High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca sativa).

CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4), a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4, were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T1) and 368 T2 plants by deep amplicon sequencing revealed that 57% of T1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T1 and more than 100 T2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature

CRISPR/Cas9-mediated mutagenesis of CAROTENOID CLEAVAGE DIOXYGENASE 8 in tomato provides resistance against the parasitic weed Phelipanche aegyptiaca.

Broomrapes (Phelipanche aegyptiaca and Orobanche spp.) are obligate plant parasites that cause extreme damage to crop plants. The parasite seeds have strict requirements for germination, involving preconditioning and exposure to specific chemicals strigolactones [SLs] exuded by the host roots. SLs are plant hormones derived from plant carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase 8 (CCD8). Having no effective means to control parasitic weeds in most crops, and with CRISPR/Cas9 being an effective gene-editing tool, here we demonstrate that CRISPR/Cas9-mediated mutagenesis of the CCD8 gene can be used to develop host resistance to the parasitic weed P. aegyptiaca. Cas9/single guide (sg) RNA constructs were targeted to the second exon of CCD8 in tomato (Solanum lycopersicum L.) plants. Several CCD8Cas9 mutated tomato lines with variable insertions or deletions in CCD8 were obtained with no identified off-targets. Genotype analysis of T1 plants showed that the introduced CCD8 mutations are inherited. Compared to control tomato plants, the CCD8Cas9 mutant had morphological changes that included dwarfing, excessive shoot branching and adventitious root formation. In addition, SL-deficient CCD8Cas9 mutants showed a significant reduction in parasite infestation compared to non-mutated tomato plants. In the CCD8Cas9 mutated lines, orobanchol (SL) content was significantly reduced but total carotenoids level and expression of genes related to carotenoid biosynthesis were increased, as compared to control plants. Taking into account, the impact of plant parasitic weeds on agriculture and difficulty to constitute efficient control methods, the current study offers insights into the development of a new, efficient method that could be combined with various collections of resistant tomato rootstocks

Modelling Salmonella transmission among pigs from farm to slaughterhouse: Interplay between management variability and epidemiological uncertainty

International audienceSalmonella carriage and cutaneous contamination of pigs at slaughter are a major risk for carcass contamination. They depend on Salmonella prevalence at farm, but also on transmission and skin soiling among pigs during their journey from farm to slaughterhouse. To better understand and potentially control what influences Salmonella transmission within a pig batch during this transport and lairage step, we proposed a compartmental, discrete-time and stochastic model. We calibrated the model using pork chain data from Brittany. We carried out a sensitivity analysis to evaluate the impact of the variability in management protocols and of the uncertainty in epidemiological parameters on three model outcomes: prevalence of infection, average cutaneous contamination and number of new infections at slaughter. Each outcome is mainly influenced by a single management factor: prevalence at slaughter mainly depends on the prevalence at farm, cutaneous contamination on the contamination of lairage pens and new infections on the total duration of transport and lairage. However, these results are strongly affected by the uncertainty in epidemiological parameters. Re-excretion of carriers due to stress does not have a major impact on the number of new infections

A dual sgRNA approach for functional genomics in Arabidopsis thaliana

Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase theta (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research

The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis

Most living organisms developed systems to efficiently time environmental changes. The plant-clock acts in coordination with external signals to generate output responses determining seasonal growth and flowering time. Here, we show that two Arabidopsis thaliana transcription factors, FAR1 RELATED SEQUENCE 7 (FRS7) and FRS12, act as negative regulators of these processes. These proteins accumulate particularly in short-day conditions and interact to form a complex. Loss-of-function of FRS7 and FRS12 results in early flowering plants with overly elongated hypocotyls mainly in short days. We demonstrate by molecular analysis that FRS7 and FRS12 affect these developmental processes in part by binding to the promoters and repressing the expression of GIGANTEA and PHYTOCHROME INTERACTING FACTOR 4 as well as several of their downstream signalling targets. Our data reveal a molecular machinery that controls the photoperiodic regulation of flowering and growth and offer insight into how plants adapt to seasonal changes

