High Genomic Stability Research Articles

Characterization of a natural 'dead-end' variant of Schmallenberg virus.

Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family Peribunyaviridae, genus Orthobunyavirus and is transmitted by Culicoides biting midges. Infection of naïve ruminants in a critical phase of gestation may lead to severe congenital malformations. Sequence analysis from viremic animals revealed a very high genome stability. In contrast, sequence variations are frequently described for SBV from malformed fetuses. In addition to S segment mutations, especially within the M segment encoding the major immunogen Gc, point mutations or genomic deletions are also observed. Analysis of the SBV_D281/12 isolate from a malformed fetus revealed multiple point mutations in all three genome segments. It also has a large genomic deletion in the antigenic domain encoded by the M segment compared to the original SBV reference strain 'BH80/11' isolated from viremic blood in 2011. Interestingly, SBV_D281/12 showed a marked replication deficiency in vitro in Culicoides sonorensis cells (KC cells), but not in standard baby hamster kidney cells (BHK-21). We therefore generated a set of chimeric viruses of rSBV_D281/12 and wild-type rSBV_BH80/11 by reverse genetics, which were characterized in both KC and BHK-21 cells. It could be shown that the S segment of SBV_D281/12 is responsible for the replication deficit and that it acts independently from the large deletion within Gc. In addition, a single point mutation at position 111 (S to N) of the nucleoprotein was identified as the critical mutation. Our results suggest that virus variants found in malformed fetuses and carrying characteristic genomic mutations may have a clear 'loss of fitness' for their insect hosts in vitro. It can also be concluded that such mutations lead to virus variants that are no longer part of the natural transmission cycle between mammalian and insect hosts. Interestingly, analysis of a series of SBV sequences confirmed the S111N mutation exclusively in samples of malformed fetuses and not in blood from viremic animals. The characterization of these changes will allow the definition of protein functions that are critical for only one group of hosts.

Molecular subtyping of skin cutaneous melanoma based on inflammatory response

The inflammatory response plays a crucial role in determining the prognosis and therapeutic response of skin cutaneous melanoma (SKCM). However, the molecular subtypes based on the inflammatory response and their clinical significance in SKCM have not been extensively studied. Clustering analyses to identify inflammation subtypes of SKCM based on the expression levels of inflammation response gene. We identified three subtypes: Inflammation_H, Inflammation_M, and Inflammation_L, which offer a more nuanced understanding of the complex relationship between inflammation and SKCM. The Inflammation_H subtype is associated with the most favourable prognosis, and is characterised by high levels of immune infiltrates and PD-L1 expression, low levels of stemness, high differentiation, and high genomic stability. In contrast, the Inflammation_L subtype has the least favourable prognosis, with the lowest levels of immune infiltrates and PD-L1 expression, high levels of stemness, low differentiation, and low genomic stability. In addition, the Inf-score, which is a linear risk scoring model based on the expression levels of inflammatory response genes, can be a useful tool for clinicians to assess SKCM prognosis and guide therapeutic decisions. This scoring model shows promise for clinical use in predicting patient outcomes and helps clinicians tailor treatments for individual patients.In conclusion, these findings represent a significant contribution to our understanding of the molecular subtypes of SKCM based on the levels of inflammatory response genes and their potential clinical significance. However, further studies are necessary to validate these findings and explore the underlying mechanisms of different subtypes.

Genomic diversity and clinical relevance of Mycobacterium simiae.

