Spatial modeling of biological patterns shows multiscale organization of Arabidopsis thaliana heterochromatin.

The spatial organization in the cell nucleus is tightly linked to genome functions such as gene regulation. Similarly, specific spatial arrangements of biological components such as macromolecular complexes, organelles and cells are involved in many biological functions. Spatial interactions among elementary components of biological systems define their relative positioning and are key determinants of spatial patterns. However, biological variability and the lack of appropriate spatial statistical methods and models limit our current ability to analyze these interactions. Here, we developed a framework to dissect spatial interactions and organization principles by combining unbiased statistical tests, multiple spatial descriptors and new spatial models. We used plant constitutive heterochromatin as a model system to demonstrate the potential of our framework. Our results challenge the common view of a peripheral organization of chromocenters, showing that chromocenters are arranged along both radial and lateral directions in the nuclear space and obey a multiscale organization with scale-dependent antagonistic effects. The proposed generic framework will be useful to identify determinants of spatial organizations and to question their interplay with biological functions.

Evaluation of ELISA kits for the screening of four nitrofuran metabolites in aquaculture products according to the European guideline for the validation of screening methods.

The administration of nitrofurans to livestock to treat or prevent animal diseases has been banned in the EU for the production of food of animal origin. The corresponding marker residues are tissue-related metabolites AMOZ, AHD, SEM, and AOZ. The MRPL (minimum required performance limit)/RPA (Reference point for action) was set at 1 µg kg-1 in the EU. Thus, all the laboratories involved in the control of nitrofuran metabolites must detect at least at this analytical limit of performance. The objectives of the work reported here were to evaluate the performance of ELISA kits from two different manufacturers (R-Biopharm, Germany; Europroxima, the Netherlands) for the individual screening of the four nitrofuran metabolites (AOZ, AMOZ; AHD; and SEM) in aquaculture products (fish, shrimps), and then to validate the kits according to the European Decision EC/2002/657 and to the European guideline for the validation of screening methods. The false positive rates were below 9 % for the kits from both manufacturers. The detection capabilities CCß determined were all below the current RPA (1 µg/kg). However, regarding the updated RPA at 0.5 µg/kg that shall apply in 2022, the AMOZ and SEM kits from R-Biopharm and the SEM kit from Europroxima will not be able to reach it.

Evaluation of three ELISA kits for the screening of colistin residue in porcine and poultry muscle according to the European guideline for the validation of screening methods.

Colistin is a polypeptide antibiotic mainly used in porcine and poultry to treat gastrointestinal infections. It has been included by the World Health Organisation (WHO) in the list of critically important human antibiotics of high priority for antimicrobial resistance since 2017. Therefore, it is necessary to develop specific and sensitive screening methods for this molecule. Screening for colistin with immunoassays is an interesting alternative to LC-MS/MS screening methods. The performance of three commercially available ELISA kits was evaluated in poultry and porcine muscles for the detection of colistin in regards to its European maximum residue limit (MRL) (150 µg/kg). The applicability of the three ELISA kits to the detection of colistin at or below the MRL in porcine and poultry muscles was demonstrated. The detection capabilities (CCß) of two kits were or lower than or equal to the MRL (150 µg/kg). The lowest detection capability (30 µg/kg) was achieved with the third ELISA kit. The specificity of the three kits was very satisfactory (false positive rates 0%). The three kits are very specific for the detection of colistin (colistin A and B) and polymyxin B.

Tidying-up the plant nuclear space: domains, functions, and dynamics.

Understanding how the packaging of chromatin in the nucleus is regulated and organized to guide complex cellular and developmental programmes, as well as responses to environmental cues is a major question in biology. Technological advances have allowed remarkable progress within this field over the last years. However, we still know very little about how the 3D genome organization within the cell nucleus contributes to the regulation of gene expression. The nuclear space is compartmentalized in several domains such as the nucleolus, chromocentres, telomeres, protein bodies, and the nuclear periphery without the presence of a membrane around these domains. The role of these domains and their possible impact on nuclear activities is currently under intense investigation. In this review, we discuss new data from research in plants that clarify functional links between the organization of different nuclear domains and plant genome function with an emphasis on the potential of this organization for gene regulation.

