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Tissues comprise a multiplicity of specialized cell types that must coordinate state changes in order to function collectively. These state changes are orchestrated by coordinated direct cellular interactions and indirect responses to microenvironmental and systemic cues. Consequently, chronic perturbations to this collective behavior can result in disease states that are difficult to reprogram such as autoimmunity and cancer. As such, studying the self-reinforced dynamics of tissue function can benefit from a systems biology approach where the aim is to understand how individual components of biological systems interact to give rise to emergent properties. The recent growth in the availability of single-cell resolution genomics platforms has further expanded biologists’ ability to do this kind of unbiased inquiry. However, despite the increasing ease of generating these high-dimensional datasets, analyzing these data still presents significant computational challenges because of their noise and sparsity, which are further exacerbated on the level of individual cells and genes. As such, there is a need to develop computational methods that enable scientists to extract systems-level biological insight from noisy high dimensional data. This dissertation introduces DECIPHER, a machine learning framework tailored for network inference in systems biology, with a focus on applications to single-cell RNA sequencing data. Chapter 2 details the DECIPHER algorithm and its implementation for the R computing environment, deciphR, that is designed to reconstruct cell state networks from high-dimensional molecular profiles. Chapter 3 applies DECIPHER to unveil cell-cell interaction networks in the human breast, elucidating how state changes on the cell-level propagate throughout tissue in response to hormonal fluctuations. Chapter 4 extends DECIPHER's application to investigate peripheral immune dysregulation in a rare pediatric autoimmune disease, revealing underlying immune imb

Understanding the genomic architecture of speciation remains a challenge in evolutionary biology. Among broadcast-spawning marine invertebrates, reproductive isolation is thought to be established and maintained by the divergence of gamete recognition proteins located on the surfaces of sperm and egg cells. However, it remains unclear whether gametic isolation has been an effective barrier to gene flow during and/or following speciation. In this dissertation, I characterized the history of introgression among the North Pacific sea urchin species of the family Strongylocentrotidae to deepen our understanding of their diversification and evaluate the importance of gametic isolation in speciation. Using whole-genome sequencing data from each strongylocentrotid species and cutting-edge phylogenomic approaches, I documented widespread introgression in both extant taxa and ancestral lineages, demonstrating that gametic isolation did not effectively limit introgression. I implemented a phylogenetic hidden Markov model to locate the specific regions of the genome affected by introgression, finding evidence of strong selection against introgression across much of the genome. Although introgressed variation has predominantly persisted in slowly evolving, low-divergence genomic regions, numerous protein-coding genes showed both introgression and historical positive selection, suggesting an adaptive role for introgression. Finally, I showed that the two gamete recognition proteins responsible for species-selectivity in sea urchin fertilization, sperm protein bindin and its egg receptor, EBR1, have experienced historical adaptive introgression, a pattern inconsistent with expectations for barrier loci. My findings contribute to the body of literature evaluating the biological consequences of introgression and question the importance of gamete recognition proteins in the evolution of reproductive isolation among incipient strongylocentrotid sea urchin species.

The tomato industry and buffalo farming generate waste, including sludge (BS) and tomato pomace (TP), which can significantly impact their economic and environmental sustainability. The case study tracked changes in microflora composition after a thermal shock during anaerobic co-digestion. The inoculum-to-substrate ratio was 0.5 based on volatile solid content under mesophilic conditions. An Automatic Methane Potential Test System was used to monitor the process before and after thermal stress (50°C) occurred for three days. Next-generation sequencing analyzed the bacterial and archaeal communities. The pH decreased, and methane production plateaued due to the high volatile solid content (87 g/L). After thermal stress, the pH returned to neutral, and the batch resumed biogas production. The cumulative CH4 production reached 3,115 Nml. The biogas had a maximum methane peak of 78.5% compared to 58.4% in BS. The taxonomic classification showed that Firmicutes (51.7%) and Bacteroidetes (29.9%) represented 81.6% of the total OTUs among the bacteria. Fonticella, the most abundant Clostridiaceae (average 4.3%), was absent in BS and increased (up to 17.1%) in TP during methane production. Methanocorpusculum was the most abundant in the archaeal community. However, Metanosarcina showed a stronger correlation with methane production. Brief thermal stress significantly altered bacterial and archaeal populations and allowed to resume biogas production.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has been evolving rapidly causing emergence of new variants and health uncertainties. Monitoring the evolution of the virus was of the utmost importance for public health interventions and the development of national and global mitigation strategies. Here, we report national data on the emergence of new variants, their distribution, and dynamics in a 3-year study conducted from March 2020 to the end of January 2023 in the Republic of Serbia. Nasopharyngeal and oropharyngeal swabs from 2,398 COVID-19-positive patients were collected and sequenced using three different next generation technologies: Oxford Nanopore, Ion Torrent, and DNBSeq. In the subset of 2,107 SARS-CoV-2 sequences which met the quality requirements, detection of mutations, assignment to SARS-CoV-2 lineages, and phylogenetic analysis were performed. During the 3-year period, we detected three variants of concern, namely, Alpha (5.6%), Delta (7.4%), and Omicron (70.3%) and one variant of interest—Omicron recombinant “Kraken” (XBB1.5) (<1%), whereas 16.8% of the samples belonged to other SARS-CoV-2 (sub)lineages. The detected SARS-CoV-2 (sub)lineages resulted in eight COVID-19 pandemic waves in Serbia, which correspond to the pandemic waves reported in Europe and the United States. Wave dynamics in Serbia showed the most resemblance with the profile of pandemic waves in southern Europe, consistent with the southeastern European location of Serbia. The samples were assigned to sixteen SARS-CoV-2 Nextstrain clades: 20A, 20B, 20C, 20D, 20E, 20G, 20I, 21J, 21K, 21L, 22A, 22B, 22C, 22D, 22E, and 22F and six different Omicron recombinants (XZ, XAZ, XAS, XBB, XBF, and XBK). The 10 most common mutations detected in the coding and untranslated regions of the SARS-CoV-2 genomes included four mutations affecting the spike protein (S:D614G, S:T478K, S:P681H, and S:S477N) and one mutation at each of the followin, The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has been evolving rapidly causing emergence of new variants and health uncertainties. Monitoring the evolution of the virus was of the utmost importance for public health interventions and the development of national and global mitigation strategies. Here, we report national data on the emergence of new variants, their distribution, and dynamics in a 3-year study conducted from March 2020 to the end of January 2023 in the Republic of Serbia. Nasopharyngeal and oropharyngeal swabs from 2,398 COVID-19- positive patients were collected and sequenced using three different next generation technologies: Oxford Nanopore, Ion Torrent, and DNBSeq. In the subset of 2,107 SARS-CoV-2 sequences which met the quality requirements, detection of mutations, assignment to SARS-CoV-2 lineages, and phylogenetic analysis were performed. During the 3-year period, we detected three variants of concern, namely, Alpha (5.6%), Delta (7.4%), and Omicron (70.3%) and one variant of interest—Omicron recombinant “Kraken” (XBB1.5) (<1%), whereas 16.8% of the samples belonged to other SARS-CoV-2 (sub)lineages. The detected SARS-CoV-2 (sub)lineages resulted in eight COVID-19 pandemic waves in Serbia, which correspond to the pandemic waves reported in Europe and the United States. Wave dynamics in Serbia showed the most resemblance with the profile of pandemic waves in southern Europe, consistent with the southeastern European location of Serbia. The samples were assigned to sixteen SARS-CoV-2 Nextstrain clades: 20A, 20B, 20C, 20D, 20E, 20G, 20I, 21J, 21K, 21L, 22A, 22B, 22C, 22D, 22E, and 22F and six different Omicron recombinants (XZ, XAZ, XAS, XBB, XBF, and XBK). The 10 most common mutations detected in the coding and untranslated regions of the SARS-CoV-2 genomes included four mutations affecting the spike protein (S:D614G, S:T478K, S:P681H, and S:S477N) and one mutation at each of the followi

Study of the diet of squid is hampered by the fact that these animals masticate their prey prior to ingestion and do not necessarily ingest the hard parts that allow the identification of prey remains present in the stomach. The result is a large percentage of prey in stomach contents that can be identified only according to broad categories (e.g., “teleost fish”). This is widely described in the literature and has also been found in samples from the English Channel of the two loliginid squid fished in this area (Loligo forbesii and Loligo vulgaris). New biological samples of both species were collected at the Port-en-Bessin fish market during the 2019–2020 fishing season. Stomach contents were studied through DNA metabarcoding targeting a small fragment of the COI mitochondrial gene selected by PCR and sequenced using high-throughput next generation sequencing. The DNA metabarcoding results were compared with databases available online. These preliminary results validated a protocol based on commercial samples kept frozen (−20 °C) before analysis. Sequences allowed 34 different types of prey to be identified to the species level, including 17 teleost fish species. Results revealed the high occurrence of cuttlefish eaten by both species of squid. Food composition was analysed in order to make comparisons between species, between seasons (in L. vulgaris) and according to the size of the predator. Differences in diet of L. vulgaris between November and March samples were greater than the difference between L. vulgaris and L. forbesii, which is consistent with opportunistic behaviour in these squids. Differences in diet related to predator size do not suggest a change in trophic level during the growth of recruited stages

Background: Mpox virus (MPXV) has recently spread outside of sub-Saharan Africa. This large multicentre study was conducted in Lombardy, the most densely populated Italian region accounting for more than 40% of Italian cases. The present study aims to: i) evaluate the presence and the shedding duration of MPXV DNA in different body compartments correlating the MPXV viability with the time to onset of symptoms; ii) provide evidence of MPXV persistence in different body compartment as a source of infection and iii) characterize the MPXV evolution by whole genome sequencing (WGS) during the outbreak occurred in Italy. Material and methods: The study included 353 patients with a laboratory-confirmed diagnosis of MPXV infection screened in several clinical specimens in the period May 24th - September 1st, 2022. Viral isolation was attempted from different biological matrices and complete genome sequencing was performed for 61 MPXV strains. Results: MPXV DNA detection was more frequent in the skin (94.4%) with the longest median time of viral clearance (16 days). The actively-replicating virus in cell culture was obtained for 123/377 (32.6%) samples with a significant higher viral quantity on isolation positive samples (20 vs 31, p < 0.001). The phylogenetic analysis highlighted the high genetic identity of the MPXV strains collected, both globally and within the Lombardy region. Conclusion: Skin lesion is gold standard material and the high viral load and the actively-replicating virus observed in genital sites confirms that sexual contact plays a key role in the viral transmission.

