range of BRAF mutated and wildtype cell line models, we could show that BRAF V600E causes a strong upregulation of MIG-6, which was mediated at the transcriptional level via the RAS-ERK pathway and resulted in downregulation of EGFR activation. This feedback loop is operational in tumors, as shown by the analysis of almost 400 patients with papillary thyroid cancer (PTC). Presence of BRAF V600E correlated with increased MIG-6 expression on the one hand, and with inactivation of the EGFR and of PI3K/AKT signaling on the other hand. Importantly, we also observed a more aggressive disease phenotype when BRAF V600E coexisted with low MIG-6 expression. Finally, analysis of methylation data was performed and revealed that higher methylation of MIG-6 correlated to its decreased expression. Taken together, we demonstrate that MIG-6 efficiently reduces cellular transformation driven by oncogenic BRAF by orchestrating a negative feedback circuit directed towards the EGFR.

Multidrug-resistant Salmonella enterica serovar Typhimurium ST313 has emerged in sub-Saharan Africa causing severe infections in humans. Therefore, it has been speculated that this specific sequence type, ST313, carries factors associated with increased pathogenicity. We assessed the role in virulence of a gene with a yet unknown function, st313-td, detected in ST313 through comparative genomics. Additionally, the structure of the genomic island ST313-GI, harbouring the gene was determined. The gene st313-td was cloned into wild type S. Typhimurium 4/74 (4/74-C) as well as knocked out in S. Typhimurium ST313 02-03/002 (Δst313-td) followed by complementation (02-03/002-C). Δst313-td was less virulent in mice following i.p. challenge than the wild type and this phenotype could be partly complemented in trans, indicating that st313-td plays a role during systemic infection. The gene st313-td was shown not to affect invasion of cultured epithelial cells, while the absence of the gene significantly affects uptake and intracellular survival within macrophages. The gene st313-td was proven to be strongly associated to invasiveness, harboured by 92.5% of S. Typhimurium blood isolates (n = 82) and 100% of S. Dublin strains (n = 50) analysed. On the contrary, S. Typhimurium isolates of animal and food origin (n = 82) did not carry st313-td. Six human, non-blood isolates of S. Typhimurium from Belarus, China and Nepal harboured the gene and belonged to sequence types ST398 and ST19. Our data showed a global presence of the st313-td gene and in other sequence types than ST313. The gene st313-td was shown to be expressed during logarithmic phase of growth in 14 selected Salmonella strains carrying the gene. This study reveals that st313-td plays a role in S. Typhimurium ST313 pathogenesis and adds another chapter to understanding of the virulence of S. Typhimurium and in particular of the emerging sequence type ST313.

Escherichia coli carrying IncK-blaCMY-2 plasmids mediating resistance to extended-spectrum cephalosporins (ESC) has been frequently described in food-producing animals and in humans. This study aimed to characterize IncK-blaCMY-2-positive ESC-resistant E. coli isolates from poultry production systems in Denmark, Finland, and Germany, as well as from Danish human blood infections, and further compare their plasmids. Whole-genome sequencing (Illumina) of all isolates (n = 46) confirmed the presence of the blaCMY-2 gene. Minimum inhibitory concentration (MIC) testing revealed a resistant phenotype to cefotaxime as well as resistance to ≥3 antibiotic classes. Conjugative transfer of the blaCMY-2 gene confirmed the resistance being on mobile plasmids. Pangenome analysis showed only one-third of the genes being in the core with the remainder being in the large accessory gene pool. Single nucleotide polymorphism (SNP) analysis on sequence type (ST) 429 and 1286 isolates showed between 0–60 and 13–90 SNP differences, respectively, indicating vertical transmission of closely related clones in the poultry production, including among Danish, Finnish, and German ST429 isolates. A comparison of 22 ST429 isolates from this study with 80 ST429 isolates in Enterobase revealed the widespread geographical occurrence of related isolates associated with poultry production. Long-read sequencing of a representative subset of isolates (n = 28) allowed further characterization and comparison of the IncK-blaCMY-2 plasmids with publicly available plasmid sequences. This analysis revealed the presence of highly similar plasmids in ESC-resistant E. coli from Denmark, Finland, and Germany pointing to the existence of common sources. Moreover, the analysis presented evidence of global plasmid transmission and evolution. Lastly, our results indicate that IncK-blaCMY-2 plasmids and their carriers had been circulating in the Danish production chain with an associated risk of spreading to humans, as exemplified by the similarity of the clinical ST429 isolate to poultry isolates. Its persistence may be driven by co-selection since most IncK-blaCMY-2 plasmids harbor resistance factors to drugs used in veterinary medicine. [ABSTRACT FROM AUTHOR]