Who Rules the Cell? An Epi-Tale of Histone, DNA, RNA, and the Metabolic Deep State.

Epigenetics refers to the mode of inheritance independent of mutational changes in the DNA. Early evidence has revealed methylation, acetylation, and phosphorylation of histones, as well as methylation of DNA as part of the underlying mechanisms. The recent awareness that many human diseases have in fact an epigenetic basis, due to unbalanced diets, has led to a resurgence of interest in how epigenetics might be connected with, or even controlled by, metabolism. The Next-Generation genomic technologies have now unleashed torrents of results exposing a wondrous array of metabolites that are covalently attached to selective sites on histones, DNA and RNA. Metabolites are often cofactors or targets of chromatin-modifying enzymes. Many metabolites themselves can be acetylated or methylated. This indicates that the acetylome and methylome can actually be deep and pervasive networks to ensure the nuclear activities are coordinated with the metabolic status of the cell. The discovery of novel histone marks also raises the question on the types of pathways by which their corresponding metabolites are replenished, how they are corralled to the specific histone residues and how they are recognized. Further, atypical cytosines and uracil have also been found in eukaryotic genomes. Although these new and extensive connections between metabolism and epigenetics have been established mostly in animal models, parallels must exist in plants, inasmuch as many of the basic components of chromatin and its modifying enzymes are conserved. Plants are chemical factories constantly responding to stress. Plants, therefore, should lend themselves readily for identifying new endogenous metabolites that are also modulators of nuclear activities in adapting to stress.

The Growing Interest in Development of Innovative Optical Aptasensors for the Detection of Antimicrobial Residues in Food Products.

The presence of antimicrobial residues in food-producing animals can lead to harmful effects on the consumer (e.g., allergies, antimicrobial resistance, toxicological effects) and cause issues in food transformation (i.e., cheese, yogurts production). Therefore, to control antimicrobial residues in food products of animal origin, screening methods are of utmost importance. Microbiological and immunological methods (e.g., ELISA, dipsticks) are conventional screening methods. Biosensors are an innovative solution for the development of more performant screening methods. Among the different kinds of biosensing elements (e.g., antibodies, aptamers, molecularly imprinted polymers (MIP), enzymes), aptamers for targeting antimicrobial residues are in continuous development since 2000. Therefore, this review has highlighted recent advances in the development of aptasensors, which present multiple advantages over immunosensors. Most of the aptasensors described in the literature for the detection of antimicrobial residues in animal-derived food products are either optical or electrochemical sensors. In this review, I have focused on optical aptasensors and showed how nanotechnologies (nanomaterials, micro/nanofluidics, and signal amplification techniques) largely contribute to the improvement of their performance (sensitivity, specificity, miniaturization, portability). Finally, I have explored different techniques to develop multiplex screening methods. Multiplex screening methods are necessary for the wide spectrum detection of antimicrobials authorized for animal treatment (i.e., having maximum residue limits).

Extensive nuclear reprogramming and endoreduplication in mature leaf during floral induction.

BACKGROUND: The floral transition is a complex developmental event, fine-tuned by various environmental and endogenous cues to ensure the success of offspring production. Leaves are key organs in sensing floral inductive signals, such as a change in light regime, and in the production of the mobile florigen. CONSTANS and FLOWERING LOCUS T are major players in leaves in response to photoperiod. Morphological and molecular events during the floral transition have been intensively studied in the shoot apical meristem. To better understand the concomitant processes in leaves, which are less described, we investigated the nuclear changes in fully developed leaves during the time course of the floral transition. RESULTS: We highlighted new putative regulatory candidates of flowering in leaves. We observed differential expression profiles of genes related to cellular, hormonal and metabolic actions, but also of genes encoding long non-coding RNAs and new natural antisense transcripts. In addition, we detected a significant increase in ploidy level during the floral transition, indicating endoreduplication. CONCLUSIONS: Our data indicate that differentiated mature leaves, possess physiological plasticity and undergo extensive nuclear reprogramming during the floral transition. The dynamic events point at functionally related networks of transcription factors and novel regulatory motifs, but also complex hormonal and metabolic changes.