For cervical cancer (CC), circulating cell-free HPV DNA (ccfHPV) may establish disease severity. Furthermore, HPV integration has been correlated to viral load and survival. In this study, pre-treatment plasma from 139 CC cases (50 primary surgery patients, 22 primary surgery+adjuvant oncological therapy patients, and 67 primary oncological therapy patients) was collected (2018-2020). Furthermore, plasma from 25 cervical intraepithelial neoplasia grade 3 (CIN3) patients and 15 healthy women (negative controls) were collected. Two next-generation sequencing (NGS) panels were used to establish ccfHPV presence and human papillomavirus type 16 (HPV16) integration status. ccfHPV was detected in four primary surgery (8.0%), eight primary surgery+adjuvant oncology (36.4%), and 54 primary oncology (80.6%) patients. For primary oncology patients with HPV16-related cancer (n=37), more ccfHPVneg than ccfHPVpos patients had HPV16 integration (p=0.04), and, in patients with HPV16 integration (n=13), ccfHPVpos patients had higher disease stages than ccfHPVneg patients (p=0.05). In summary, ccfHPV presence is related to disease severity and may add to the debated Sedlis criteria used for identifying patients for adjuvant oncological therapy. However, ccfHPV detection is influenced by HPV integration status and disease stage, and these factors need to be considered in ccfHPVneg patients.

Background: The increased attention in agri-food quality and safety has led to the progress of DNA-based tools aimed to fight adulteration issues. Among the various molecular approaches, those ones based on molecular markers and DNA barcoding have been adequately validated, whereas new tools such as droplet digital PCR (ddPCR), isothermal amplification and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated (Cas) (CRISPR/Cas) system are starting to overtake the performances of the former ones and to be applied in the agri-food sector. Scope and approach: Herein, an overview of the recent advances and exploitations of the DNA-based techniques for the authenticity and traceability of fresh and processed plant-derived food, feed and medicinal products is provided, including research about the monitoring of contaminant and allergen presence. Moreover, the potentials and flaws of these molecular tools are also discussed. Key finding and conclusions: DNA-based technologies represent valuable tools for preventing agri-food frauds and adulteration of several vegetable products present on the market such as spices, extra virgin olive oil, wine, cocoa, and medicinal plants. The application of these methods can contribute to the protection of consumers and all stakeholders involved in the agri-food production and distribution.

Avian colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is a disease of significant economic importance for the global poultry industry. Controlling APEC infections through vaccination is an effective strategy that can also contribute to reducing antibiotic use, an important goal in an era of ever-growing antibiotic resistance. An aroA mutant of E. coli serotype O78 has previously been commercialised as a live attenuated vaccine against avian colibacillosis. This vaccine provides O-antigen-specific protection against a homologous challenge but is ineffective against a heterologous challenge. This thesis aimed to develop a vaccine that can provide a broader protection against APECs. Four live attenuated vaccine candidates were constructed by deleting the aroA gene or the tonB and oppD genes in genetically distinct APEC strains. The presence of tonB is essential for the activity of outer membrane iron siderophore receptors/transporters that are common in all APEC strains. The deletion of tonB gene is expected to cause a substantial depletion of the pool of intra-bacterial iron, which in turn will suppress the inhibition of iron uptake by the Fur protein. The de-repression of the Fur regulon would increase the expression of siderophore receptors on the surface of the APEC cell. Therefore, it is hypothesized that the immune response against overexpressed iron siderophore receptors could induce serotype-independent, cross-protective immune responses following vaccination. The deletion of the oligopeptide transporter gene oppD was introduced in the tonB mutants as a second virulence attenuation step, to reduce the probability of a reversion to a wildtype phenotype. In parallel to these studies, the aromatic amino acid pathway aroA gene was deleted in APEC strains of the same genetic background, to compare the protective efficacy of aroA against tonB/oppD deletion mutants. The vaccine candidate strains APEC 102026 (O78) and APEC 10-578 (O127) were selecte

Ecological speciation within the mormyrid genus Campylomormyrus resulted in sympatric species exhibiting divergence in their feeding apparatus and electric organ discharge (EOD). This study documents the overall diet of the genus Campylomormyrus and examines the hypothesis that the Campylomormyrus radiation is caused by adaptation to different food sources. We performed diet assessment of five sympatric Campylomormyrus species (C. alces, C. compressirostris, C. curvirostris, C. tshokwe, C. numenius) and their sister taxon Gnathonemus petersii with markedly different snout morphologies and EODs using hybrid capture/HTS DNA metabarcoding of their stomach contents. Our approach allowed for high taxonomic resolution of prey items, including benthic invertebrates, allochthonous invertebrates and vegetation. Comparisons of the diet compositions using quantitative measures and diet overlap indices revealed that all species are able to exploit multiple food niches in their habitats, that is fauna at the bottom, the water surface and the water column. A major part of the diet is larvae of aquatic insects, such as dipterans, coleopterans and trichopterans, known to occur in holes and interstitial spaces of the substrate. The results indicate that different snout morphologies and the associated divergence in the EOD could translate into different prey spectra. This suggests that the diversification in EOD and/or morphology of the feeding apparatus could be under functional adaptation., Ministry of Higher Education of the Arab Republic of Egypt, Universität Potsdam http://dx.doi.org/10.13039/501100004238, Peer Reviewed

Fanconi anemia (FA) patients display an exacerbated risk of oral squamous cell carcinoma (OSCC) and oral potentially malignant lesions (OPMLs) at early ages. As patients have defects in their DNA repair mechanisms, standard-of-care treatments for OSCC such as radiotherapy and chemotherapy, give rise to severe toxicities. New methods for early diagnosis are urgently needed to allow for treatment in early disease stages and achieve better clinical outcomes. We conducted a prospective, longitudinal study wherein liquid biopsies from sixteen patients with no clinical diagnoses of OPML and/or OSCC were analyzed for the presence of mutations in cancer genes. The DNA from saliva and plasma were sequentially collected and deep-sequenced, and the clinical evaluation followed over a median time of approximately 2 years. In 9/16 FA patients, we detected mutations in cancer genes (mainly TP53) with minor allele frequencies (MAF) of down to 0.07%. Importantly, all patients that had mutations and clinical follow-up data after mutation detection (n = 6) developed oral precursor lesions or OSCC. The lead-time between mutation detection and tumor diagnosis ranged from 23 to 630 days. Strikingly, FA patients without mutations displayed a significantly lower risk of developing precursor lesions or OSCCs. Therefore, our diagnostic approach could help to stratify FA patients into risk groups, which would allow for closer surveillance for OSCCs or precursor lesions., Instituto de Salud Carlos III, European Comission, Fundación española de Anemia de Fanconi, Ministerio de Ciencia e Innovación (España), Comunidad de Madrid, Depto. de Medicina, Fac. de Medicina, TRUE, pub, Descuento UCM

Mitochondrial disorders are the most common group of inherited metabolic disorders affecting children. Patients suffer from debilitating chronic conditions that are often life-threatening. Comprising more than 360 monogenic disorders, inherited through all modes of inheritance, this group of conditions can manifest at any age and can affect any tissue or organ system. Their high clinical and genetic heterogeneity means genomic diagnostic yields are typically only ~50%. This study aimed to extend this incomplete understanding of the genetic and mutational spectrum of mitochondrial disease through the study of a cohort of patients with a suspected mitochondrial disease who have remained undiagnosed following extensive molecular investigations, specifically exome sequencing, using new -Omics approaches. Three overlapping retrospective cohorts of paediatric mitochondrial disease patients (Leigh syndrome, Complex I deficiency or Population-based) were combined, forming a combined cohort comprising 263 patients. Among them, 77% had previously received a firm genetic diagnosis through extensive molecular and exome analyses, surpassing the typical diagnostic rate of 50% published in other studies. The remaining 61 undiagnosed individuals formed the cohort for this current study, referred to as the ‘exome-negative’ cohort, and underwent one or more of: expanded exome analysis, genome sequencing, RNA sequencing or quantitative proteomics. The analysis gene list comprised a recently updated list of genes encoding the mitochondrial proteome and the Mendeliome. At the conclusion of this PhD, 34 ‘exome-negative’ patients (56%) now have a firm genetic diagnosis; with an additional 12 requiring further functional validation of strong candidate genes. Pathogenic variants were identified in 21 different genes including 15 phenocopy genes and six mitochondrial disease genes, one of which is a novel disease gene (MRPL39). The new diagnoses consisted of structural variants that

