Benchmarking short-, long- and hybrid-read assemblers for metagenome sequencing of complex microbial communities.

Metagenome community analyses, driven by the continued development in sequencing technology, is rapidly providing insights in many aspects of microbiology and becoming a cornerstone tool. Illumina, Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) are the leading technologies, each with their own advantages and drawbacks. Illumina provides accurate reads at a low cost, but their length is too short to close bacterial genomes. Long reads overcome this limitation, but these technologies produce reads with lower accuracy (ONT) or with lower throughput (PacBio high-fidelity reads). In a critical first analysis step, reads are assembled to reconstruct genomes or individual genes within the community. However, to date, the performance of existing assemblers has never been challenged with a complex mock metagenome. Here, we evaluate the performance of current assemblers that use short, long or both read types on a complex mock metagenome consisting of 227 bacterial strains with varying degrees of relatedness. We show that many of the current assemblers are not suited to handle such a complex metagenome. In addition, hybrid assemblies do not fulfil their potential. We conclude that ONT reads assembled with CANU and Illumina reads assembled with SPAdes offer the best value for reconstructing genomes and individual genes of complex metagenomes, respectively.

Utilizing genomic signatures to gain insights into the dynamics of SARS-CoV-2 through Machine and Deep Learning techniques.

The global spread of the SARS-CoV-2 pandemic, originating in Wuhan, China, has had profound consequences on both health and the economy. Traditional alignment-based phylogenetic tree methods for tracking epidemic dynamics demand substantial computational power due to the growing number of sequenced strains. Consequently, there is a pressing need for an alignment-free approach to characterize these strains and monitor the dynamics of various variants. In this work, we introduce a swift and straightforward tool named GenoSig, implemented in C++. The tool exploits the Di and Tri nucleotide frequency signatures to delineate the taxonomic lineages of SARS-CoV-2 by employing diverse machine learning (ML) and deep learning (DL) models. Our approach achieved a tenfold cross-validation accuracy of 87.88% (± 0.013) for DL and 86.37% (± 0.0009) for Random Forest (RF) model, surpassing the performance of other ML models. Validation using an additional unexposed dataset yielded comparable results. Despite variations in architectures between DL and RF, it was observed that later clades, specifically GRA, GRY, and GK, exhibited superior performance compared to earlier clades G and GH. As for the continental origin of the virus, both DL and RF models exhibited lower performance than in predicting clades. However, both models demonstrated relatively higher accuracy for Europe, North America, and South America compared to other continents, with DL outperforming RF. Both models consistently demonstrated a preference for cytosine and guanine over adenine and thymine in both clade and continental analyses, in both Di and Tri nucleotide frequencies signatures. Our findings suggest that GenoSig provides a straightforward approach to address taxonomic, epidemiological, and biological inquiries, utilizing a reductive method applicable not only to SARS-CoV-2 but also to similar research questions in an alignment-free context.

Correction: Sallam et al. DNA Methylation Alterations in Fractionally Irradiated Rats and Breast Cancer Patients Receiving Radiotherapy. Int. J. Mol. Sci. 2022, 23, 16214.

Radia Tamarat and Susana Constantino Rosa Santos were not included as authors in the original publication [...].

A comprehensive analysis of genetic risk for metabolic syndrome in the Egyptian population via allele frequency investigation and Missense3D predictions.

Diabetes mellitus (DM) represents a major health problem in Egypt and worldwide, with increasing numbers of patients with prediabetes every year. Numerous factors, such as obesity, hyperlipidemia, and hypertension, which have recently become serious concerns, affect the complex pathophysiology of diabetes. These metabolic syndrome diseases are highly linked to genetic variability that drives certain populations, such as Egypt, to be more susceptible to developing DM. Here we conduct a comprehensive analysis to pinpoint the similarities and uniqueness among the Egyptian genome reference and the 1000-genome subpopulations (Europeans, Ad-Mixed Americans, South Asians, East Asians, and Africans), aiming at defining the potential genetic risk of metabolic syndromes. Selected approaches incorporated the analysis of the allele frequency of the different populations' variations, supported by genotypes' principal component analysis. Results show that the Egyptian's reference metabolic genes were clustered together with the Europeans', Ad-Mixed Americans', and South-Asians'. Additionally, 8563 variants were uniquely identified in the Egyptian cohort, from those, two were predicted to cause structural damage, namely, CDKAL1: 6_21065070 (A > T) and PPARG: 3_12351660 (C > T) utilizing the Missense3D database. The former is a protein coding gene associated with Type 2 DM while the latter is a key regulator of adipocyte differentiation and glucose homeostasis. Both variants were detected heterozygous in two different Egyptian individuals from overall 110 sample. This analysis sheds light on the unique genetic traits of the Egyptian population that play a role in the DM high prevalence in Egypt. The proposed analysis pipeline -available through GitHub- could be used to conduct similar analysis for other diseases across populations.

