Real-time Screening of Foods Using Repetitive Element PCR Reveals a DNA Marker Characteristic for Enterotoxigenic Bacillus Species

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables, and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic, including not just food-associated gastrointestinal toxicoinfections, but human endophthalmitis as well. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Repetitive element PCR was used to compare the banding patterns of each sample against B. cereus ATCC 14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe and were capable of causing gastroenteritis in consumers.

The Ecology of Palm Genomes: Repeat-associated genome size expansion is constrained by aridity

Genome size varies 2,400-fold across plants, influencing their evolution through changes in cell size and cell division rates which impact plants' environmental stress tolerance. Repetitive element expansion explains much genome size diversity, and the processes structuring repeat 'communities' are analogous to those structuring ecological communities. However, which environmental stressors influence repeat community dynamics has not yet been examined from an ecological perspective. We measured genome size and leveraged climatic data for 91% of genera within the ecologically diverse palm family (Arecaceae). We then generated genomic repeat profiles for 141 palm species, and analysed repeats using phylogenetically-informed linear models to explore relationships between repeat dynamics and environmental factors. We show that palm genome size and repeat 'community' composition are best explained by aridity. Specifically, EnSpm CACTA repeats were more abundant in palm species from wetter environments, which generally had larger genomes (>2.15Gbp/1C), suggesting amplification. In contrast, Ty1-copia Angela elements were more abundant in drier environments. Our results suggest water stress inhibits the expansion of repeats through selection on upper genome size limits. However, Ty1-copia Angela elements, which may associate with stress-response genes, have amplified in arid-adapted palm species. Overall, we provide novel evidence of climate influencing the assembly of repeat 'communities'.

ADAR and hnRNPC deficiency synergize in activating endogenous dsRNA-induced type I IFN responses

Cytosolic double-stranded RNA (dsRNA) initiates type I IFN responses. Endogenous retroelements, notably Alu elements, constitute a source of dsRNA. Adenosine-to-inosine (A-to-I) editing by ADAR induces mismatches in dsRNA and prevents recognition by MDA5 and autoinflammation. To identify additional endogenous dsRNA checkpoints, we conducted a candidate screen in THP-1 monocytes and found that hnRNPC and ADAR deficiency resulted in synergistic induction of MDA5-dependent IFN responses. RNA-seq analysis demonstrated dysregulation of Alu-containing introns in hnRNPC-deficient cells via utilization of unmasked cryptic splice sites, including introns containing ADAR-dependent A-to-I editing clusters. These putative MDA5 ligands showed reduced editing in the absence of ADAR, providing a plausible mechanism for the combined effects of hnRNPC and ADAR. This study contributes to our understanding of the control of repetitive element–induced autoinflammation and suggests that patients with hnRNPC-mutated tumors might maximally benefit from ADAR inhibition-based immunotherapy.

The transcriptional trajectories of pluripotency and differentiation comprise genes with antithetical architecture and repetitive-element content

Background Extensive molecular differences exist between proliferative and differentiated cells. Here, we conduct a meta-analysis of publicly available transcriptomic datasets from preimplantation and differentiation stages examining the architectural properties and content of genes whose abundance changes significantly across developmental time points. Results Analysis of preimplantation embryos from human and mouse showed that short genes whose introns are enriched in Alu (human) and B (mouse) elements, respectively, have higher abundance in the blastocyst compared to the zygote. These highly expressed genes encode ribosomal proteins or metabolic enzymes. On the other hand, long genes whose introns are depleted in repetitive elements have lower abundance in the blastocyst and include genes from signaling pathways. Additionally, the sequences of the genes that are differentially expressed between the blastocyst and the zygote contain distinct collections of pyknon motifs that differ between up- and down-regulated genes. Further examination of the genes that participate in the stem cell-specific protein interaction network shows that their introns are short and enriched in Alu (human) and B (mouse) elements. As organogenesis progresses, in both human and mouse, we find that the primarily short and repeat-rich expressed genes make way for primarily longer, repeat-poor genes. With that in mind, we used a machine learning-based approach to identify gene signatures able to classify human adult tissues: we find that the most discriminatory genes comprising these signatures have long introns that are repeat-poor and include transcription factors and signaling-cascade genes. The introns of widely expressed genes across human tissues, on the other hand, are short and repeat-rich, and coincide with those with the highest expression at the blastocyst stage. Conclusions Protein-coding genes that are characteristic of each trajectory, i.e., proliferation/pluripotency or differentiation, exhibit antithetical biases in their intronic and exonic lengths and in their repetitive-element content. While the respective human and mouse gene signatures are functionally and evolutionarily conserved, their introns and exons are enriched or depleted in organism-specific repetitive elements. We posit that these organism-specific repetitive sequences found in exons and introns are used to effect the corresponding genes’ regulation.