Osteosarcoma is an aggressive malignancy characterized by high genomic complexity. Identification of few recurrent mutations in protein coding genes suggests that somatic copy-number aberrations (SCNA) are the genetic drivers of disease. Models around genomic instability conflict—it is unclear whether osteosarcomas result from pervasive ongoing clonal evolution with continuous optimization of the fitness landscape or an early catastrophic event followed by stable maintenance of an abnormal genome. We address this question by investigating SCNAs in >12,000 tumor cells obtained from human osteosarcomas using single-cell DNA sequencing, with a degree of precision and accuracy not possible when inferring single-cell states using bulk sequencing. Using the CHISEL algorithm, we inferred allele- and haplotype-specific SCNAs from this whole-genome single-cell DNA sequencing data. Surprisingly, despite extensive structural complexity, these tumors exhibit a high degree of cell-cell homogeneity with little subclonal diversification. Longitudinal analysis of patient samples obtained at distant therapeutic timepoints (diagnosis, relapse) demonstrated remarkable conservation of SCNA profiles over tumor evolution. Phylogenetic analysis suggests that the majority of SCNAs were acquired early in the oncogenic process, with relatively few structure-altering events arising in response to therapy or during adaptation to growth in metastatic tissues. These data further support the emerging hypothesis that early catastrophic events, rather than sustained genomic instability, give rise to structural complexity, which is then preserved over long periods of tumor developmental time. Significance: Chromosomally complex tumors are often described as genomically unstable. However, determining whether complexity arises from remote time-limited events that give rise to structural alterations or a progressive accumulation of structural events in persistently unstable tumors has implications f

Producción Científica, The study of microbial communities associated with different plants of agronomic interest has allowed, in recent years, to answer a number of questions related to the role and influence of certain microbes in key aspects of their autoecology, such as improving the adaptability of the plant host to different abiotic or biotic stresses. In this study, we present the results of the characterization, through both high-throughput sequencing and classical microbiological methods, of the fungal microbial communities associated with grapevine plants in two vineyards of different ages and plant genotypes located in the same biogeographical unit. The study is configured as an approximation to the empirical demonstration of the concept of “microbial priming” by analyzing the alpha- and beta-diversity present in plants from two plots subjected to the same bioclimatic regime to detect differences in the structure and taxonomic composition of the populations. The results were compared with the inventories of fungal diversity obtained by culture-dependent methods to establish, where appropriate, correlations between both microbial communities. Metagenomic data showed a differential enrichment of the microbial communities in the two vineyards studied, including the populations of plant pathogens. This is tentatively explained due to factors such as the different time of exposure to microbial infection, different plant genotype, and different starting phytosanitary situation. Thus, results suggest that each plant genotype recruits differential fungal communities and presents different profiles of associated potential microbial antagonists or communities of pathogenic species., Esta investigación fue financiada por la Cátedra Agrobank bajo el programa “IV Convocatoria de Ayudas de la Cátedra AgroBank para la transferencia del conocimiento al sector agroalimentario”

Fipronil is a broad-spectrum insecticide with remarkable efficacy that is widely used to control insect pests around the world. However, its extensive use has led to increasing soil and water contamination. This fact is of concern and makes it necessary to evaluate the risk of undesirable effects on non-target microorganisms, such as the microbial community in water and/or soil. Studies using the metagenomic approach to assess the effects of fipronil on soil microbial communities are scarce. In this context, the present study was conducted to identify microorganisms that can biodegrade fipronil and that could be of great environmental interest. For this purpose, the targeted metabarcoding approach was performed in soil microcosms under two environmental conditions: fipronil exposure and control (without fipronil). After a 35-day soil microcosm period, the 16S ribosomal RNA (rRNA) gene of all samples was sequenced using the ion torrent personal genome machine (PGM) platform. Our study showed the presence of Proteobacteria, Actinobacteria, and Firmicutes in all of the samples; however, the presence of fipronil in the soil samples resulted in a significant increase in the concentration of bacteria from these phyla. The statistical results indicate that some bacterial genera benefited from soil exposure to fipronil, as in the case of bacteria from the genus Thalassobacillus, while others were affected, as in the case of bacteria from the genus Streptomyces. Overall, the results of this study provide a potential contribution of fipronil-degrading bacteria.

Variable phenotypes, including developmental encephalopathy with (DEE) or without seizures and myoclonic epilepsy and ataxia due to potassium channel mutation, are caused by pathogenetic variants in KCNC1, encoding for Kv3.1 channel subunits. In vitro, channels carrying most KCNC1 pathogenic variants display loss-of-function features. Here, we describe a child affected by DEE with fever-triggered seizures, caused by a novel de novo heterozygous missense KCNC1 variant (c.1273G>A; V425M). Patch-clamp recordings in transiently transfected CHO cells revealed that, compared to wild-type, Kv3.1 V425M currents (1) were larger, with membrane potentials between −40 and +40 mV; (2) displayed a hyperpolarizing shift in activation gating; (3) failed to inactivate; and (4) had slower activation and deactivation kinetics, consistent with a mixed functional pattern with prevalent gain-of-function effects. Exposure to the antidepressant drug fluoxetine inhibited currents expressed by both wild-type and mutant Kv3.1 channels. Treatment of the proband with fluoxetine led to a rapid and prolonged clinical amelioration, with the disappearance of seizures and an improvement in balance, gross motor skills, and oculomotor coordination. These results suggest that drug repurposing based on the specific genetic defect may provide an effective personalized treatment for KCNC1-related DEEs.

Background: Identification of genomic alterations (e.g., EGFR, ALK, ROS1, BRAF, NTRK, and MET) is essential for initiating targeted therapy in patients with advanced non-small-cell lung cancer (aNSCLC). This study estimated the budget impact of using the sequential single-gene (SSG) test, which tests for each mutation one at a time, versus next-generation sequencing (NGS), which tests for all mutations at the same time, among newly diagnosed patients with aNSCLC from a Japanese healthcare payer's perspective. Methods: A budget impact model (BIM) was used to determine the expected budget impact associated with NGS for newly diagnosed aNSCLC in Japan over a 3-year period. The BIM compared the total costs (biopsy, testing, and treatment) and average turnaround time of future NGS and current NGS versus SSG testing. Results: The adoption of current NGS over SSG testing had a budget impact of-0.24%, but adoption of future NGS over SSG testing had a budget impact of thorn 4.33% across a 3-year time horizon on the Japanese budget for aNSCLC treatment. The adoption of current or future NGS over SSG testing would shorten the average turnaround time for testing. Conclusions: The adoption of current NGS over SSG testing would slightly decrease the yearly costs. However, the adoption of future or current NGS over SSG testing would shorten the average turnaround time, enabling faster identification of genomic alterations and earlier initiation of treatment for aNSCLC patients in Japan. (c) 2022 The Authors. Published by Elsevier B.V. on behalf of The Japanese Respiratory So-ciety. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Paleogenetics has significantly changed since its inception almost forty years ago. Initially, molecular techniques available to the researchers offered minimal possibilities for ancient DNA analysis. The subsequent expansion of the scientific tool cabinet allowed for more remarkable achievements, combined has with the newfound popularity of this budding field of science. Finally, a breakthrough was made with the development of next-generation sequencing (NGS) technologies and the update of DNA isolation protocols, through which even very fragmented aDNA samples could be used to sequence whole genomes. In this paper, we review the achievements made thus far and compare the methodologies utilized in this field of science, discussing their benefits and challenges.

Water reuse has become a promising alternative to alleviate stress on conventional freshwater resources in the face of population growth, sea level rise, source water depletion, eutrophication of water bodies, and climate change. Potable water reuse intentionally looks to purify wastewater effluent to drinking water quality or better through the development and implementation of advanced treatment trains. While membrane-based treatment has become a widely-adopted treatment step to meet this purpose, there is growing interest in implementing treatment trains that harness microorganisms as a more sustainable and less energy-intensive means of removing contaminants of emerging concern (CECs), through biological degradation or transformation. In this dissertation, various aspects of the operation of a microbially-active carbon-based advanced treatment train producing water intended for potable reuse are examined, including fate of dissolved organic carbon, underlying microbial populations, and functional genes are explored. Further, dynamics associated with antibiotic resistance genes (ARGs), identified as a microbially-relevant CECs, are also assessed. Overall, this dissertation advances understanding associated with the interplay between and within treatment processes as they relate to removal of various organic carbon fractions, microbially community dynamics, functional genes, and ARGs. Further, when relevant, these insights are contextualized to operational conditions, process upsets, water quality parameters, and other intended water uses within the water industry with the goal of broadening the application of advanced molecular tools beyond the scope of academic research. Specifically, this dissertation illuminates relationships among organic carbon fractions and molecular markers within an advanced treatment train employing flocculation, coagulation, and sedimentation (FlocSed), ozonation, biologically active carbon (BAC) filtration, granular active carbon (GAC)

In this study, predominant bacterial and archaeal populations and their roles during anaerobic mono-digestion of food waste (FW) and co-digestion of FW with straw pellets (SP) at thermophilic temperature (53 ± 1 °C) were assessed by Next Generation Sequencing (NGS) analysis at organic loading rates (OLRs) of 3.0 and 7.0 gVS/L/d. Depending on the seed; results revealed that Firmicutes, Bacteroidetes, and Proteobacteria were, respectively the most prevalent bacterial phyla at both OLRs investigated. On the other hand, Euryarchaeota was dominated by methanogens playing crucial role in biogas production and correlated mainly with the activities of Methanobacteria and Methanomicrobia at class level. Acetoclastic Methanosaetae was the predominant genus at OLR = 3.0 gVS/L/d; however, shared the same predominance with hydrogenotrophic methanogens Methanospirillium at the highest OLR. Although no clear effect in response to straw addition at OLR of 3.0 gVS/L/d could be seen in terms of methanogenic archaea at genus level, hydrogenotrophic methanogens revealed some shift from Methanobacterium to Methanospirillium at higher OLR. Nevertheless, no prominent microbial shift in the presence of wheat straw at increased OLR was likely due to adapted inoculation at start-up which was also demonstrated by relatively stable biogas yields during co-digestion.