Mycobacterium simiae is a slow-growing non-tuberculous mycobacterium that can cause non-tuberculous mycobacterium (NTM) pulmonary disease and extrapulmonary infections. Until now, detailed genomic and clinical characteristics, as well as possible transmission routes of this rare pathogen remain largely unknown. We conducted whole genome sequencing of available M. simiae isolates collected at a tertiary care centre in Central Germany from 2006 to 2020 and set them into context with publicly available M. simiae complex sequences through phylogenetic analysis. Resistance, virulence and stress genes, as well as known Mycobacteriaceae plasmid sequences were detected in whole genome raw reads. Clinical data and course were retrieved and correlated with genomic data. We included 33 M. simiae sensu stricto isolates from seven patients. M. simiae showed low clinical relevance with only two patients fulfilling American Thoracic Society (ATS) criteria in our cohort and three receiving NTM-effective therapy. The bacterial populations were highly stable over time periods of up to 14 years, and no instances of mixed or re-infections with other strains of M. simiae were observed. Clustering with <12 single nucleotide polymorphisms distance was evident among isolates from different patients; however, proof for human-to-human transmission could not be established from epidemiological data. Overall, the available sequence data for M. simiae complex was significantly extended and new insights into its pathogenomic traits were obtained. We demonstrate high longitudinal genomic stability within single patients. Although we cannot exclude human-to-human transmission, we consider it unlikely in the light of available epidemiological data.

Induced pluripotent stem cells reprogramming overcomes technical limitations for highly pigmented adult melanocyte amplification and integration in 3D skin model.

Understanding pigmentation regulations taking into account the original skin color type is important to address pigmentary disorders. Biological models including adult melanocytes from different phenotypes allow to perform fine-tuned explorative studies and support discovery of treatments adapted to populations' skin color. However, technical challenges arise when trying to not only isolate but also amplify melanocytes from highly pigmented adult skin. To bypass the initial isolation and growth of cutaneous melanocytes, we harvested and expanded fibroblasts from light and dark skin donors and reprogrammed them into iPSC, which were then differentiated into melanocytes. The resulting melanocyte populations displayed high purity, genomic stability, and strong proliferative capacity, the latter being a critical parameter for dark skin cells. The iPSC-derived melanocyte strains expressed lineage-specific markers and could be successfully integrated into reconstructed skin equivalent models, revealing pigmentation status according to the native phenotype. In both monolayer cultures and 3D skin models, the induced melanocytes demonstrated responsiveness to promelanogenic stimuli. The data demonstrate that the iPSC-derived melanocytes with high proliferative capacity maintain their pigmentation genotype and phenotypic properties up to a proper integration into 3D skin equivalents, even for highly pigmented cells.

Rates of Mutations and Transcript Errors in the Foodborne Pathogen Salmonella enterica subsp. enterica.

Because errors at the DNA level power pathogen evolution, a systematic understanding of the rate and molecular spectra of mutations could guide the avoidance and treatment of infectious diseases. We thus accumulated tens of thousands of spontaneous mutations in 768 repeatedly bottlenecked lineages of 18 strains from various geographical sites, temporal spread, and genetic backgrounds. Entailing over ∼1.36 million generations, the resultant data yield an average mutation rate of ∼0.0005 per genome per generation, with a significant within-species variation. This is one of the lowest bacterial mutation rates reported, giving direct support for a high genome stability in this pathogen resulting from high DNA-mismatch-repair efficiency and replication-machinery fidelity. Pathogenicity genes do not exhibit an accelerated mutation rate, and thus, elevated mutation rates may not be the major determinant for the diversification of toxin and secretion systems. Intriguingly, a low error rate at the transcript level is not observed, suggesting distinct fidelity of the replication and transcription machinery. This study urges more attention on the most basic evolutionary processes of even the best-known human pathogens and deepens the understanding of their genome evolution.