Dissecting the role of the gut microbiome and fecal microbiota transplantation in radio- and immunotherapy treatment of colorectal cancer.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and poses a major burden on the human health worldwide. At the moment, treatment of CRC consists of surgery in combination with (neo)adjuvant chemotherapy and/or radiotherapy. More recently, immune checkpoint blockers (ICBs) have also been approved for CRC treatment. In addition, recent studies have shown that radiotherapy and ICBs act synergistically, with radiotherapy stimulating the immune system that is activated by ICBs. However, both treatments are also associated with severe toxicity and efficacy issues, which can lead to temporary or permanent discontinuation of these treatment programs. There's growing evidence pointing to the gut microbiome playing a role in these issues. Some microorganisms seem to contribute to radiotherapy-associated toxicity and hinder ICB efficacy, while others seem to reduce radiotherapy-associated toxicity or enhance ICB efficacy. Consequently, fecal microbiota transplantation (FMT) has been applied to reduce radio- and immunotherapy-related toxicity and enhance their efficacies. Here, we have reviewed the currently available preclinical and clinical data in CRC treatment, with a focus on how the gut microbiome influences radio- and immunotherapy toxicity and efficacy and if these treatments could benefit from FMT.

Are Arabidopsis thaliana plants able to recover from exposure to gamma radiation? A molecular perspective.

Most plant research focuses on the responses immediately after exposure to ionizing irradiation (IR). However, it is as important to investigate how plants recover after exposure since this has a profound effect on future plant growth and development and hence on the long-term consequences of exposure to stress. This study aimed to investigate the IR-induced responses after exposure and during recovery by exposing 1-week old A. thaliana seedlings to gamma dose rates ranging from 27 to 103.7 mGy/h for 2 weeks and allowing them to recover for 4 days. A high-throughput RNAsequencing analysis was carried out. An enrichment of GO terms related to the metabolism of hormones was observed both after irradiation and during recovery at all dose rates. While plants exposed to the lowest dose rate activate defence responses after irradiation, they recover from the IR by resuming normal growth during the recovery period. Plants exposed to the intermediate dose rate invest in signalling and defence after irradiation. During recovery, in the plants exposed to the highest dose rate, fundamental metabolic processes such as photosynthesis and RNA modification were still affected. This might lead to detrimental effects in the long-term or in the next generations of those irradiated plants.

Modeling the Effects of Cypermethrin Toxicity on Ovalbumin-Induced Allergic Pneumonitis Rats: Macrophage Phenotype Differentiation and p38/STAT6 Signaling Are Candidate Targets of Pirfenidone Treatment.