Chicken amyloid arthropathy is a debilitating disease with a major impact on animal welfare. Since the disease is triggered by bacterial infection, preventative treatment also contributes to the widespread overuse of antibiotics. Bacterial infection initiates an acute phase response including increased serum amyloid A (SAA) production by the liver. SAA accumulates at sites of infection and in particular in large joints of affected birds. Interestingly, white egg-laying chickens (WL) are resistant to the disease whilst brown egg-laying chickens (BL) are most affected. Disease susceptibility has an immunological basis but the possible contribution of underlying genetic risk factors is not understood. Using a whole genome sequencing approach, we discovered a novel variant in the SAA gene in WL, which is predicted to result in an arginine to serine substitution at position 90 (SAA.R90S). Surprisingly, when overexpressed in chicken hepatocellular carcinoma cells, SAA.R90S was expressed at a higher rate and secreted to a greater degree than the wild-type SAA protein. Moreover, RNASeq analysis showed that the R90S mutant exerted a differential effect on the expression of core transcription factors linked to cell fate determination and cell differentiation. Comparative analysis of gene expression in murine CD4 T-cells stimulated with IL-6/SAA, suggests that SAA.R90S might block an induced cell fate change toward pro-inflammatory T helper 17 cells, which are required for immunological protection against pathogenic bacteria during an acute phase response. Our results provide first mechanistic insights into the genetic resistance of WL to amyloid arthropathy and could be applied to commercial layer breeding programs to improve animal welfare and reduce the negative effects of the overuse of antibiotics.

In this study, predominant bacterial and archaeal populations and their roles during anaerobic mono-digestion of food waste (FW) and co-digestion of FW with straw pellets (SP) at thermophilic temperature (53 ± 1 °C) were assessed by Next Generation Sequencing (NGS) analysis at organic loading rates (OLRs) of 3.0 and 7.0 gVS/L/d. Depending on the seed; results revealed that Firmicutes, Bacteroidetes, and Proteobacteria were, respectively the most prevalent bacterial phyla at both OLRs investigated. On the other hand, Euryarchaeota was dominated by methanogens playing crucial role in biogas production and correlated mainly with the activities of Methanobacteria and Methanomicrobia at class level. Acetoclastic Methanosaetae was the predominant genus at OLR = 3.0 gVS/L/d; however, shared the same predominance with hydrogenotrophic methanogens Methanospirillium at the highest OLR. Although no clear effect in response to straw addition at OLR of 3.0 gVS/L/d could be seen in terms of methanogenic archaea at genus level, hydrogenotrophic methanogens revealed some shift from Methanobacterium to Methanospirillium at higher OLR. Nevertheless, no prominent microbial shift in the presence of wheat straw at increased OLR was likely due to adapted inoculation at start-up which was also demonstrated by relatively stable biogas yields during co-digestion.

Chicken amyloid arthropathy is a debilitating disease with a major impact on animal welfare. Since the disease is triggered by bacterial infection, preventative treatment also contributes to the widespread overuse of antibiotics. Bacterial infection initiates an acute phase response including increased serum amyloid A (SAA) production by the liver. SAA accumulates at sites of infection and in particular in large joints of affected birds. Interestingly, white egg-laying chickens (WL) are resistant to the disease whilst brown egg-laying chickens (BL) are most affected. Disease susceptibility has an immunological basis but the possible contribution of underlying genetic risk factors is not understood. Using a whole genome sequencing approach, we discovered a novel variant in the SAA gene in WL, which is predicted to result in an arginine to serine substitution at position 90 (SAA.R90S). Surprisingly, when overexpressed in chicken hepatocellular carcinoma cells, SAA.R90S was expressed at a higher rate and secreted to a greater degree than the wild-type SAA protein. Moreover, RNASeq analysis showed that the R90S mutant exerted a differential effect on the expression of core transcription factors linked to cell fate determination and cell differentiation. Comparative analysis of gene expression in murine CD4 T-cells stimulated with IL-6/SAA, suggests that SAA.R90S might block an induced cell fate change toward pro-inflammatory T helper 17 cells, which are required for immunological protection against pathogenic bacteria during an acute phase response. Our results provide first mechanistic insights into the genetic resistance of WL to amyloid arthropathy and could be applied to commercial layer breeding programs to improve animal welfare and reduce the negative effects of the overuse of antibiotics.

Chicken amyloid arthropathy is a debilitating disease with a major impact on animal welfare. Since the disease is triggered by bacterial infection, preventative treatment also contributes to the widespread overuse of antibiotics. Bacterial infection initiates an acute phase response including increased serum amyloid A (SAA) production by the liver. SAA accumulates at sites of infection and in particular in large joints of affected birds. Interestingly, white egg-laying chickens (WL) are resistant to the disease whilst brown egg-laying chickens (BL) are most affected. Disease susceptibility has an immunological basis but the possible contribution of underlying genetic risk factors is not understood. Using a whole genome sequencing approach, we discovered a novel variant in the SAA gene in WL, which is predicted to result in an arginine to serine substitution at position 90 (SAA.R90S). Surprisingly, when overexpressed in chicken hepatocellular carcinoma cells, SAA.R90S was expressed at a higher rate and secreted to a greater degree than the wild-type SAA protein. Moreover, RNASeq analysis showed that the R90S mutant exerted a differential effect on the expression of core transcription factors linked to cell fate determination and cell differentiation. Comparative analysis of gene expression in murine CD4 T-cells stimulated with IL-6/SAA, suggests that SAA.R90S might block an induced cell fate change toward pro-inflammatory T helper 17 cells, which are required for immunological protection against pathogenic bacteria during an acute phase response. Our results provide first mechanistic insights into the genetic resistance of WL to amyloid arthropathy and could be applied to commercial layer breeding programs to improve animal welfare and reduce the negative effects of the overuse of antibiotics.

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

Introduction: Non-infectious inflammatory diseases of the central nervous system in dogs, such as steroid responsive meningitis-arteritis (SRMA) and meningoencephalitis of unknown origin (MUO), represent a common clinical challenge that needs extensive and multimodal work-up to reach a presumptive diagnosis. Both diseases are presumably caused by dysregulations of the immune system, but further research is needed in order to understand the molecular mechanisms behind each disease and to optimize treatment. Methods: By next-generation sequencing and subsequent quantitative real-time PCR (qPCR) verification, we designed a prospective case–control pilot study to analyze the small RNA profiles of cerebrospinal fluid from dogs suffering from MUO (N = 5), dogs suffering from SRMA (N = 8), and healthy dogs (N = 5) presented for elective euthanasia used as the Control group. Results: Our results showed an overall enrichment in Y-RNA fragments across all samples, followed by microRNAs (miRNAs) and ribosomal RNAs as the major findings. Additional traces of short RNA reads mapped to long non-coding RNAs and protein-coding genes were also found. From the detected canine miRNAs, miR-21, miR-486, miR-148a, miR-99a, miR-191 and miR-92a were among the most abundant. Dogs with SRMA showed higher differences in miRNA abundance than dogs with MUO when compared to healthy dogs, and miR-142-3p was consistently detected as differentially upregulated in both diseases, although at a low concentration. Moreover, miR-405-5p and miR-503-5p showed different profiles between SRMA and MUO dogs. Subsequent qPCR analyses confirmed miR-142-5p, miR-191-5p and miR-92a-3p as significantly upregulated miRNAs in dogs with SRMA and/or MUO. Discussion: Cerebrospinal fluid is a challenging biological material to use for profiling miRNAs due to the low content of circulating RNAs. Despite this, we could confirm several miRNAs being differentially abundant when comparing healthy dogs and dogs with MUO and SR

Die folgende Doktorarbeit soll neue Erkenntnisse über die Nahrungsaufnahme von Laufkäfern mit Hilfe von DNA-basierten Techniken zur Nahrungsanalyse liefern, damit das Potenzial von Laufkäfern als biologische Bekämpfungsmittel für Nacktschnecken und Unkrautsamen in Zukunft besser beurteilt werden kann. Das Wissen über das Fressverhalten von Carabiden an Schnecken beruht aktuell häufig noch auf Laborexperimenten, sodass detailliertere Kenntnisse über die Schneckenprädation von Carabiden und deren Wirksamkeit als potenzielle Schneckenjäger im Freiland fehlen. Somit fällt es schwer, genaue Aussagen über das Fressverhalten einzelner Carabidenarten an Nacktschnecken und deren Eiern im Agrarland zu treffen. Ebenso wäre ein präziserer Wissensstand darüber wünschenswert, welche Carabidenarten auf den Feldern den Samenfraß bevorzugen und die Samen welcher Pflanzenarten die jeweiligen Carabidenarten verzehren, um dadurch zu klären, welche Carabidenarten für einen zuverlässigen Fraß von Unkrautsamen und damit für einen Rückgang der Ackerunkräuter sorgen könnten. Ein genaueres Bild von der Ernährung der einzelnen Carabidenarten als bisher bekannt wäre daher zur Einteilung der jeweiligen Art in genau definierte trophische Funktionsgruppen von Nöten. Zur Erforschung der aufgenommenen Nahrung und des Fressverhaltens von in Feldern Mitteleuropas (Frankreich und Österreich) präsenten Carabidenarten war es zunächst erforderlich, die Darminhalte von Carabiden mittels DNA-basierter Techniken im Hinblick auf die potenziellen Nahrungsquellen Nacktschnecke und Unkrautsamen analysieren zu können. Zu diesem Zweck wurden neue DNA-basierte Ansätze für die molekulare Analyse des Darminhalts entwickelt, um Nacktschnecken-DNA durch Multiplex-Polymerasekettenreaktion (PCR) nachweisen und eine gezielte Amplikon-Sequenzierung (TAS) von Pflanzenarten mit anschließender detaillierter Datenanalyse durchführen zu können (Kapitel 2 und 3). Hochempfindliche Primerpaare für die Nacktschnecken Arion distinc, The following PhD thesis aims to provide new insights into the food consumption of carabid beetles using DNA-based techniques for dietary analysis, so that the potential of ground beetles to be used as biological control agents for slugs and weed seeds can be better assessed in the future. Knowledge about the feeding behaviour of carabids on slugs is currently often still based on laboratory experiments, so that more detailed information about slug predation by carabids and their effectiveness as potential slug predators in the field is lacking. It is therefore difficult to make precise statements about the feeding behaviour of individual carabid species on slugs and their eggs in agricultural land. Similarly, it would be desirable to have more precise knowledge of which carabid species prefer seed feeding in the fields and of which plant species the respective carabid species consume seeds from, in order to clarify which carabid species could provide reliable feeding on weed seeds and consequently a decline in field weeds. A more precise picture of the diet of the individual carabid species than known so far would thus be necessary to classify each species into well-defined trophic functional groups. In order to investigate the ingested food and feeding behaviour of carabid species present in Central European fields (France and Austria), it was first necessary to be able to analyse the gut contents of the carabids using DNA-based techniques with regard to the potential food sources slugs and weed seeds. For this purpose, new DNA-based approaches for molecular gut content analysis were developed to detect slug DNA by multiplex polymerase chain reaction (PCR) and to perform a targeted amplicon sequencing (TAS) approach of plant species with subsequent detailed data analysis (chapters 2 and 3). Highly sensitive primer pairs for the slug species Arion distinctus (Mabille, 1868) (Stylommatophora: Arionidae) and Deroceras reticulatum (Müller, 1774) (Stylommatophora: Agri, Abweichender Titel laut Übersetzung der Verfasserin/des Verfassers, Dissertation Universität Innsbruck 2023