Comparative pangenome analysis of capsulated Haemophilus influenzae serotype f highlights their high genomic stability

Haemophilus influenzae is an opportunistic pathogen adapted to the human respiratory tract. Non-typeable H. influenzae are highly heterogeneous, but few studies have analysed the genomic variability of capsulated strains. This study aims to examine the genetic diversity of 37 serotype f isolates from the Netherlands, Portugal, and Spain, and to compare all capsulated genomes available on public databases. Serotype f isolates belonged to CC124 and shared few single nucleotide polymorphisms (SNPs) (n = 10,999), but a high core genome (> 80%). Three main clades were identified by the presence of 75, 60 and 41 exclusive genes for each clade, respectively. Multi-locus sequence type analysis of all capsulated genomes revealed a reduced number of clonal complexes associated with each serotype. Pangenome analysis showed a large pool of genes (n = 6360), many of which were accessory genome (n = 5323). Phylogenetic analysis revealed that serotypes a, b, and f had greater diversity. The total number of SNPs in serotype f was significantly lower than in serotypes a, b, and e (p < 0.0001), indicating low variability within the serotype f clonal complexes. Capsulated H. influenzae are genetically homogeneous, with few lineages in each serotype. Serotype f has high genetic stability regardless of time and country of isolation.

One Cell, Two Gears: Extensive Somatic Genome Plasticity Accompanies High Germline Genome Stability in Paramecium.

Mutation accumulation (MA) experiments are conventionally employed to study spontaneous germline mutations. However, MA experiments can also shed light on somatic genome plasticity in a habitual and genetic drift-maximizing environment. Here, we revisit an MA experiment that uncovered extraordinary germline genome stability in Paramecium tetraurelia, a single-celled eukaryote with nuclear dimorphism. Our re-examination of isogenic P. tetraurelia MA lines propagated in nutrient-rich medium for >40 sexual cycles reveals that their polyploid somatic genome accrued hundreds of intervening DNA segments (IESs), which are normally eliminated during germline-soma differentiation. These IESs frequently occupy a fraction of the somatic DNA copies of a given locus, producing IES excision/retention polymorphisms, and preferentially fall into a class of epigenetically controlled sequences. Relative to control lines, retained IESs are flanked by stronger cis-acting signals and interrupt an excess of highly expressed coding exons. These findings suggest that P. tetraurelia’s elevated germline DNA replication fidelity is associated with pervasive somatic genome plasticity. They show that MA regimes are powerful tools for investigating the role that developmental plasticity, somatic mutations, and epimutations have in ecology and evolution.

Diverse \u2018just-right\u2019 levels of chromosomal instability and their clinical implications in neoadjuvant treated gastric cancer

BackgroundThe Cancer Genome Atlas (TCGA) consortium described EBV positivity(+), high microsatellite instability (MSI-H), genomic stability (GS) and chromosomal instability (CIN) as molecular subtypes in gastric carcinomas (GC). We investigated the predictive and prognostic value of these subtypes with emphasis on CIN in the context of neoadjuvant chemotherapy (CTx) in GC.MethodsTCGA subgroups were determined for 612 resected adenocarcinomas of the stomach and gastro-oesophageal junction (291 without, 321 with CTx) and 143 biopsies before CTx.EBV and MSI-H were analysed by standard assays. CIN was detected by multiplex PCRs analysing 22 microsatellite markers. Besides the TCGA classification, CIN was divided into four CIN-subgroups: low, moderate, substantial, high. Mutation profiling was performed for 52 tumours by next-generation sequencing.ResultsEBV(+) (HR, 0.48; 95% CI, 0.23–1.02), MSI-H (HR, 0.56; 95% CI, 0.35–0.89) and GS (HR, 0.72; 95% CI, 0.45–1.13) were associated with increased survival compared to CIN in the resected tumours. Considering the extended CIN-classification, CIN-substantial was a negative prognostic factor in uni- and multivariable analysis in resected tumours with CTx (each p < 0.05). In biopsies before CTx, CIN-high predicted tumour regression (p = 0.026), but was not prognostically relevant.ConclusionA refined CIN classification reveals tumours with different biological characteristics and potential clinical implications.