Although the classic form of asthma is characterized by chronic pneumonitis with eosinophil infiltration and steroid responsivity, asthma has multifactorial pathogenesis and various clinical phenotypes. Previous studies strongly suggested that chemical exposure could influence the severity and course of asthma and reduce its steroid responsiveness. Cypermethrin (CYP), a common pesticide used in agriculture, was investigated for the possible aggravation of the ovalbumin (OVA)-induced allergic pneumonitis and the possible induction of steroid resistance in rats. Additionally, it was investigated whether pirfenidone (PFD) could substitute dexamethasone, as an alternative treatment option, for the induced steroid resistance. Fifty-six male Wistar albino rats were randomly divided into seven groups: control, PFD alone, allergic pneumonitis, CYP alone, allergic pneumonitis/CYP-exposed, allergic pneumonitis/CYP/dexamethasone (Dex), and allergic pneumonitis/CYP/PFD-treated groups. Allergic pneumonitis was induced by three intraperitoneal OVA injections administered once a week, followed by an intranasal OVA instillation challenge. CYP (25 mg/kg/d), Dex (1 mg/kg/d), and PFD (100 mg/kg/d) were administered orally from day 15 to the end of the experiment. Bronchoalveolar lavage fluid (BALF) was analyzed for cytokine levels. Hematoxylin and eosin (H&E) and periodic acid Schiff (PAS)-stained lung sections were prepared. Immunohistochemical identification of p38 MAPK and lung macrophages was performed. The inflammatory/oxidative status of the lung and PCR-quantification of the STAT6, p38 MAPK, MUC5AC, and IL-13 genes were carried out. The allergic pneumonitis-only group showed eosinophil-mediated inflammation (p < 0.05). Further CYP exposure aggravated lung inflammation and showed steroid-resistant changes, p38 activation, neutrophil-mediated, M1 macrophage-related inflammation (p < 0.05). All changes were reversed (p < 0.05) by PFD, meanwhile not by dexamethasone treatment. Pirfenidone could replace dexamethasone treatment in the current rat model of CYP-induced severe steroid-resistant asthma via inhibiting the M1 macrophage differentiation through modulation of the STAT6/p38 MAPK pathway.

A cellular screening platform, stably expressing DENV2 NS5, defines a novel anti-DENV mechanism of action of Apigenin based on STAT2 activation.

Type I interferon (IFN-I) evasion by Dengue virus (DENV) is key in DENV pathogenesis. The non-structural protein 5 (NS5) antagonizes IFN-I response through the degradation of the signal transducer and activator of transcription 2 (STAT2). We developed a K562 cell-based platform, for high throughput screening of compounds potentially counteracting the NS5-mediated antagonism of IFN-I signaling. Upon a screening with a library of 1220 approved drugs, 3 compounds previously linked to DENV inhibition (Apigenin, Chrysin, and Luteolin) were identified. Luteolin and Apigenin determined a significant inhibition of DENV2 replication in Huh7 cells and the restoration of STAT2 phosphorylation in both cell systems. Apigenin and Luteolin were able to stimulate STAT2 even in the absence of infection. Despite the "promiscuous" and "pan-assay-interfering" nature of Luteolin, Apigenin promotes STAT2 Tyr 689 phosphorylation and activation, highlighting the importance of screening for compounds able to interact with host factors, to counteract viral proteins capable of dampening innate immune responses.

Limnospira indica PCC 8005 Supplementation Prevents Pelvic Irradiation-Induced Dysbiosis but Not Acute Inflammation in Mice.

Pelvic irradiation-induced mucositis secondarily leads to dysbiosis, which seriously affects patients' quality of life after treatment. No safe and effective radioprotector or mitigator has yet been approved for clinical therapy. Here, we investigated the potential protective effects of fresh biomass of Limnospira indica PCC 8005 against ionizing irradiation-induced mucositis and dysbiosis in respect to benchmark probiotic Lacticaseibacillus rhamnosus GG ATCC 53103. For this, mice were supplemented daily before and after 12 Gy X-irradiation of the pelvis. Upon sacrifice, food supplements' efficacy was assessed for intestinal barrier protection, immunomodulation and changes in the microbiota composition. While both could not confer barrier protection or significant immunomodulatory effects, 16S microbial profiling revealed that L. indica PCC 8005 and L. rhamnosus GG could prevent pelvic irradiation-induced dysbiosis. Altogether, our data show that-besides benchmarked L. rhamnosus GG-L. indica PCC 8005 is an interesting candidate to further explore as a radiomitigator counteracting pelvic irradiation-induced dysbiosis in the presented in vivo irradiation-gut-microbiota platform.

DNA methylation in clonal duckweed (Lemna minor L.) lineages reflects current and historical environmental exposures.