Multiplex immunoassay based on biochip technology for the screening of antibiotic residues in milk: validation according to the European guideline.

The Infiniplex for milk® (IPM) kit is a quick method for the simultaneous and qualitative detection of more than 100 molecules including antibiotic residues, mycotoxins, anti-inflammatories and antiparasitic drugs into a single test that does not require milk treatment. The IPM® kit was validated according to the European decision EC/2002/657 and according to the European guideline for the validation of screening methods (2010). Our validation was focused only on antibiotic residues. The washing step was identified as the most critical step of the assay. Insufficient washes could cause a significant background noise that prevents imaging. Positive controls have to be freshly prepared each day (insufficient stability). The method was specific with a low false-positive rate of 1.7% on 5 discrete test regions (DTR) ((beta-lactams, lincomycin, virginiamycin, quinolones and sulphonamides)) and a false-positive rate of 0% on the 26 other DTR. During our validation, the 42 determined detection capabilities CCß for 12 antibiotic families (aminoglycosides, cephalosporins, lincosamides, macrolides, miscellaneous antibiotics, penicillins, phenolated polymixins, polypeptide antibiotics, quinolones, sulphonamides, tetracyclines) were at between once and twice the decision levels stated by the manufacturer. Forty CCß determined were lower than the respective regulatory limits (i.e. MRL, RC, MRPL) in milk, except for tilmicosin (1.5 times the MRL) and neospiramycin (>1.25 times the MRL). The estimated CCß of thiamphenicol, cloxacillin, danofloxacin, sulphathiazol, ceftiofur and sulphamonomethoxine were lower than or at the MRL. However, it was difficult to approach an accurate CCß with only qualitative results. It is impossible to know whether or not we were close to the cut-off value. The software could be improved by differentiating between low-positive and high-positive results. The results of our participation in three qualitative proficiency tests in 2016 and 2017 for the detection of quinolones, tetracyclines and sulphonamides in cows' milk were very satisfactory.

A Method for Testing Random Spatial Models on Nuclear Object Distributions.

The cell nucleus is a structurally complex and dynamic organelle ensuring key biological functions. Complex relationships between nuclear structure and functions require a better understanding of the three-dimensional organization of the genome and of the subnuclear compartments. Quantitative image analysis coupled with spatial statistics and modeling is a relevant approach to address these questions. In this chapter, we describe a step-by-step procedure to process images and to test a spatial random model for the distribution of nuclear objects using chromocenters as an example. More elaborate models can be designed on the basis of the random model by introducing additional and more complex constraints to better fit observations and to question determinants of these spatial organizations.

State of the art in the validation of screening methods for the control of antibiotic residues: is there a need for further development?

Screening methods are used as a first-line approach to detect the presence of antibiotic residues in food of animal origin. The validation process guarantees that the method is fit-for-purpose, suited to regulatory requirements, and provides evidence of its performance. This article is focused on intra-laboratory validation. The first step in validation is characterisation of performance, and the second step is the validation itself with regard to pre-established criteria. The validation approaches can be absolute (a single method) or relative (comparison of methods), overall (combination of several characteristics in one) or criterion-by-criterion. Various approaches to validation, in the form of regulations, guidelines or standards, are presented and discussed to draw conclusions on their potential application for different residue screening methods, and to determine whether or not they reach the same conclusions. The approach by comparison of methods is not suitable for screening methods for antibiotic residues. The overall approaches, such as probability of detection (POD) and accuracy profile, are increasingly used in other fields of application. They may be of interest for screening methods for antibiotic residues. Finally, the criterion-by-criterion approach (Decision 2002/657/EC and of European guideline for the validation of screening methods), usually applied to the screening methods for antibiotic residues, introduced a major characteristic and an improvement in the validation, i.e. the detection capability (CCß). In conclusion, screening methods are constantly evolving, thanks to the development of new biosensors or liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS) methods. There have been clear changes in validation approaches these last 20 years. Continued progress is required and perspectives for future development of guidelines, regulations and standards for validation are presented here.