Multi-platform profiling characterizes molecular subgroups and resistance networks in chronic lymphocytic leukemia

Knowledge of the genomic landscape of chronic lymphocytic leukemia (CLL) grows increasingly detailed, providing challenges in contextualizing the accumulated information. To define the underlying networks, we here perform a multi-platform molecular characterization. We identify major subgroups characterized by genomic instability (GI) or activation of epithelial-mesenchymal-transition (EMT)-like programs, which subdivide into non-inflammatory and inflammatory subtypes. GI CLL exhibit disruption of genome integrity, DNA-damage response and are associated with mutagenesis mediated through activation-induced cytidine deaminase or defective mismatch repair. TP53 wild-type and mutated/deleted cases constitute a transcriptionally uniform entity in GI CLL and show similarly poor progression-free survival at relapse. EMT-like CLL exhibit high genomic stability, reduced benefit from the addition of rituximab and EMT-like differentiation is inhibited by induction of DNA damage. This work extends the perspective on CLL biology and risk categories in TP53 wild-type CLL. Furthermore, molecular targets identified within each subgroup provide opportunities for new treatment approaches.

Improved strategy for jet-in-air cell sorting with high purity, yield, viability, and genome stability.

Flow cytometric sorting is a vital tool in biological research and clinical diagnostics. Theoretically, a high‐speed jet‐in‐air sorter is a fluorescent‐activated cell sorting sorter that ideally processes cells with high purity, yield, and viability. However, high‐speed jet‐in‐air sorting is a complex process due to its inherent requirements for high fluidic stability and electronic and timing precision. Here, we report that an additional manual correction of drop delay leads to improved cell yield. Adding 2% FBS to the loading buffer had no significant effect on the fate of sorted cells in 4 h. However, the addition of a suitable concentration of FBS/BSA in the collecting buffer resulted in a notable increase in cell count and proliferation and a significant decrease in cell apoptosis for cell lines and primary cells. Moreover, the level of gene expression remained steady in the 5% FBS collecting buffer. In summary, here we demonstrate techniques that can be easily followed to refine sorted yields of healthy cells.

Unexpected Discovery of Hypermutator Phenotype Sounds the Alarm for Quality Control Strains.

Microbial strains with high genomic stability are particularly sought after for testing the quality of commercial microbiological products, such as biological media and antibiotics. Yet, using mutation–accumulation experiments and de novo assembled complete genomes based on Nanopore long-read sequencing, we find that the widely used quality-control strain Shewanella putrefaciens ATCC-8071, also a facultative pathogen, is a hypermutator, with a base-pair substitution mutation rate of 2.42 × 10−8 per nucleotide site per cell division, ∼146-fold greater than that of the wild-type strain CGMCC-1.6515. Using complementation experiments, we confirm that mutL dysfunction, which was a recent evolutionary event, is the cause for the high mutation rate of ATCC-8071. Further analyses also give insight into possible relationships between mutation and genome evolution in this important bacterium. This discovery of a well-known strain being a hypermutator necessitates screening the mutation rate of bacterial strains before any quality control or experiments.

Lemna minor, a hyperaccumulator shows elevated levels of Cd accumulation and genomic template stability in binary application of Cd and Ni: a physiological and genetic approach

In this study, to determine whether having potential to be used as hyperaccumulator for Cd and Ni, numerous experiments were designed for conducting assessments for physiological and genotoxic changes along with defining possible alterations on mineral nutrient status of Lemna minor L. by applying Cd-Ni binary treatments (0, 100, 200 and 400 µM). Our study revealed that there were increases in the concentrations of B, Cr, Fe, K, Mg, and Mn whereas decreases were noticed in the concentrations of Na and Zn and the levels of Ca were inversely proportional to Cd-Ni applications showing tendency to increase at the low concentration and to decrease at the high concentration. Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) analyses revealed that rather than band losses and new band formations, mostly intensity changes in the band profiles, and low polymorphism and high genomic template stability (GTS) were observed. Although, to date, L. minor was defined as an efficient hyperaccumulator/potential accumulator or competent phytoremedial agent by researchers. Our research revealed that L. minor showing high accumulation capability for Cd and having low polymorphism rate and high genomic template stability is a versatile hyperaccumulator, especially for Cd; therefore, highly recommended by us for decontamination of water polluted with Cd. NOVELTY STATEMENT Many studies have been focused on the effects of individual metal ions. However, heavy metal contaminants usually exist as their mixtures in natural aquatic environments. Especially, Cd and Ni coexist in industrial wastes. In this study, the accumulation properties of Lemna minor for both Cd and Ni were investigated and the effects of Cd and Ni on the bioaccumulation of B, Ca, Cu, Fe, Mg, K, Mn, Na, Pb and Zn in L. minor were also determined. This study furthermore aimed to assess the genotoxic effects of Cd and Ni found in being extended concentrations on DNA using the Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) method.