Immunopathogenesis in pulmonary tuberculosis : impact of immunomodulation and diabetes co-morbidity

Even in the 21st century, tuberculosis (TB) remains a major global health threat, primarily due to the emergence of antibiotic resistance. Presence of co-morbidities such as diabetes mellitus (DM) has worsened the current situation and made it more difficult to treat this deadly disease, especially in resource-poor settings. It is well-known that Mtb (Mycobacterium tuberculosis) bacilli can manipulate both innate and adaptive arms of the human immune system, but how Mtb evade host antimicrobial mechanism is not fully understood. Therefore, a deeper understanding of the immunomodulation caused by Mtb, with and without co-existing illnesses, is essential to develop more effective treatment strategies. The work in this thesis was intended to uncover Mtb-mediated immune alterations, particularly in TB-DM disease, and to examine the feasibility of novel host-directed therapy (HDT). In Paper I, we set out to study the efficacy of HDT using phenylbutyrate (PBA) and vitamin D (vitD) to strengthen host immune defenses upon administration to pulmonary TB patients. In a randomized controlled trial conducted in Bangladesh, we previously reported positive effects on clinical as well as microbiological TB outcomes upon daily PBA and vitD treatment together with standard chemotherapy for 8 weeks. Stored samples obtained from the clinical trial subjects were now used to assess secondary outcomes including cytokine/chemokine secretion by peripheral blood mononuclear cell (PBMC) cultures (Luminex assay), endoplasmic reticulum (ER) stress markers expressed in monocyte-derived-macrophages (MDMs) (quantitative real-time PCR), and activation of LC3-dependent autophagy in Mtb-infected MDMs (confocal microscopy). We observed a marked reduction in the concentration of inflammatory mediators including tumor necrosis factor (TNF)-α, CC motif chemokine ligand (CCL)-11 and CCL5 after 8 weeks of PBA treatment compared to the placebo group. Similarly, vitD treatment effectively reduced CCL11, C-X-C motif chemokine ligand (CXCL)-10 and PDGF concentrations after 8 weeks of treatment. Both PBA- and vitD-treatment contributed to reduced mRNA levels of the ER stress marker, x-box binding protein1spliced (XBP1sp)-l. Autophagy was enhanced in MDMs obtained from all intervention groups after 8 weeks of treatment as compared to placebo. These findings suggested that the improvement of primary outcomes observed in the clinical trial, were associated with reduced inflammation and ER stress and instead enhanced autophagy in Mtb-infected patient cells. In paper II, we aimed to explore DM-associated immune alterations of clinical, radiological, and immunological outcomes in TB disease using TB and TB-DM study cohorts collected in Bangladesh. Clinical samples from peripheral blood and sputum from patients and controls were analyzed (blood chemistry, Luminex, quantitative real-time PCR) along with clinical data (composite clinical TB score and demographics) and chest radiography (chest X-ray score) before and after 1, 2 and 6 months of standard anti-TB treatment. TB-DM patients were significantly older, had higher body mass index (BMI), were less anemic and from a better socio-economic background compared to TB patients. Intriguingly, clinical TB symptoms and time to bacterial clearance in sputum were similar comparing TB and TB-DM patients. Even so, TB-DM patients had poorly managed glycemic control throughout the study period and glycemic status was positively associated with BMI. Importantly, the TB-DM cohort showed reduced resolution of inflammation in the middle and lower lung zones compared with TB patients, which was correlated to plasma leptin concentrations at all time points. These changes were associated with upregulated mRNA expression of inflammatory TNF-α and IL-1β in PBMCs as well as higher CD8 mRNA levels but downregulated CD4 and IL-10 transcripts in sputum cells after standard treatment in TB-DM compared to TB patients. Additionally, glycemic status in TB-DM patients was inversely correlated to sputum IL-10 transcript levels observed after start of anti-TB treatment. These results indicate that TB-DM disease is characterized by low-grade inflammation that persist even after completion of successful anti-TB chemotherapy. In Paper III, we developed a protocol for assessment of M1/M2 polarization of human myeloid-derived cells using 10-color flow cytometry of adherent macrophages infected with green fluorescent protein (GFP)-expressing Mtb. The experimental protocol involved in vitro polarization of MDMs into classically activated (M1) or alternatively activated (M2) macrophages and assessment of phenotype and function before, and 4 to 24 hours after Mtb infection. M1 or M2 cells were successfully differentiated with granulocyte monocyte colony stimulating factor (GM-CSF) or monocyte colony stimulating factor (M-CSF), followed by polarization with interferon (IFN)-γ and lipopolysaccharide (LPS), or interleukin (IL)-4, respectively. This protocol allowed us to polarize and define M1 cells by elevated levels of CD64 and CD86 co-expression, while M2 cells were characterized by a high CD163 and CD200R co-expression. The level of Mtb infection was generally higher in M2 as compared to M1 cells, although the relative increase in infected cells from 4 to 24 hours was higher in M1- compared with M2-polarized cells. Manual gating as well as unsupervised analysis using dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph clustering, showed that Mtb infection altered the expression of M1 and M2 markers after 24 hours and generated clearly separated cell clusters of different sizes. This M1/M2 flow cytometry protocol could be used as a backbone in Mtb-macrophage research and be adopted for special needs including assessment of cells cultured in vitro or obtained ex vivo from clinical patient samples