Environmentally induced DNA methylation variants may mediate gene expression responses to environmental changes. If such induced variants are transgenerationally stable, there is potential for expression responses to persist over multiple generations. Our current knowledge in plants, however, is almost exclusively based on studies conducted in sexually reproducing species where the majority of DNA methylation changes are subject to resetting in germlines, limiting the potential for transgenerational epigenetics stress memory. Asexual reproduction circumvents germlines, and may therefore be more conducive to long-term inheritance of epigenetic marks. Taking advantage of the rapid clonal reproduction of the common duckweed Lemna minor, we hypothesize that long-term, transgenerational stress memory from exposure to high temperature can be detected in DNA methylation profiles. Using a reduced representation bisulphite sequencing approach (epiGBS), we show that temperature stress induces DNA hypermethylation at many CG and CHG cytosine contexts but not CHH. Additionally, differential methylation in CHG context that was observed was still detected in a subset of cytosines, even after 3-12 generations of culturing in a common environment. This demonstrates a memory effect of stress reflected in the methylome and that persists over multiple clonal generations. Structural annotation revealed that this memory effect in CHG methylation was enriched in transposable elements. The observed epigenetic stress memory is probably caused by stable transgenerational persistence of temperature-induced DNA methylation variants across clonal generations. To the extent that such epigenetic memory has functional consequences for gene expression and phenotypes, this result suggests potential for long-term modulation of stress responses in asexual plants.

Meta-Analysis of RNA-Seq Datasets Identifies Novel Players in Glioblastoma.

Glioblastoma is a devastating grade IV glioma with poor prognosis. Identification of predictive molecular biomarkers of disease progression would substantially contribute to better disease management. In the current study, we performed a meta-analysis of different RNA-seq datasets to identify differentially expressed protein-coding genes (PCGs) and long non-coding RNAs (lncRNAs). This meta-analysis aimed to improve power and reproducibility of the individual studies while identifying overlapping disease-relevant pathways. We supplemented the meta-analysis with small RNA-seq on glioblastoma tissue samples to provide an overall transcriptomic view of glioblastoma. Co-expression correlation of filtered differentially expressed PCGs and lncRNAs identified a functionally relevant sub-cluster containing DANCR and SNHG6, with two novel lncRNAs and two novel PCGs. Small RNA-seq of glioblastoma tissues identified five differentially expressed microRNAs of which three interacted with the functionally relevant sub-cluster. Pathway analysis of this sub-cluster identified several glioblastoma-linked pathways, which were also previously associated with the novel cell death pathway, ferroptosis. In conclusion, the current meta-analysis strengthens evidence of an overarching involvement of ferroptosis in glioblastoma pathogenesis and also suggests some candidates for further analyses.

DNA Methylation Alterations in Fractionally Irradiated Rats and Breast Cancer Patients Receiving Radiotherapy.

Radiation-Induced CardioVascular Disease (RICVD) is an important concern in thoracic radiotherapy with complex underlying pathophysiology. Recently, we proposed DNA methylation as a possible mechanism contributing to RICVD. The current study investigates DNA methylation in heart-irradiated rats and radiotherapy-treated breast cancer (BC) patients. Rats received fractionated whole heart X-irradiation (0, 0.92, 6.9 and 27.6 Gy total doses) and blood was collected after 1.5, 3, 7 and 12 months. Global and gene-specific methylation of the samples were evaluated; and gene expression of selected differentially methylated regions (DMRs) was validated in rat and BC patient blood. In rats receiving an absorbed dose of 27.6 Gy, DNA methylation alterations were detected up to 7 months with differential expression of cardiac-relevant DMRs. Of those, SLMAP showed increased expression at 1.5 months, which correlated with hypomethylation. Furthermore, E2F6 inversely correlated with a decreased global longitudinal strain. In BC patients, E2F6 and SLMAP exhibited differential expression directly and 6 months after radiotherapy, respectively. This study describes a systemic radiation fingerprint at the DNA methylation level, elucidating a possible association of DNA methylation to RICVD pathophysiology, to be validated in future mechanistic studies.

Introduction to the principles and methods underlying the recovery of metagenome-assembled genomes from metagenomic data.