The bone marrow failure syndromes (BMFS) are a heterogeneous group of diseases with differing underlying pathogenic processes and treatment approaches. Diagnosis remains challenging as these conditions can mimic each other, both in clinical presentation, on morphological examination of the bone marrow and their shared potential to transform to haematological malignancy. The BMFS are associated with poor outcomes and there remain many gains to be made in improving both diagnostic and treatment strategies. Both comprehensive clinical data interrogation and genomic analysis by next generation sequencing (NGS) were employed in an effort to examine the clinical, genomic and molecular determinants of outcome in patients with BMFS. The focus of this thesis was to integrate genomics into diagnostic and management strategies to improve outcomes for patients with BMFS. Initially, the utility of comprehensive genomic sequencing to attain accurate diagnosis in patients presenting with bone marrow failure (BMF) was analysed. This work was a prospective demonstration evaluation study. The observed utility of germline and somatic genetic testing in patients presenting with hypocellular BMF and hereditary predisposition to haematological malignancy (HPHM) led to availability of clinical accredited BMF testing, the uptake and outcomes of which were then analysed in the real world setting. The comprehensive dataset of the Australian Aplastic Anaemia and other Bone Marrow Failure Syndromes Registry (the AAR) was interrogated to examine the treatments applied in and outcomes of patients with immune aplastic anaemia (iAA). The AAR was modernised to facilitate enrolment of patients with both traditional inherited BMF (IBMF) and also novel germline conditions, including those recently discovered and described as conferring a HPHM. Consideration of whether a HPHM exists is now considered to represent a crucial component of new leukaemia diagnostic work up. The AAR provides an opportunity t

Fipronil is a broad-spectrum insecticide with remarkable efficacy that is widely used to control insect pests around the world. However, its extensive use has led to increasing soil and water contamination. This fact is of concern and makes it necessary to evaluate the risk of undesirable effects on non-target microorganisms, such as the microbial community in water and/or soil. Studies using the metagenomic approach to assess the effects of fipronil on soil microbial communities are scarce. In this context, the present study was conducted to identify microorganisms that can biodegrade fipronil and that could be of great environmental interest. For this purpose, the targeted metabarcoding approach was performed in soil microcosms under two environmental conditions: fipronil exposure and control (without fipronil). After a 35-day soil microcosm period, the 16S ribosomal RNA (rRNA) gene of all samples was sequenced using the ion torrent personal genome machine (PGM) platform. Our study showed the presence of Proteobacteria, Actinobacteria, and Firmicutes in all of the samples; however, the presence of fipronil in the soil samples resulted in a significant increase in the concentration of bacteria from these phyla. The statistical results indicate that some bacterial genera benefited from soil exposure to fipronil, as in the case of bacteria from the genus Thalassobacillus, while others were affected, as in the case of bacteria from the genus Streptomyces. Overall, the results of this study provide a potential contribution of fipronil-degrading bacteria.

Background: Thymic epithelial tumors (TETs) are prone to developing in East Asian populations. However, little is known about the genomic profile of TETs in East Asian populations, and the genomic aberrations in TETs have not yet been fully clarified. Thus, molecular targeted therapies for patients with TETs have not been established. This prospective study was conducted to explore the genetic abnormalities of surgically resected TETs in a Japanese cohort and to identify clues for carcinogenesis and potential therapeutic targets in TETs. Methods: Genetic profiles of TETs were investigated using fresh-frozen specimens resected from operable cases with TETs. DNA sequencing was performed using a next-generation sequencing (NGS) gene panel test with Ion Reporter™ and CLC Genomics Workbench 11.0. The mutation sites were further confirmed by Sanger sequencing, digital droplet polymerase chain reaction (ddPCR), and TA cloning for validation. Results: Among 43 patients diagnosed with anterior mediastinal tumors between January 2013 and March 2019, NGS and validation analyses were performed in 31 patients [29 thymomas and two thymic cancers (TCs)] who met the study criteria. Of these, 12 cases of thymoma types A, AB, B1, and B2 harbored the general transcription factor 2-I (GTF2I) mutation (L424H). Conversely, the mutation was not detected in type B3 thymoma or TC, suggesting that the GTF2I mutation existed in indolent types of TETs. Rat sarcoma viral oncogene (RAS) mutations were detected in three cases [Harvey RAS (HRAS) in two cases of type AB thymoma and neuroblastoma RAS (NRAS)] in one case of type B1 thymoma), and additional sex combs like 1 (ASXL1) mutation was present in one case of TC. All RAS mutations were observed in GTF2I-mutated cases. Conclusions: The GTF2I mutation (L424H) is the most frequently occurring mutation in the limited histology of thymoma, consistent with those in the non-Asian population. HRAS and NRAS mutations co-occurred in cases harboring the, Translational Lung Cancer Research, 12(4), pp.707-718; 2023

Microfluidics is a versatile tool with many applications in biology. Its ability to manipulate small volumes of liquid precisely has led to the development of many microfluidic assay platforms. They could handle small amounts of samples and carry out analysis with high sensitivity and throughput. Microfluidic assays have provided new insights into scarce biological samples at higher resolution. In this thesis, we developed microfluidic tools to conduct low input ChIP-seq and ChIRP-seq. We applied them to a variety of samples profiling different targets. The native MOWChIP-seq platform was developed to map RNA polymerase II, transcription factors and histone deacetylase binding in 1,000-50,000 cells. We examined mouse prefrontal cortex and cerebellum using this technology. We found extensive differences that correlated with distinct neurological functions of the brain regions. The same platform and workflow were used to profile five key histone modifications in human lung tumor and normal tissue samples. Integrative analysis with gene expression data revealed extensive chromatin remodeling in lung tumor. Spatial histone modification mapping was conducted in mouse neocortex in a similar fashion. We generated an epigenomic tomography that demonstrated the molecular state of the brain in 3D. Lastly, we developed a microfluidic version of the ChIRP-seq process which successfully conducted the assay using only 500K cells. This improvement makes ChIRP-seq in tissue samples feasible.

A stem-pitting isolate of citrus tristeza virus (CTV), spreading in Hunan province of  China (HU-PSTS), was sequenced and indexed on indicator plants. Biological assays showed that HU-PSTS is a highly aggressive stem pitting isolate belonging to biogroup 5. Viral small RNAs (18-26 nt) of the isolate were deep sequenced by Illumina technology to gain genomic information on the CTV strain infecting the source plant. The reads mapping with 17 CTV reference genomes enabled us to re-assemble the genomes of VT, T68, T30 and T3 strains. Among the VT, the highest number of mapped reads (47-41%) was with SG29, T318A, CT11A, Nuaga and AT-1 genomes, whereas T68, T30 and T3 genomes were less represented (28-20%). Alignments with genomes belonging to T36 and RB strains revealed percentage of mapped reads ranging from 10 to 12%. This is the first sequenced genome of a CTV isolate from Hunan province. According to the results, further sequencing and bioindexing need to be developed to explore the potential of a local cross protective strategy.