Double Materiality In CSRD : transcending Challenges, Uncovering Opportunities

The political leadership worldwide is changing significantly in 2024, amid geopolitical conflicts. The World Economic Forum's 2024 Global Risks Report highlights that five out of the top ten global risks are environmental. The EU, committed to sustainability, ramped up its efforts with the EU Green Deal and sustainable finance strategies. A key tool in these efforts is the regulation of sustainability reporting, notably through the CSRD (Corporate Sustainability Reporting Directive) and the ESRS (European Sustainability Reporting Standards). This regulatory change aims to direct investment towards sustainable enterprises, impacting EU companies, foreign companies operating in the EU, and other economic actors collaborating with EU businesses. The thesis consists of an article published in HAMK Unlimited (Journal) in English language. The article explores the implementation of the double materiality assessment (DMA) required by the CSRD in Finnish companies. The article explores how sustainability reporting has changed from voluntary to mandatory and aims at understanding how Finnish companies conduct the DMA required by the CSRD. The study underlining the article was conducted by applying qualitative methods consisting of semi-structured interviews with sustainability professionals and experts. A total of six participants were interviewed during the year 2024. According to their background, work position, and expertise, three are classified as experts and the other as sustainability professionals. Key findings relate to the matters in which the companies focus when conducting a DMA. The results show that the participants focus on effectiveness when conducting a DMA. Other focus area is that of change and compliance. The results show that a significant challenge when conducting the DMA is linked to resourcing. Another challenge is related to finding the expertise needed to conduct the assessment. Analysing the salient concerns for those conducting a DMA provides insights into the priorities of the organizations and their sustainability management strategy

Comparative Study Of The Incidence Of Family Entrepreneurship And Social Business Development In Two Areas Of La Libertad, 2022

The objective of this study was to determine the incidence between family entrepreneurship and business social development in two areas of Libertad, 2022, for which I work with the applied method, quantitative approach, of a non-experimental design of a causal comparative level, where false As a population, 300 entrepreneurs from the footwear sector and 250 from Trujillo manufacturing, therefore, as a sample, there were 118 and 110 respectively for each business category, as a technique adapted the questionnaire for both cases. Results, with greater relevance in the family entrepreneurship variable, with 72% good in the footwear category and 62% regular manufacturing; Similarly, in the social aspect of development, 73% are good and 58% regular; respectively for each category. Therefore, it was concluded that the significant incidence with family entrepreneurship and business social development in the footwear and manufacturing areas of La Libertad will be determined, with (rho=0.810**) and (rho=0.759**) and p < 0.05. In this manner, the Ho is refuted and the Ha is admitted. For this reason, the relationship between the variables is evidenced, in addition, the linear regression, in both items it was obtained 75.20% and 53.70%, respectively, the rest is related to other factors