Adaptive response to iterative passages of five Lactobacillus species in simulated vaginal fluid

BackgroundMicrobiome and metagenomic studies have given rise to a new understanding of microbial colonization of various human tissues and their ability to impact our health. One human microbiome growing in notoriety, the vaginal microbiome, stands out given its importance for women’s health, and is peculiar in terms of its relative bacterial composition, including its simplicity and typical domination by a small number of Lactobacillus species. The loss of Lactobacillus dominance is associated with disorders such as bacterial vaginosis, and efforts are now underway to understand the ability of Lactobacillus species to colonize the vaginal tract and adapt to this dynamic and acidic environment. Here, we investigate how various Lactobacillus species often isolated from the vaginal and intestinal cavities genomically and transcriptionally respond to iterative growth in simulated vaginal fluid.ResultsWe determined the genomes and transcriptomes of L. acidophilus, L. crispatus, L. fermentum, L. gasseri, and L. jensenii and compared profiles after 50, 100, 500, and 1000 generations of iterative passages in synthetic vaginal fluid. In general, we identified relatively few genetic changes consisting of single nucleotide polymorphisms, with higher counts occurring more frequently in non-vaginal isolated species. Transcriptional profiles were more impacted over time and tended to be more extensive for species that typically do not dominate the vaginal tract, reflecting a more extensive need to adapt to a less familiar environment.ConclusionsThis study provides insights into how vaginal and non-vaginal Lactobacillus species respond and adapt to a simulated vaginal environment. Overall, trends indicate high genomic stability for all species involved, with more variability in the transcriptome especially for non-dominant species of the vaginal tract.

Functional Oocytes Derived from Granulosa Cells

The generation of genomically stable and functional oocytes has great potential for preserving fertility and restoring ovarian function. It remains elusive whether functional oocytes can be generated from adult female somatic cells through reprogramming to germline-competent pluripotent stem cells (gPSCs) by chemical treatment alone. Here, we show that somatic granulosa cells isolated from adult mouse ovaries can be robustly induced to generate gPSCs by a purely chemical approach, with additional Rock inhibition and critical reprogramming facilitated by crotonic sodium or acid. These gPSCs acquired high germline competency and could consistently be directed to differentiate into primordial-germ-cell-like cells and form functional oocytes that produce fertile mice. Moreover, gPSCs promoted by crotonylation and the derived germ cells exhibited longer telomeres and high genomic stability like PGCs invivo,providing additional evidence supporting the safety and effectiveness of chemical induction, which is particularly important for germ cells in genetic inheritance.

Codon Usage Bias Analysis of Citrus tristeza Virus: Higher Codon Adaptation to Citrus reticulata Host.