Background: Identification of genomic alterations (e.g., EGFR, ALK, ROS1, BRAF, NTRK, and MET) is essential for initiating targeted therapy in patients with advanced non-small-cell lung cancer (aNSCLC). This study estimated the budget impact of using the sequential single-gene (SSG) test, which tests for each mutation one at a time, versus next-generation sequencing (NGS), which tests for all mutations at the same time, among newly diagnosed patients with aNSCLC from a Japanese healthcare payer's perspective. Methods: A budget impact model (BIM) was used to determine the expected budget impact associated with NGS for newly diagnosed aNSCLC in Japan over a 3-year period. The BIM compared the total costs (biopsy, testing, and treatment) and average turnaround time of future NGS and current NGS versus SSG testing. Results: The adoption of current NGS over SSG testing had a budget impact of-0.24%, but adoption of future NGS over SSG testing had a budget impact of thorn 4.33% across a 3-year time horizon on the Japanese budget for aNSCLC treatment. The adoption of current or future NGS over SSG testing would shorten the average turnaround time for testing. Conclusions: The adoption of current NGS over SSG testing would slightly decrease the yearly costs. However, the adoption of future or current NGS over SSG testing would shorten the average turnaround time, enabling faster identification of genomic alterations and earlier initiation of treatment for aNSCLC patients in Japan. (c) 2022 The Authors. Published by Elsevier B.V. on behalf of The Japanese Respiratory So-ciety. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Background: Thymic epithelial tumors (TETs) are prone to developing in East Asian populations. However, little is known about the genomic profile of TETs in East Asian populations, and the genomic aberrations in TETs have not yet been fully clarified. Thus, molecular targeted therapies for patients with TETs have not been established. This prospective study was conducted to explore the genetic abnormalities of surgically resected TETs in a Japanese cohort and to identify clues for carcinogenesis and potential therapeutic targets in TETs. Methods: Genetic profiles of TETs were investigated using fresh-frozen specimens resected from operable cases with TETs. DNA sequencing was performed using a next-generation sequencing (NGS) gene panel test with Ion Reporter™ and CLC Genomics Workbench 11.0. The mutation sites were further confirmed by Sanger sequencing, digital droplet polymerase chain reaction (ddPCR), and TA cloning for validation. Results: Among 43 patients diagnosed with anterior mediastinal tumors between January 2013 and March 2019, NGS and validation analyses were performed in 31 patients [29 thymomas and two thymic cancers (TCs)] who met the study criteria. Of these, 12 cases of thymoma types A, AB, B1, and B2 harbored the general transcription factor 2-I (GTF2I) mutation (L424H). Conversely, the mutation was not detected in type B3 thymoma or TC, suggesting that the GTF2I mutation existed in indolent types of TETs. Rat sarcoma viral oncogene (RAS) mutations were detected in three cases [Harvey RAS (HRAS) in two cases of type AB thymoma and neuroblastoma RAS (NRAS)] in one case of type B1 thymoma), and additional sex combs like 1 (ASXL1) mutation was present in one case of TC. All RAS mutations were observed in GTF2I-mutated cases. Conclusions: The GTF2I mutation (L424H) is the most frequently occurring mutation in the limited histology of thymoma, consistent with those in the non-Asian population. HRAS and NRAS mutations co-occurred in cases harboring the, Translational Lung Cancer Research, 12(4), pp.707-718; 2023

Introducción: Las enfermedades genéticas constituyen una parte notable de la patología pediátrica. El cribado neonatal es un programa de salud pública cuyo objetivo es identificar a los recién nacidos presintomáticos con trastornos en los que un tratamiento precoz evitaría el desarrollo de daños graves e irreversibles. El rápido avance de las técnicas de secuenciación masiva ha supuesto una revolución a nivel médico, planteándose su implementación en el cribado metabólico actual. Sin embargo, la decisión de adoptar un programa de este tipo debe abordar previamente el análisis de varias consideraciones. Objetivos: Obtener la evidencia disponible hasta la fecha acerca de los aspectos técnicos, éticos y sociales de la implementación del cribado genético neonatal como prueba de primer nivel. Material y métodos: Esta revisión resume la evidencia disponible sobre el empleo de diferentes métodos de NGS en un entorno de cribado neonatal. Para ello, se realizó una búsqueda en diferentes bases de datos siguiendo la declaración PRISMA, estableciéndose previamente criterios de inclusión y exclusión utilizando el sistema PICOs. Resultados: Finalmente fueron incluidos 7 estudios, que emplearon WES, WGS o paneles genéticos como método de secuenciación. En general, demostraron una reducción de la sensibilidad respecto al cribado neonatal convencional, así como la identificación de una gran cantidad de hallazgos secundarios. Discusión: La cantidad de información generada mostró hallazgos incidentales y variantes relacionadas con el estado de portador, que suponen un desafío en cuanto a su interpretación y divulgación al no demostrar utilidad clínica inmediata; pudiendo suscitar estrés familiar y una sobrecarga en el circuito asistencial, requiriendo además el desarrollo de protocolos de seguimiento adecuados en el caso de la identificación de variantes de significado incierto. Conclusiones: La NGS no debería reemplaza al cribado metabólico actual, pudiendo resultar de utilidad como, Introdución: As enfermedades xenéticas constitúen unha parte notable da patoloxía pediátrica. O cribado neonatal é un programa de saúde pública cuxo obxectivo é identificar aos neonatos sans presintomáticos con trastornos nos que un tratamento precoz evitaría o desenvolvemento de danos graves e irreversibles. O rápido avance das técnicas de secuenciación masiva supuxo unha revolución a nivel médico, plantexándose a súa implementación no cribado metabólico actual. Sen embargo, a decisión de adoptar un programa deste tipo debe abordar previamente a análise de varias consideracións. Obxectivos: Obter a evidencia dispoñible ata a data acerca dos aspectos técnicos, éticos e sociais da implementación do cribado xenético neonatal como proba de primeiro nivel. Material e métodos: Esta revisión resume a evidencia dispoñible sobre o emprego de diferentes métodos de secuenciación nun entorno de cribado neonatal. Para iso, realizouse unha búsqueda nas diferentes bases de datos seguindo a declaración PRISMA, establecéndose previamente criterios de inclusión e exclusión utilizando o sistema PICOs. Resultados: Finalmente foron incluidos 7 estudios, que empregaron WES, WGS ou paneis xenéticos como métodos de secuenciación. En xeral, demostrouse unha redución da sensibilidade con respecto ao cribado neonatal convencional, así como a identicación dunha gran cantidade de achados secundarios. Discusión: A cantidade de información xerada mostrou achados incidentais e variantes relacionadas co estado de portador, que amosan un desafío en canto a súa interpretación e divulgación ao non amosar utilidade clínica inmediata; poidendo suscitar estrés familiar e unha sobrecarga no circuito asistencial, esixindo ademais o desenvolvemento de protocolos de seguimento adecuados no caso de identificación de variantes de significado incerto. Conclusións: A NGS non debería reemplazar ao cribado metabólico actual, aínda que pode ser útil como método complementario; e é necesario o desenvolvemento de es, Introduction: Genetic diseases constitute an important part of pediatric pathology. Neonatal screening is a public health program whose objective is to identify presymptomatic newborns with disorders in which early treatment would prevent the development of serious and irreversible damage. The rapid advance of massive sequencing techniques has meant a revolution at the medical level, considering its implementation in current metabolic screening. However, the decision to adopt a program of this type must first address the analysis of several considerations. Aims: Obtain the evidence available to date about the technical, ethical, and social aspects of the implementation of neonatal genetic screening as a first level test. Material and methods: This review summarizes the available evidence on the use of different NGS methods in a newborn screening setting. For this, a search was carried out in different databases following the PRISMA statement, establishing inclusion and exclusion criteria using the PICOs system. Results: Finally, 7 studies were included, which used WES, WGS or genetic panels as sequencing methods. In general, a decrease in sensitivity compared to conventional neonatal screening was demonstrated, as well as the identification of many secondary findings. Discussion: The amount of information generated showed incidental findings and variants related to the carrier state, which involve a challenge in terms of their interpretation and dissemination as they do not demonstrate immediate clinical utility; may cause family stress and an overload in the care circuit, also requiring the development of adequate follow-up protocols in the case of identification of uncertain significance variants. Conclusions: The NGS should not replace the current metabolic newborn screening, although it may be useful as a complementary method; and the development of future studies is necessary to further investigate the technical, ethical, and psychosocial factors associated wit

Svaka bolest čija je učestalost manja od 1 u 2000 ljudi definiše se kao retka bolest. Iz tog razloga, broj retkih bolesti je veliki. Do sada je opisano preko 6000 različitih retkih bolesti. Preko 80% retkih bolesti ima genetičku osnovu i to je razlog zašto su znanja iz molekularne biologije od neprocenjivog značaja za istraživanje molekularne osnove retkih bolesti, postavljanje tačne dijagnoze i razvoj inovativnih terapeutika. Cilj ovog rada je da objasni važnost otkrivanja molekularno-genetičke osnove retkih bolesti i da prikaže desetogodišnje iskustvo primene sekvenciranja nove generacije u Srbiji (2014.-2023.) u tu svrhu. U prethodnom periodu za istraživanje retkih bolesti korišćeni su sekvenciranje kliničkog egzoma, sekvenciranje kompletnog egzoma i sekvenciranje kompletnog genoma. Takođe, date su i perspektive za budućnost gde će genomika biti kompletirana tehnologijom sekvenciranja dugačkih fragmenata i komplementirana upotrebom transkriptomike, proteomike, metabolomike i drugih „omika“., Any disease found in less than 1 person out of 2000 people is defined as a rare disease. For this reason, the number of rare diseases is high. Over 6,000 different rare diseases have been described so far. More than 80% of rare diseases have a genetic basis, and this is the reason why knowledge of molecular biology is invaluable for research into the molecular basis of rare diseases, establishing an accurate diagnosis and developing innovative therapeutics. The aim of this paper is to explain the importance of discovering the molecular genetic basis of rare diseases and to present the ten-year experience of applying new generation sequencing in Serbia (2014-2023) for this purpose. During this period, clinical exome sequencing, complete exome sequencing and complete genome sequencing were used for research of rare diseases. In the future, it is expected that genomics, which until now was based mainly on the technology of shortread fragments, will be broaden with the long-reads sequencing technology, and complemented by the use of transcriptomics, proteomics, metabolomics and other omics.

Fipronil is a broad-spectrum insecticide with remarkable efficacy that is widely used to control insect pests around the world. However, its extensive use has led to increasing soil and water contamination. This fact is of concern and makes it necessary to evaluate the risk of undesirable effects on non-target microorganisms, such as the microbial community in water and/or soil. Studies using the metagenomic approach to assess the effects of fipronil on soil microbial communities are scarce. In this context, the present study was conducted to identify microorganisms that can biodegrade fipronil and that could be of great environmental interest. For this purpose, the targeted metabarcoding approach was performed in soil microcosms under two environmental conditions: fipronil exposure and control (without fipronil). After a 35-day soil microcosm period, the 16S ribosomal RNA (rRNA) gene of all samples was sequenced using the ion torrent personal genome machine (PGM) platform. Our study showed the presence of Proteobacteria, Actinobacteria, and Firmicutes in all of the samples; however, the presence of fipronil in the soil samples resulted in a significant increase in the concentration of bacteria from these phyla. The statistical results indicate that some bacterial genera benefited from soil exposure to fipronil, as in the case of bacteria from the genus Thalassobacillus, while others were affected, as in the case of bacteria from the genus Streptomyces. Overall, the results of this study provide a potential contribution of fipronil-degrading bacteria.