The rise of metagenomics offers a leap forward for understanding the genetic diversity of microorganisms in many different complex environments by providing a platform that can identify potentially unlimited numbers of known and novel microorganisms. As such, it is impossible to imagine new major initiatives without metagenomics. Nevertheless, it represents a relatively new discipline with various levels of complexity and demands on bioinformatics. The underlying principles and methods used in metagenomics are often seen as common knowledge and often not detailed or fragmented. Therefore, we reviewed these to guide microbiologists in taking the first steps into metagenomics. We specifically focus on a workflow aimed at reconstructing individual genomes, that is, metagenome-assembled genomes, integrating DNA sequencing, assembly, binning, identification and annotation.

Host Immunity Influences the Composition of Murine Gut Microbiota.

The influence of gut microbiota on host immunity is widely studied, and its disturbance has been linked to several immune-mediated disorders. Conversely, whether and how inherently disturbed canonical Th1 (pro-inflammatory) and/or Th2 (anti-inflammatory) immune pathways modify the host microbiome is not sufficiently investigated. Here, we characterized the humoral, cellular, and cytokine immunity, and associated alterations in gut microbiota of naïve wild-type mice (C57BL/6 and BALB/c), and mice with deficiencies in Th2 responses (IL-4Rα and IL-33 knockout mice) or in both Th1 and Th2 responses (NOD scid gamma, NSG mice). A global analysis by de novo clustering of 16S rRNA profiles of the gut microbiota independently grouped wild-type immunocompetent (C57BL/6 and BALB/c), Th2-deficient (IL-4Rα-/- and IL-33-/-), and severely immunodeficient (NSG) mice; where wild-type mice, but not Th2 or severely immunodeficient mice, were enriched in gut bacteria that produce short-chain fatty acids. These include members of phyla Firmicutes, Verrucomicrobia, and Bacteroidetes such as Lactobacillus spp., Akkermansia muciniphila, and Odoribacter spp. Further comparison of the two naïve wild-type mouse strains showed higher microbial diversity (Shannon), primarily linked to higher richness (Chao1), as well as a distinct difference in microbial composition (weighted UniFrac) in BALB/c mice compared to C57BL/6. T-cell and blood cytokine analyses demonstrated a Th1-polarization in naïve adaptive immunity in C57BL/6 animals compared to BALB/c mice, and an expected Th2 deficient cellular response in IL-4Rα-/- and IL-33-/- mice compared to its genetic background BALB/c strain. Together, these data suggest that alterations in the Th1/Th2 balance or a complete ablation of Th1/Th2 responses can lead to major alterations in gut microbiota composition and function. Given the similarities between the human and mouse immune systems and gut microbiota, our finding that immune status is a strong driver of gut microbiota composition has important consequences for human immunodeficiency studies.

Accurate prediction of metagenome-assembled genome completeness by MAGISTA, a random forest model built on alignment-free intra-bin statistics.

BACKGROUND: Although the total number of microbial taxa on Earth is under debate, it is clear that only a small fraction of these has been cultivated and validly named. Evidently, the inability to culture most bacteria outside of very specific conditions severely limits their characterization and further studies. In the last decade, a major part of the solution to this problem has been the use of metagenome sequencing, whereby the DNA of an entire microbial community is sequenced, followed by the in silico reconstruction of genomes of its novel component species. The large discrepancy between the number of sequenced type strain genomes (around 12,000) and total microbial diversity (106-1012 species) directs these efforts to de novo assembly and binning. Unfortunately, these steps are error-prone and as such, the results have to be intensely scrutinized to avoid publishing incomplete and low-quality genomes. RESULTS: We developed MAGISTA (metagenome-assembled genome intra-bin statistics assessment), a novel approach to assess metagenome-assembled genome quality that tackles some of the often-neglected drawbacks of current reference gene-based methods. MAGISTA is based on alignment-free distance distributions between contig fragments within metagenomic bins, rather than a set of reference genes. For proper training, a highly complex genomic DNA mock community was needed and constructed by pooling genomic DNA of 227 bacterial strains, specifically selected to obtain a wide variety representing the major phylogenetic lineages of cultivable bacteria. CONCLUSIONS: MAGISTA achieved a 20% reduction in root-mean-square error in comparison to the marker gene approach when tested on publicly available mock metagenomes. Furthermore, our highly complex genomic DNA mock community is a very valuable tool for benchmarking (new) metagenome analysis methods.