Citrus tristeza virus (CTV), a member of the aphid-transmitted closterovirus group, is the causal agent of the notorious tristeza disease in several citrus species worldwide. The codon usage patterns of viruses reflect the evolutionary changes for optimization of their survival and adaptation in their fitness to the external environment and the hosts. The codon usage adaptation of CTV to specific citrus hosts remains to be studied; thus, its role in CTV evolution is not clearly comprehended. Therefore, to better explain the host–virus interaction and evolutionary history of CTV, the codon usage patterns of the coat protein (CP) genes of 122 CTV isolates originating from three economically important citrus hosts (55 isolate from Citrus sinensis, 38 from C. reticulata, and 29 from C. aurantifolia) were studied using several codon usage indices and multivariate statistical methods. The present study shows that CTV displays low codon usage bias (CUB) and higher genomic stability. Neutrality plot and relative synonymous codon usage analyses revealed that the overall influence of natural selection was more profound than that of mutation pressure in shaping the CUB of CTV. The contribution of high-frequency codon analysis and codon adaptation index value show that CTV has host-specific codon usage patterns, resulting in higheradaptability of CTV isolates originating from C. reticulata (Cr-CTV), and low adaptability in the isolates originating from C. aurantifolia (Ca-CTV) and C. sinensis (Cs-CTV). The combination of codon analysis of CTV with citrus genealogy suggests that CTV evolved in C. reticulata or other Citrus progenitors. The outcome of the study enhances the understanding of the factors involved in viral adaptation, evolution, and fitness toward their hosts. This information will definitely help devise better management strategies of CTV.

Abstract P3-01-09: Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient

Abstract Background: Copy number variation analysis has been shown as a valuable tool to infer clonal evolution of tumor cells. While bulk genomic analysis of FFPE tissue can provide information about the characteristics of a primary tumor or a metastatic nodule, these biopsies are rarely repeated due to their invasive nature. Liquid biopsies on the other hand, provide minimally invasive access to tumor derived cells and DNA and can therefore be used to monitor genomic changes of the tumor longitudinally and in real time. Here we evaluate the kinetics of liquid biopsy biomarkers and monitor genomic changes in single circulating tumor cells (CTCs) and cell-free DNA (cfDNA) from 17 longitudinal blood draws collected from a metastatic breast cancer patient over seven treatment regimes. FFPE tissue from the primary tumor and two metastatic sites in liver and bone are available for comparative analysis. Methods: We used the high definition – single cell assay (HD-SCA) platform to detect and enumerate CTCs of the blood of one metastatic breast cancer patient followed for 4 years. CTCs were characterized morphometrically and single CTCs of each draw were sequenced for copy number variation (CNV). cfDNA was extracted from plasma and tissue samples of the primary breast, bone and liver metastasis were micro-dissected. Extracted DNA from tissue samples and plasma underwent CNV analysis and were compared with CNV data of CTCs. Results: CTCs were identified in 14/17 draws and varied from 0 to &amp;gt;3000 cells/ml, spiking at two different times concordant with clinical progression. Changes in cell free tumor DNA fraction (ctDNA) correlated strongly with CTC count. Comparative CNV analysis of CTCs, cfDNA and FFPE tissue confirmed the tumorous origin of both cells and cfDNA. Single cell CNV analysis found four genomically related sub-clones in the patient's blood, three of which were present at time of enrollment, while the fourth clone emerged 3.5 years later concordant with the decline in the patient's health. Genomic copy number losses included tumor suppressor genes such as ARID1A, FOXP1, ATM, BRCA2, RB1, CDH11 and CDH1. Genomic gains were limited but included the proto-oncogenes BCL9, ABL2 and MDM4. It is striking that the sub-clonal structure persisted nearly unchanged for 3.5 years throughout various hormone and cytotoxic treatments, indicating high genomic stability. Despite their genomic differences, cells from the different clones are indistinguishable by immunofluorescence or morphometric analysis. CNV profiles of cfDNA reflect the most abundant CTC clone at each time-point. Conclusion: Using the HD-SCA assay cellular (CTC), and soluble (cfDNA) fractions of liquid biopsies can be compared at the molecular level over a long time course, and verified by comparison with tissue biopsy. This work confirms that liquid biopsies can be used to access the molecular state of a patient's cancer in near real-time and provide insight into the response to treatment years after the original characterization of the primary tissue. Here we show in a single patient example the power of single CTC analysis and highlight how CNV analysis of liquid biopsies could serve as minimally invasive tool to monitor tumor evolution longitudinally. Citation Format: Welter L, Xu L, McKinley D, Dago AE, Restrepo-Vassalli S, Rodriguez Lee M, Kolatkar A, Nieva J, Hicks J, Kuhn P. Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-09.