Strategies for the screening of antibiotic residues in eggs: comparison of the validation of the classical microbiological method with an immunobiosensor method.

Efficient screening methods are needed to control antibiotic residues in eggs. A microbiological kit (Explorer® 2.0 test (Zeu Inmunotech, Spain)) and an immunobiosensor kit (Microarray II (AM® II) on Evidence Investigator™ system (Randox, UK)) have been evaluated and validated for screening of antibiotic residues in eggs, according to the European decision EC/2002/657 and to the European guideline for the validation of screening methods. The e-reader™ system, a new automatic incubator/reading system, was coupled to the Explorer 2.0 test. The AM II kit can detect residues of six different families of antibiotics in different matrices including eggs. For both tests, a different liquid/liquid extraction of eggs had to be developed. Specificities of the Explorer 2.0 and AM II kit were equal to 8% and 0% respectively. The detection capabilities were determined for 19 antibiotics, with representatives from different families, for Explorer 2.0 and 12 antibiotics for the AM II kit. For the nine antibiotics having a maximum residue limit (MRL) in eggs, the detection capabilities CCß of Explorer 2.0 were below the MRL for four antibiotics, equal to the MRL for two antibiotics and between 1 and 1.5 MRLs for the three remaining antibiotics (tetracyclines). For the antibiotics from other families, the detection capabilities were low for beta-lactams and sulfonamides and satisfactory for dihydrostreptomycin (DHS) and fluoroquinolones, which are usually difficult to detect with microbiological tests. The CCß values of the AM II kit were much lower than the respective MRLs for three detected antibiotics (tetracycline, oxytetracycline, tylosin). Concerning the nine other antibiotics, the detection capabilities determined were low. The highest CCß was obtained for streptomycin (100 µg kg-1).

Advances in biosensor development for the screening of antibiotic residues in food products of animal origin - A comprehensive review.

Antibiotic residues may be found in food of animal origin, since veterinary drugs are used for preventive and curative purposes to treat animals. The control of veterinary drug residues in food is necessary to ensure consumer safety. Screening methods are the first step in the control of antibiotic residues in food of animal origin. Conventional screening methods are based on different technologies, microbiological methods, immunological methods or physico-chemical methods (e.g. thin-layer chromatography, HPLC, LC-MS/MS). Screening methods should be simple, quick, inexpensive and specific, with low detection limits and high sample throughput. Biosensors can meet some of these requirements. Therefore, the development of biosensors for the screening of antibiotic residues has been increasing since the 1980s. The present review provides extensive and up-to-date findings on biosensors for the screening of antibiotic residues in food products of animal origin. Biosensors are constituted of a bioreceptor and a transducer. In the detection of antibiotic residues, even though antibodies were the first bioreceptors to be used, new kinds of bioreceptors are being developed more and more (enzymes, aptamers, MIPs); their advantages and drawbacks are discussed in this review. The different categories of transducers (electrochemical, mass-based biosensors, optical and thermal) and their potential applications for the screening of antibiotic residues in food are presented. Moreover, the advantages and drawbacks of the different types of transducers are discussed. Lastly, outlook and the future development of biosensors for the control of antibiotic residues in food are highlighted.

The Arabidopsis hnRNP-Q Protein LIF2 and the PRC1 Subunit LHP1 Function in Concert to Regulate the Transcription of Stress-Responsive Genes.