We developed an efficient approach to diagnostic copy number analysis of targeted gene panel or whole exome sequence (WES) data. Here we present CNV-Z as a new tool for detection of copy number variants (CNVs). Deletions and duplications of chromosomal regions are widely implicated in both genomic evolution and genetic disorders. However, calling CNVs from targeted or exome sequence data is challenging. In most cases, the copy number of a chromosomal region is estimated as the depth of reads mapping to a certain bin or sliding window divided by the expected number of reads derived from a set of reference samples. This approach will inevitably miss smaller CNVs on an irregular basis, and quite frequently results in a disturbing number of false positive CNVs. We developed an alternative approach to detect CNVs based on deviation from expected read depth per position, instead of region. Cautiously used, the cohort of samples in the same run will do as a reference. With appropriate filtering, given high quality DNA and a set of suitable reference samples, CNV-Z detects CNVs ranging in length from one nucleotide to an entire chromosome, with few false positives. Performance is proved by benchmarking using both in-house targeted gene panel NGS data and a publicly available NGS dataset, both sets with multiplex ligation-dependent amplification probe (MLPA) validated CNVs. The outcome shows that CNV-Z detects single- and multi-exonic CNVs with high specificity and sensitivity using different kind of NGS data. On gene level, CNV-Z shows both excellent sensitivity and specificity. Compared to competing CNV callers, CNV-Z shows higher specificity and positive predictive value for detecting exonic CNVs.

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

Die folgende Doktorarbeit soll neue Erkenntnisse über die Nahrungsaufnahme von Laufkäfern mit Hilfe von DNA-basierten Techniken zur Nahrungsanalyse liefern, damit das Potenzial von Laufkäfern als biologische Bekämpfungsmittel für Nacktschnecken und Unkrautsamen in Zukunft besser beurteilt werden kann. Das Wissen über das Fressverhalten von Carabiden an Schnecken beruht aktuell häufig noch auf Laborexperimenten, sodass detailliertere Kenntnisse über die Schneckenprädation von Carabiden und deren Wirksamkeit als potenzielle Schneckenjäger im Freiland fehlen. Somit fällt es schwer, genaue Aussagen über das Fressverhalten einzelner Carabidenarten an Nacktschnecken und deren Eiern im Agrarland zu treffen. Ebenso wäre ein präziserer Wissensstand darüber wünschenswert, welche Carabidenarten auf den Feldern den Samenfraß bevorzugen und die Samen welcher Pflanzenarten die jeweiligen Carabidenarten verzehren, um dadurch zu klären, welche Carabidenarten für einen zuverlässigen Fraß von Unkrautsamen und damit für einen Rückgang der Ackerunkräuter sorgen könnten. Ein genaueres Bild von der Ernährung der einzelnen Carabidenarten als bisher bekannt wäre daher zur Einteilung der jeweiligen Art in genau definierte trophische Funktionsgruppen von Nöten. Zur Erforschung der aufgenommenen Nahrung und des Fressverhaltens von in Feldern Mitteleuropas (Frankreich und Österreich) präsenten Carabidenarten war es zunächst erforderlich, die Darminhalte von Carabiden mittels DNA-basierter Techniken im Hinblick auf die potenziellen Nahrungsquellen Nacktschnecke und Unkrautsamen analysieren zu können. Zu diesem Zweck wurden neue DNA-basierte Ansätze für die molekulare Analyse des Darminhalts entwickelt, um Nacktschnecken-DNA durch Multiplex-Polymerasekettenreaktion (PCR) nachweisen und eine gezielte Amplikon-Sequenzierung (TAS) von Pflanzenarten mit anschließender detaillierter Datenanalyse durchführen zu können (Kapitel 2 und 3). Hochempfindliche Primerpaare für die Nacktschnecken Arion distinc, The following PhD thesis aims to provide new insights into the food consumption of carabid beetles using DNA-based techniques for dietary analysis, so that the potential of ground beetles to be used as biological control agents for slugs and weed seeds can be better assessed in the future. Knowledge about the feeding behaviour of carabids on slugs is currently often still based on laboratory experiments, so that more detailed information about slug predation by carabids and their effectiveness as potential slug predators in the field is lacking. It is therefore difficult to make precise statements about the feeding behaviour of individual carabid species on slugs and their eggs in agricultural land. Similarly, it would be desirable to have more precise knowledge of which carabid species prefer seed feeding in the fields and of which plant species the respective carabid species consume seeds from, in order to clarify which carabid species could provide reliable feeding on weed seeds and consequently a decline in field weeds. A more precise picture of the diet of the individual carabid species than known so far would thus be necessary to classify each species into well-defined trophic functional groups. In order to investigate the ingested food and feeding behaviour of carabid species present in Central European fields (France and Austria), it was first necessary to be able to analyse the gut contents of the carabids using DNA-based techniques with regard to the potential food sources slugs and weed seeds. For this purpose, new DNA-based approaches for molecular gut content analysis were developed to detect slug DNA by multiplex polymerase chain reaction (PCR) and to perform a targeted amplicon sequencing (TAS) approach of plant species with subsequent detailed data analysis (chapters 2 and 3). Highly sensitive primer pairs for the slug species Arion distinctus (Mabille, 1868) (Stylommatophora: Arionidae) and Deroceras reticulatum (Müller, 1774) (Stylommatophora: Agri, presented by: Yasemin Günay-Greunke, M.Sc., Zusammenfassung in deutscher Sprache, Kumulative Dissertation aus fünf Artikeln, Dissertation University of Innsbruck 2023

The Inter European Union Reference Laboratories (EURLs) Working Group on Next Generation Sequencing (NGS) involves eight EURLs for microbiological food and feed hazards and has been working since 2017 to promote the adoption of NGS by the National Reference Laboratories (NRLs) in the European Union. This work illustrates the results of the first 5 years of activity. By working together, the EURLs involved have released guidance documents for assisting NRLs in all the steps of NGS, helping the transition from classical molecular methods towards whole genome sequencing while ensuring harmonization, with the final aim of improving preparedness in the use of NGS to characterize microbial hazards and trace the sources of infection.

[ES] Antecedentes: Los avances en el campo de la genética han servido para desarrollar la denominada autopsia molecular desde Sanger, hasta la Next Generation Sequencing (NGS). Conocemos un entramado de genes que codifican unas proteínas esenciales a la hora de generar el Potencial de Acción (PA) cardiaco correcto. Pero no todo son soluciones. Nos encontramos con obstáculos a la hora de hacer siempre una interpretación clínica coherente de los resultados que ayude clínicamente y que no nos ocasione más problemas de los que pretendemos resolver. Objetivo principal: Recopilar, revisar y combinar los resultados de estudios publicados sobre el uso de las nuevas plataformas diagnósticas en diferentes probandos clínicos con Variantes de Significado Incierto (VUS) generadas mediante NGS y determinar su utilidad práctica en la canalopatía más frecuente el Síndrome de QT Largo (LQTS). Material y Métodos: Revisión sistemática de la literatura en la base de datos PubMed de los estudios que utilizan células pluripotenciales humanas y la edición del genoma para analizar VUS. Resultados. Se revisaron 29 artículos en los que se usan nuevas tecnologías como human induced Pluripotent Stem Cells (hiPSCs) y/o la edición del genoma mediante Clustered Regurlarly Interspaced Short Palindromic Repeats (CRISPR/cas9) con las que se ha podido delinear la patogenicidad de VUS usando la edición del genoma para corregir la variante en el gen y normalizándose de este modo el fenotipo celular. Limitaciones: La inmadurez de las células, la necesidad de modelos 3D (3 Dimensiones), el tiempo de generación. Conclusiones: hiPSCs y/o CRISPR/cas9 son herramientas muy útiles para predecir in vitro el comportamiento de las VUS. Estas tecnologías junto con el uso de la Inteligencia Artificial (AI) nos permitirá avanzar en la medicina de precisión., [EN] Background: Advances in the field of genetics have served to develop the so-called molecular autopsy from Sanger, to the Next Generation Sequencing (NGS). We know that there is a network of genes that encodes proteins that are essential for generating the correct cardiac action potential. Not everything is a solution. We encounter obstacles when it comes to always making a coherent clinical interpretation of the results that help clinically and do not cause more problems than those we are trying to solve. Main objective: To collect, review and combine the results of published studies on the use of new diagnostic platforms in different clinical probands with Variants of Uncertain Significance (VUS) generated by NGS and to determine their practical utility in the most frequent channelopathy, the Long QT Syndrome (LQTS). Material and Methods: Systematic literature review in the PubMed database of studies using human pluripotent cells and genome editing to analyze VUS. Results: 29 articles were reviewed using new technologies such as human induced Pluripotent Stem Cells (hiPSCs) and/or genome editing using Clustered Regurlarly Interspaced Short Palindromic Repeats (CRISPR/cas9) which has made possible to delineate the pathogenicity of VUS using genome editing to correct the variant in the gene and thereby normalizing the cellular phenotype. Limitations: Immaturity of cells, need for 3D models, generation time. Conclusions: hiPSCs and/or CRISPR/cas9 are very useful tools for predicting VUS behavior in vitro. These technologies, together with the use of Artificial Intelligence (AI), will allow us to advance in precision medicine.