Comparative genome analysis reveals key genetic factors associated with probiotic property in Enterococcus faecium strains

BackgroundEnterococcus faecium though commensal in the human gut, few strains provide a beneficial effect to humans as probiotics while few are responsible for the nosocomial infection. Comparative genomics of E. faecium can decipher the genomic differences responsible for probiotic, pathogenic and non-pathogenic properties. In this study, we compared E. faecium strain 17OM39 with a marketed probiotic, non-pathogenic non-probiotic (NPNP) and pathogenic strains.ResultsE. faecium 17OM39 was found to be closely related with marketed probiotic strain T110 based on core genome analysis. Strain 17OM39 was devoid of known vancomycin, tetracycline resistance and functional virulence genes. Moreover, E. faecium 17OM39 genome was found to be more stable due to the absence of frequently found transposable elements. Genes imparting beneficial functional properties were observed to be present in marketed probiotic T110 and 17OM39 strains. Genes associated with colonization and survival within gastrointestinal tract was also detected across all the strains.ConclusionsBeyond shared genetic features; this study particularly identified genes that are responsible for imparting probiotic, non-pathogenic and pathogenic features to the strains of E. faecium. Higher genomic stability, absence of known virulence factors and antibiotic resistance genes and close genomic relatedness with marketed probiotics makes E. faecium 17OM39 a potential probiotic candidate. The work presented here demonstrates that comparative genome analyses can be applied to large numbers of genomes, to find potential probiotic candidates.

Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication.

The mismatch repair (MMR) system, exemplified by the MutS/MutL proteins, is widespread in Bacteria and Eukarya. However, molecular mechanisms how numerous archaea and bacteria lacking the mutS/mutL genes maintain high replication fidelity and genome stability have remained elusive. EndoMS is a recently discovered hyperthermophilic mismatch-specific endonuclease encoded by nucS in Thermococcales. We deleted the nucS from the actinobacterium Corynebacterium glutamicum and demonstrated a drastic increase of spontaneous transition mutations in the nucS deletion strain. The observed spectra of these mutations were consistent with the enzymatic properties of EndoMS in vitro. The robust mismatch-specific endonuclease activity was detected with the purified C. glutamicum EndoMS protein but only in the presence of the β-clamp (DnaN). Our biochemical and genetic data suggest that the frequently occurring G/T mismatch is efficiently repaired by the bacterial EndoMS-β−clamp complex formed via a carboxy-terminal sequence motif of EndoMS proteins. Our study thus has great implications for understanding how the activity of the novel MMR system is coordinated with the replisome and provides new mechanistic insight into genetic diversity and mutational patterns in industrially and clinically (e.g. Mycobacteria) important archaeal and bacterial phyla previously thought to be devoid of the MMR system.

Sulfolobus acidocaldarius Transports Pentoses via a Carbohydrate Uptake Transporter 2 (CUT2)-Type ABC Transporter and Metabolizes Them through the Aldolase-Independent Weimberg Pathway.