Genotoxicity effects of medicinal plants extracts against bacterial species, Mycoplasma hominis

Aim: To assess the antimicrobial activity and genotoxicity of three medicinal plants used by Saudi Arabian people as traditional medicine against Mycoplasma hominis. Methodology: Different concentrations of Syzygium aromaticum (clove), Vachellia nilotica (acacia), and Thyme vulgaris (thyme) extracts were used as antimicrobial agents against M. hominis, and their lethal effects on Mycoplasma genome DNA were analyzed using repetitive element PCR(Rep-PCR). Results: The aqueous extracts of clove and Acacia at 3.125 mg ml-1 were found to be active antimicrobials against three tested Mycoplasm. Thyme extract exhibited antimicrobial activity at 12.5 mg ml-1. Moreover, this extract revealed potent lethal activities as growth turbidity decreased with increasing concentration or exposure time as compared to untreated Mycoplasma. The results of Rep-PCR clearly indicate that changes occured in the number of genetic bands in treated Mycoplasma at certain concentrations as compared to untreated Mycoplasma. Interpretation: These results indicate the possibility of using these extracts as a source of antibacterial compounds for treating infections caused by Mycoplasma. Key words: Antimicrobial activity, Genotoxicity, Mycoplasma hominis, Medicinal plants, S. aromaticum

A real-time quantitative polymerase chain reaction for the specific detection of Hammondia hammondi and its differentiation from Toxoplasma gondii

Introduction Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites, but only T. gondii is zoonotic. Both species use felids as definitive hosts and cannot be differentiated by oocyst morphology. In T. gondii, a 529-base pair (bp) repetitive element (TgREP-529) is of utmost diagnostic importance for polymerase chain reaction (PCR) diagnostic tests. We identified a similar repetitive region in the H. hammondi genome (HhamREP-529). Methods Based on reported sequences, primers and probes were selected in silico and optimal primer probe combinations were explored, also by including previously published primers. The analytical sensitivity was tested using serial dilutions of oocyst DNA. For testing analytical specificity, DNA isolated from several related species was used as controls. The newly established TaqMan PCR (Hham-qPCR1) was applied to tissues collected from H. hammondi-infected gamma-interferon gene knockout (GKO) mice at varying time points post-infection. Results Ten forward and six reverse primers were tested in varying combinations. Four potentially suitable dual-labelled probes were selected. One set based on the primer pair (Hham275F, Hham81R) and the probe (Hham222P) yielded optimal results. In addition to excellent analytic specificity, the assay revealed an analytical sensitivity of genome equivalents of less than one oocyst. Investigation of the tissue distribution in GKO mice revealed the presence of parasite DNA in all examined organs, but to a varying extent, suggesting 100- to 10,000-fold differences in parasitic loads between tissues in the chronic state of infection, 42 days post-infection. Discussion The use of the 529-bp repeat of H. hammondi is suitable for establishing a quantitative real-time PCR assay, because this repeat probably exists about 200 times in the genome of a single organism, like its counterpart in T. gondii. Although there were enough sequence data available, only a few of the primers predicted in silico revealed sufficient amplification; the identification of a suitable probe was also difficult. This is in accord with our previous observations on considerable variability in the 529-bp repetitive element of H. hammondi. Conclusions The H. hammondi real-time PCR represents an important novel diagnostic tool for epidemiological and cell biological studies on H. hammondi and related parasites.