Cyclical Patterns Affect Microbial Dynamics in the Water Basin of a Nuclear Research Reactor.

The BR2 nuclear research reactor in Mol, Belgium, runs in successive phases of operation (cycles) and shutdown, whereby a water basin surrounding the reactor vessel undergoes periodic changes in physico-chemical parameters such as flow rate, temperature, and radiation. The aim of this study was to explore the microbial community in this unique environment and to investigate its long-term dynamics using a 16S rRNA amplicon sequencing approach. Results from two sampling campaigns spanning several months showed a clear shift in community profiles: cycles were mostly dominated by two Operational Taxonomic Units (OTUs) assigned to unclassified Gammaproteobacterium and Pelomonas, whereas shutdowns were dominated by an OTU assigned to Methylobacterium. Although 1 year apart, both campaigns showed similar results, indicating that the system remained stable over this 2-year period. The community shifts were linked with changes in physico-chemical parameters by Non-metric Multidimensional Scaling (NMDS) and correlation analyses. In addition, radiation was hypothesized to cause a decrease in cell number, whereas temperature had the opposite effect. Chemoautotrophic use of H2 and dead cell recycling are proposed to be used as a strategies for nutrient retrieval in this extremely oligotrophic environment.

First Description of Various Bacteria Resistant to Heavy Metals and Antibiotics Isolated from Polluted Sites in Tunisia.

Environmental bacteria belonging to various families were isolated from polluted water collected from ten different sites in Tunisia. Sites were chosen near industrial and urban areas known for their high degree of pollution. The aim of this study was to investigate cross-resistance between heavy metals (HM), i.e., silver, mercury and copper (Ag, Hg, and Cu), and antibiotics. In an initial screening, 80 isolates were selected on ampicillin, and 39 isolates, retained for further analysis, could grow on a Tris-buffered mineral medium with gluconate as carbon source. Isolates were identified based on their 16S rRNA gene sequence. Results showed the prevalence of antibiotic resistance genes, especially all isolates harbored the bla TEM gene. Some of them (15.38%) harbored bla SHV. Moreover, several were even ESBLs and MBLs-producers, which can threaten the human health. On the other hand, 92.30%, 56.41%, and 51.28% of the isolates harbored the heavy metals resistance genes silE, cusA, and merA, respectively. These genes confer resistance to silver, copper, and mercury. A cross-resistance between antibiotics and heavy metals was detected in 97.43% of our isolates.

Irradiation at Ultra-High (FLASH) Dose Rates Reduces Acute Normal Tissue Toxicity in the Mouse Gastrointestinal System.

PURPOSE: Preclinical studies using ultra-high dose rate (FLASH) irradiation have demonstrated reduced normal tissue toxicity compared with conventional dose rate (CONV) irradiation, although this finding is not universal. We investigated the effect of temporal pulse structure and average dose rate of FLASH compared with CONV irradiation on acute intestinal toxicity. MATERIALS AND METHODS: Whole abdomens of C3H mice were irradiated with a single fraction to various doses, using a 6 MeV electron linear accelerator with single pulse FLASH (dose rate = 2-6 × 106 Gy/s) or conventional (CONV; 0.25 Gy/s) irradiation. At 3.75 days postirradiation, fresh feces were collected for 16S rRNA sequencing to assess changes in the gut microbiota. A Swiss roll-based crypt assay was used to quantify acute damage to the intestinal crypts to determine how tissue toxicity was affected by the different temporal pulse structures of FLASH delivery. RESULTS: We found statistically significant improvements in crypt survival for mice irradiated with FLASH at doses between 7.5 and 12.5 Gy, with a dose modifying factor of 1.1 for FLASH (7.5 Gy, P < .01; 10 Gy, P < .05; 12.5 Gy, P < .01). This sparing effect was lost when the delivery time was increased, either by increasing the number of irradiation pulses or by prolonging the time between 2 successive pulses. Sparing was observed for average dose rates of ≥280 Gy/s. Fecal microbiome analysis showed that FLASH irradiation caused fewer changes to the microbiota than CONV irradiation. CONCLUSIONS: This study demonstrates that FLASH irradiation can spare mouse small intestinal crypts and reduce changes in gut microbiome composition compared with CONV irradiation. The higher the average dose rate, the larger the FLASH effect, which is also influenced by temporal pulse structure of the delivery.