Sulfolobus spp. possess a great metabolic versatility and grow heterotrophically on various carbon sources, such as different sugars and peptides. Known sugar transporters in Archaea predominantly belong to ABC transport systems. Although several ABC transporters for sugar uptake have been characterized in the crenarchaeon Sulfolobus solfataricus, only one homologue of these transporters, the maltose/maltooligomer transporter, could be identified in the closely related Sulfolobus acidocaldarius Comparison of the transcriptome of S. acidocaldarius MW001 grown on peptides alone and peptides in the presence of d-xylose allowed for the identification of the ABC transporter for d-xylose and l-arabinose transport and the gaining of deeper insights into pentose catabolism under the respective growth conditions. The d-xylose/l-arabinose substrate binding protein (SBP) (Saci_2122) of the ABC transporter is unique in Archaea and shares more similarity to bacterial SBPs of the carbohydrate uptake transporter-2 (CUT2) family than to any characterized archaeal one. The identified pentose transporter is the first CUT2 family ABC transporter analyzed in the domain of Archaea Single-gene deletion mutants of the ABC transporter subunits exemplified the importance of the transport system for d-xylose and l-arabinose uptake. Next to the transporter operon, enzymes of the aldolase-independent pentose catabolism branch were found to be upregulated in N-Z-Amine and d-xylose medium. The α-ketoglutarate semialdehyde dehydrogenase (KGSADH; Saci_1938) seemed not to be essential for growth on pentoses. However, the deletion mutant of the 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD [also known as KDAD]; Saci_1939) was no longer able to catabolize d-xylose or l-arabinose, suggesting the absence of the aldolase-dependent branch in S. acidocaldariusIMPORTANCE Thermoacidophilic microorganisms are emerging model organisms for biotechnological applications, as their optimal growth conditions resemble conditions used in certain biotechnologies such as industrial plant waste degradation. Because of its high genome stability, Sulfolobus acidocaldarius is especially suited as a platform organism for such applications. For use in (ligno)cellulose degradation, it was important to understand pentose uptake and metabolism in S. acidocaldarius This study revealed that only the aldolase-independent Weimberg pathway is required for growth of S. acidocaldarius MW001 on d-xylose and l-arabinose. Moreover, S. acidocaldarius employs a CUT2 ABC transporter for pentose uptake, which is more similar to bacterial than to archaeal ABC transporters. The identification of pentose-inducible promoters will expedite the metabolic engineering of S. acidocaldarius for its development into a platform organism for (ligno)cellulose degradation.

P53 Mutation and Epigenetic Imprinted IGF2/H19 Gene Analysis in Mesenchymal Stem Cells Derived from Amniotic Fluid, Amnion, Endometrium, and Wharton's Jelly.

Mesenchymal stem cells (MSC) are promising cells for medical therapy. In in vitro expansion, MSC can give rise to progeny with genomic and epigenomic alterations, resulting in senescence, loss of terminal differentiation, and transformation to cancer. However, MSC genome protects its genetic instability by a guardian function of the P53 tumor suppressor gene and epigenetic balance system during MSC culture. Mutations of P53 and epigenetic alterations have been reported to disrupt the quality and quantity of MSC and initiate tumorigenesis. We monitor P53 and epigenetic changes in MSC derived from amniotic fluid (AF-MSC), amnion membrane (AM-MSC), endometrium (EM-MSC), and Wharton's jelly (WJ-MSC) by the missense mutation analysis of the P53 gene and the expression levels of P53, and epigenetic insulin-like growth factor 2 (IGF2) and H19-imprinted genes. Our work demonstrates a variation of P53 expression among different MSC types. AF-MSC has a high P53 expression level with retaining a stability of P53 expression throughout a long culture period, whereas EM-MSC and WJ-MSC showed variation of P53 gene expression during culture. Epigenetic analysis showed a stable H19 expression pattern in AF-MSC, AM-MSC, and EM-MSC culture, whereas H19 expression fluctuated in WJ-MSC culture. We conclude that gene instability can be found during in vitro MSC expansion. With awareness to MSC quality and safety in MSC transformation risk, P53 mutation and IGF2 and H19-imprinted gene analysis should be applied to monitor in therapeutic-grade MSC. We also demonstrated that AF-MSC is one of the most interesting MSC for medical therapy because of its high genomic stability and epigenetic fidelity.