LHP1-INTERACTING FACTOR2 (LIF2), a heterogeneous nuclear ribonucleoprotein involved in Arabidopsis thaliana cell fate and stress responses, interacts with LIKE HETEROCHROMATIN PROTEIN1 (LHP1), a Polycomb Repressive Complex1 subunit. To investigate LIF2-LHP1 functional interplay, we mapped their genome-wide distributions in wild-type, lif2, and lhp1 backgrounds, under standard and stress conditions. Interestingly, LHP1-targeted regions form local clusters, suggesting an underlying functional organization of the plant genome. Regions targeted by both LIF2 and LHP1 were enriched in stress-responsive genes, the H2A.Z histone variant, and antagonistic histone marks. We identified specific motifs within the targeted regions, including a G-box-like motif, a GAGA motif, and a telo-box. LIF2 and LHP1 can operate both antagonistically and synergistically. In response to methyl jasmonate treatment, LIF2 was rapidly recruited to chromatin, where it mediated transcriptional gene activation. Thus, LIF2 and LHP1 participate in transcriptional switches in stress-response pathways.

Evaluation and validation of a multi-residue method based on biochip technology for the simultaneous screening of six families of antibiotics in muscle and aquaculture products.

The Evidence Investigator™ system (Randox, UK) is a biochip and semi-automated system. The microarray kit II (AM II) is capable of detecting several compounds belonging to different families of antibiotics: quinolones, ceftiofur, thiamphenicol, streptomycin, tylosin and tetracyclines. The performance of this innovative system was evaluated for the detection of antibiotic residues in new matrices, in muscle of different animal species and in aquaculture products. The method was validated according to the European Decision No. EC/2002/657 and the European guideline for the validation of screening methods, which represents a complete initial validation. The false-positive rate was equal to 0% in muscle and in aquaculture products. The detection capabilities CCß for 12 validated antibiotics (enrofloxacin, difloxacin, ceftiofur, desfuroyl ceftiofur cysteine disulfide, thiamphenicol, florfenicol, tylosin, tilmicosin, streptomycin, dihydrostreptomycin, tetracycline, doxycycline) were all lower than the respective maximum residue limits (MRLs) in muscle from different animal origins (bovine, ovine, porcine, poultry). No cross-reactions were observed with other antibiotics, neither with the six detected families nor with other families of antibiotics. The AM II kit could be applied to aquaculture products but with higher detection capabilities from those in muscle. The detection capabilities CCß in aquaculture products were respectively at 0.25, 0.10 and 0.5 of the respective MRL in aquaculture products for enrofloxacin, tylosin and oxytetracycline. The performance of the AM II kit has been compared with other screening methods and with the performance characteristics previously determined in honey.

The many faces of plant chromatin: Meeting summary of the 4th European workshop on plant chromatin 2015, Uppsala, Sweden.

In June 2015, the fourth European Workshop on Plant Chromatin took place in Uppsala, Sweden, bringing together 80 researchers studying various aspects of plant chromatin and epigenetics. The intricate relationships between plant chromatin dynamics and gene expression change, chromatin organization within the plant cell nucleus, and the impact of chromatin structure on plant development were discussed. Among the main highlights of the meeting were an ever-growing list of newly identified players in chromatin structure establishment and the development of novel tools and approaches to foster our understanding of chromatin-mediated gene regulation, taking into account the context of the plant cell nucleus and its architecture. In this report, we summarize some of the main advances and prospects of plant chromatin research presented at this meeting.

One, Two, Three: Polycomb Proteins Hit All Dimensions of Gene Regulation.