ACMG recognizes five different categories of sequence variants identified by next generation sequencing (pathogenic, likely pathogenic, variants of unknown significance, likely benign and benign). Sometimes, potentially relevant gene variants could be categorized as variants of unknown significance according to the level of available evidences. Because of that, detailed assessment of the phenotype-genotype correlation by the clinical geneticist in each individual case is crucially important. The interpretation and classification of a variant may change over time. Variant reinterpretation is defined as the practice of reevaluating all the evidence available about the pathogenicity of a genetic variant and taking into account any new evidence that is made available since the previous interpretation. For the last seven years, we had 168 patients with clinically suspected locus heterogeneous skeletal dysplasia. Next generation sequencing (NGS) using clinical exome sequencing or whole exome sequencing was performed for all. All patients underwent detailed phenotype-genotype correlation investigation. Molecular diagnosis by determining the pathogenic or likely pathogenic causative gene variant(s) was established for 102 out of 168 patients (60.71%). Additionally, in 10 patients (5.95%) variant of unknown significance (VUS) with good phenotype-genotype correlation was identified. These VUS variants could be potentially, and possibly are, causal, although there are no reliable evidences of their pathogenicity at the moment. In one of the positive patients in our study, the variant was initially classified as VUS, but with new evidence it was reclassified as likely pathogenic. In the present study, a potentially relevant variant of unknown significance was detected in 5.95% of patients, which is a non-negligible proportion. For all these patients, we have organized clinical follow-up with periodic reinterpretation and reclassification of the detected variants.

Human genome sequencing has revolutionized our understanding of disease susceptibility, drug metabolism and human ancestry. This course will explore how these advances have been made possible by revolutionary new sequencing methodologies that have decreased costs and increased throughput of genome analysis, making it possible to examine genetic correlates for a variety of biological processes and disorders. The course will combine discussions of primary scientific research papers with hands-on data analysis and small group presentations. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.

Monitoring of SARS-CoV-2 in wastewater (WW) is a promising tool for epidemiological surveillance, correlating not only viral RNA levels with the infection dynamics within the population, but also to viral diversity. However, the complex mixture of viral lineages in WW samples makes tracking of specific variants or lineages circulating in the population a challenging task. We sequenced sewage samples of 9 WW-catchment areas within the city of Rotterdam, used specific signature mutations from individual SARS-CoV-2 lineages to estimate their relative abundances in WW and compared them against those observed in clinical genomic surveillance of infected individuals between September 2020 and December 2021. We showed that especially for dominant lineages, the median of the frequencies of signature mutations coincides with the occurrence of those lineages in Rotterdam's clinical genomic surveillance. This, along with digital droplet RT-PCR targeting signature mutations of specific variants of concern (VOCs), showed that several VOCs emerged, became dominant and were replaced by the next VOC in Rotterdam at different time points during the study. In addition, single nucleotide variant (SNV) analysis provided evidence that spatio-temporal clusters can also be discerned from WW samples. We were able to detect specific SNVs in sewage, including one resulting in the Q183H amino acid change in the Spike gene, that was not captured by clinical genomic surveillance. Our results highlight the potential use of WW samples for genomic surveillance, increasing the set of epidemiological tools to monitor SARS-CoV-2 diversity., Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., Sanitary Engineering

Background: Identification of genomic alterations (e.g., EGFR, ALK, ROS1, BRAF, NTRK, and MET) is essential for initiating targeted therapy in patients with advanced non-small-cell lung cancer (aNSCLC). This study estimated the budget impact of using the sequential single-gene (SSG) test, which tests for each mutation one at a time, versus next-generation sequencing (NGS), which tests for all mutations at the same time, among newly diagnosed patients with aNSCLC from a Japanese healthcare payer's perspective. Methods: A budget impact model (BIM) was used to determine the expected budget impact associated with NGS for newly diagnosed aNSCLC in Japan over a 3-year period. The BIM compared the total costs (biopsy, testing, and treatment) and average turnaround time of future NGS and current NGS versus SSG testing. Results: The adoption of current NGS over SSG testing had a budget impact of-0.24%, but adoption of future NGS over SSG testing had a budget impact of thorn 4.33% across a 3-year time horizon on the Japanese budget for aNSCLC treatment. The adoption of current or future NGS over SSG testing would shorten the average turnaround time for testing. Conclusions: The adoption of current NGS over SSG testing would slightly decrease the yearly costs. However, the adoption of future or current NGS over SSG testing would shorten the average turnaround time, enabling faster identification of genomic alterations and earlier initiation of treatment for aNSCLC patients in Japan. (c) 2022 The Authors. Published by Elsevier B.V. on behalf of The Japanese Respiratory So-ciety. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Chicken amyloid arthropathy is a debilitating disease with a major impact on animal welfare. Since the disease is triggered by bacterial infection, preventative treatment also contributes to the widespread overuse of antibiotics. Bacterial infection initiates an acute phase response including increased serum amyloid A (SAA) production by the liver. SAA accumulates at sites of infection and in particular in large joints of affected birds. Interestingly, white egg-laying chickens (WL) are resistant to the disease whilst brown egg-laying chickens (BL) are most affected. Disease susceptibility has an immunological basis but the possible contribution of underlying genetic risk factors is not understood. Using a whole genome sequencing approach, we discovered a novel variant in the SAA gene in WL, which is predicted to result in an arginine to serine substitution at position 90 (SAA.R90S). Surprisingly, when overexpressed in chicken hepatocellular carcinoma cells, SAA.R90S was expressed at a higher rate and secreted to a greater degree than the wild-type SAA protein. Moreover, RNASeq analysis showed that the R90S mutant exerted a differential effect on the expression of core transcription factors linked to cell fate determination and cell differentiation. Comparative analysis of gene expression in murine CD4 T-cells stimulated with IL-6/SAA, suggests that SAA.R90S might block an induced cell fate change toward pro-inflammatory T helper 17 cells, which are required for immunological protection against pathogenic bacteria during an acute phase response. Our results provide first mechanistic insights into the genetic resistance of WL to amyloid arthropathy and could be applied to commercial layer breeding programs to improve animal welfare and reduce the negative effects of the overuse of antibiotics.

Under de senaste 30 åren har en ny metod för att få information om tidigare ekosystem utveck- lats, nämligen att analysera gammalt (“ancient”) DNA (aDNA). Denna forskningsgren är fortfarande ung men har redan hittat många områden inom vilka den kan tillämpas. Inom paleoekologin finns det två huvudsakliga ändamål med aDNA-analyser; att kartlägga en arts hela genom, samt att bestämma antal taxa och vilka taxa som finns beva- rade som aDNA i prover från olika typer av sediment. Beroende på ändamålet, skiljer sig analysmetoderna något. Genom att analysera växters aDNA i sjösediment har det gjorts nya upptäckter om vilka växtarter som fanns, till exempel under den kallaste delen av den senaste istiden (LGM) i norra Europa. Genom att analysera fyra artiklar som behandlar studier av aDNA hämtat från sjösediment, har jag sett närmare på hur studier av aDNA har bidragit till förståelsen för vegetationsutvecklingen i Nordeuropa. Generellt i de fyra studierna kan aDNA-analyser inte identifiera lika många taxa som pollenanalys kan, men i stället är metoden bättre på att identifiera arter snarare än bara släkten och familjer (högre taxonomisk upplösning). Dessutom kan aDNA-analys generellt identifiera fler taxa än makrofossilanalys. Dessa förhållanden är dock beroende av tafonomiska processer. Studier av aDNA har gett oss nya insikter om vilka växter som fanns i Nordeuropa under LGM, den senglaciala perioden och efter den senaste istiden (Weichsel). Med aDNA-analys har det identifierats nya taxa inom följande växtgrupper: vattenväxter, mossor, träd och buskar samt alger. Dessa nya upptäckter har visat att deglaciationen och klimatet i de områden som fungerade som nordliga växtrefugier under LGM var mer komplex än man tidigare trott och att ytterligare studier krävs för att få en djupare förståelse av hur vegetationen förändrades i Nordeuropa under och efter Weichsel-istiden., During the last 30 years a new method for obtaining information about past ecosystems has been devel- oped, namely to analyse old (“ancient”) DNA (aDNA). This research field is still young but has already found many areas within which it can be applied. In palaeoecology, there are two main purposes with aDNA analyses; to map the entire genome of a species, and to determine the number of taxa, and which taxa, that are preserved as aDNA in samples from different types of sediments. Depending on the purpose of the analysis, the analytical meth- ods differ slightly. By analysing plant aDNA in lake sediments there have been new discoveries about which plant species were present, for example, during the coldest part of the last glacial period (LGM) in Northern Europe. By analyzing four articles dealing with studies of aDNA which was gathered from lacustrine sediments, I have taken a closer look at how studies of aDNA have contributed to the understanding of the vegetation development in North- ern Europe. Generally in the four studies, aDNA analysis cannot identify as many taxa as pollen analysis can do, but instead the method is better at identifying species rather than just genera or families (higher taxonomic resolu- tion). In addition, aDNA analysis can overall identify more taxa than macrofossil analysis. These conditions are, however, dependent on taphonomic processes. Studies of aDNA have provided us with new insights into which plants were present in Northern Europe during the LGM, the late-glacial period and after the last glacial period (the Weichselian). With aDNA analysis, the following plant groups have been identified: water plants, mosses, trees and bushes, and algae. These new discoveries have shown that the deglaciation and the climate in northern refugia was more complex than previously believed and that further studies are required to reach a deeper understanding of how the vegetation changed in Northern Europe during and after the Weichselian glacial.

A large amount of microbial cells is found in the human gastrointestinal tract. The gut microbiota consists of microbes from different kingdoms: bacteria, viruses and fungi. Studies on the gut viruses and fungi show that they can be important in human health and therefore tools to research them are needed. In this thesis, in vitro gut fermentation models were used for research on viruses and fungi in the human gut. The survival of bacteriophages in the stomach and the small intestine, and in the colon was investigated with the use of the TIM systems. These studies show the importance of survival studies in the design of successful phage therapy. The gut fungal community, and interactions with the bacterial community were also investigated in an in vitro model of the colon (TIM-2). Fungal-bacterial interactions were furthermore studied in a large number of fecal samples. These experiments gave more insights in the gut fungal community.