Assessing the Impact of Flavophospholipol and Virginiamycin Supplementation on the Broiler Microbiota: a Prospective Controlled Intervention Study.

The antibiotic growth promoters (AGPs) flavophospholipol and virginiamycin have been widely used for decades in food animal production. AGP activity is believed to be partly modulated by gut microbial composition although exact AGP-induced changes remain unclear. In a controlled intervention study, we studied the effect of flavophospholipol and virginiamycin on the broiler chicken ileal microbiota spanning from birth to 39 days. Using 16S rRNA gene profiling and prediction of metabolic activity, we show that both AGPs result in dynamic microbial shifts that potentially increase anti-inflammatory mechanisms and bioavailability of several essential nutrients by decreasing degradation (flavophospholipol) or increasing biosynthesis (virginiamycin). Further, virginiamycin-supplemented broilers showed increased colonization with potentially pathogenic bacteria, Clostridium perfringens, Campylobacter, and Escherichia/Shigella spp. Overall, we show that both AGPs induce microbial changes potentially beneficial for growth. However, the increase in (foodborne) pathogens shown here with virginiamycin use could impact not only broiler mortality but also human health. IMPORTANCE Antibiotic growth promoters (AGPs) are commonly used within poultry farming to increase muscle growth. Microbial composition in the gut is known to be influenced by AGP use although exact AGP-induced changes remain unclear. Utilizing 16S rRNA gene profiling, this study provides a first head-to-head comparison of the effect of the two most commonly used AGPs, flavophospholipol and virginiamycin, on the broiler chicken ileum microbiota over time. We found that supplementation with both AGPs altered ileal microbial composition, thereby increasing potential bioavailability of essential nutrients and weight gain. Flavophospholipol showed a slight benefit over virginiamycin as the latter resulted in more extensive microbial perturbations including increased colonization by enteropathogens, which could impact broiler mortality.

Genetic Responses of Metabolically Active Limnospira indica Strain PCC 8005 Exposed to γ-Radiation during Its Lifecycle.

Two morphotypes of the cyanobacterial Limnospira indica (formerly Arthrospira sp.) strain PCC 8005, denoted as P2 (straight trichomes) and P6 (helical trichomes), were subjected to chronic gamma radiation from spent nuclear fuel (SNF) rods at a dose rate of ca. 80 Gy·h-1 for one mass doubling period (approximately 3 days) under continuous light with photoautotrophic metabolism fully active. Samples were taken for post-irradiation growth recovery and RNA-Seq transcriptional analysis at time intervals of 15, 40, and 71.5 h corresponding to cumulative doses of ca. 1450, 3200, and 5700 Gy, respectively. Both morphotypes, which were previously reported by us to display different antioxidant capacities and differ at the genomic level in 168 SNPs, 48 indels and 4 large insertions, recovered equally well from 1450 and 3200 Gy. However, while the P2 straight type recovered from 5700 Gy by regaining normal growth within 6 days, the P6 helical type took about 13 days to recover from this dose, indicating differences in their radiation tolerance and response. To investigate these differences, P2 and P6 cells exposed to the intermediate dose of gamma radiation (3200 Gy) were analyzed for differential gene expression by RNA-Seq analysis. Prior to batch normalization, a total of 1553 genes (887 and 666 of P2 and P6, respectively, with 352 genes in common) were selected based on a two-fold change in expression and a false discovery rate FDR smaller or equal to 0.05. About 85% of these 1553 genes encoded products of yet unknown function. Of the 229 remaining genes, 171 had a defined function while 58 genes were transcribed into non-coding RNA including 21 tRNAs (all downregulated). Batch normalization resulted in 660 differentially expressed genes with 98 having a function and 32 encoding RNA. From PCC 8005-P2 and PCC 8005-P6 expression patterns, it emerges that although the cellular routes used by the two substrains to cope with ionizing radiation do overlap to a large extent, both strains displayed a distinct preference of priorities.