Polycomb group (PcG) proteins contribute to the formation and maintenance of a specific repressive chromatin state that prevents the expression of genes in a particular space and time. Polycomb repressive complexes (PRCs) consist of several PcG proteins with specific regulatory or catalytic properties. PRCs are recruited to thousands of target genes, and various recruitment factors, including DNA-binding proteins and non-coding RNAs, are involved in the targeting. PcG proteins contribute to a multitude of biological processes by altering chromatin features at different scales. PcG proteins mediate both biochemical modifications of histone tails and biophysical modifications (e.g., chromatin fiber compaction and three-dimensional (3D) chromatin conformation). Here, we review the role of PcG proteins in nuclear architecture, describing their impact on the structure of the chromatin fiber, on chromatin interactions, and on the spatial organization of the genome in nuclei. Although little is known about the role of plant PcG proteins in nuclear organization, much is known in the animal field, and we highlight similarities and differences in the roles of PcG proteins in 3D gene regulation in plants and animals.

The Arabidopsis GAGA-Binding Factor BASIC PENTACYSTEINE6 Recruits the POLYCOMB-REPRESSIVE COMPLEX1 Component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA Motifs.

Polycomb-repressive complexes (PRCs) play key roles in development by repressing a large number of genes involved in various functions. Much, however, remains to be discovered about PRC-silencing mechanisms as well as their targeting to specific genomic regions. Besides other mechanisms, GAGA-binding factors in animals can guide PRC members in a sequence-specific manner to Polycomb-responsive DNA elements. Here, we show that the Arabidopsis (Arabidopsis thaliana) GAGA-motif binding factor protein basic pentacysteine6 (BPC6) interacts with like heterochromatin protein1 (LHP1), a PRC1 component, and associates with vernalization2 (VRN2), a PRC2 component, in vivo. By using a modified DNA-protein interaction enzyme-linked immunosorbant assay, we could show that BPC6 was required and sufficient to recruit LHP1 to GAGA motif-containing DNA probes in vitro. We also found that LHP1 interacts with VRN2 and, therefore, can function as a possible scaffold between BPC6 and VRN2. The lhp1-4 bpc4 bpc6 triple mutant displayed a pleiotropic phenotype, extreme dwarfism and early flowering, which disclosed synergistic functions of LHP1 and group II plant BPC members. Transcriptome analyses supported this synergy and suggested a possible function in the concerted repression of homeotic genes, probably through histone H3 lysine-27 trimethylation. Hence, our findings suggest striking similarities between animal and plant GAGA-binding factors in the recruitment of PRC1 and PRC2 components to Polycomb-responsive DNA element-like GAGA motifs, which must have evolved through convergent evolution.

Evaluation and validation of biochip multi-array technology for the screening of six families of antibiotics in honey according to the European guideline for the validation of screening methods for residues of veterinary medicines.

The main chemicals used against varoa are acaricides, and the antibiotics used for the control of bee bacterial diseases are mainly tetracyclines, streptomycins, sulfonamides and chloramphenicol. No maximum residue limits (MRLs) have been set for any antibiotics in honey. Therefore, in the European Union, minimum recommended concentrations (RC) for the analytical performance of methods to control a certain set of these non-authorised chemicals in honey were published by the European Union Reference Laboratory (EU-RL) in 2007. Concerning the strategy for the control for antibiotic residues in honey, there is still a great need for a cheap and single multi-residue method. Biochip array technology is an innovative assay technology for the multi-analyte screening of biological samples in a rapid and easy-to-use format. A multi-array system, called Evidence Investigator™ (Randox, Crumlin, Co., Antrim, UK), was evaluated in our laboratory. It is a semi-automated biochip system designed for research, clinical applications and veterinary use. A competitive chemiluminescent immunoassay is employed for the detection of antimicrobials. The MicroArray II kit (AM II) dedicated to the screening of six different families of antibiotic residues was validated according to the European guideline for the validation of screening methods for residues of veterinary medicines. The specificity was proven to be very satisfactory, and applicability to different kinds of honey was demonstrated. The detection capabilities (CCß) of six antibiotic residues were determined and were below the RCs when exist. The AM II kit could detect at least six quinolones, four tetracyclines and three epimers, three aminoglycosides, three macrolides, thiamphenicol, florfenicol and ceftiofur along with one of its stabilised metabolites, the desfuroylceftiofurcysteine disulfide (DCCD).