Foodborne Botulism Outbreak Associated With Commercial Nacho Cheese Sauce From a Gas Station Market.

BACKGROUND: Botulism is a rare and potentially fatal paralytic disease caused by botulinum neurotoxin (BoNT). In April 2017, 4 California residents from 2 adjacent counties were hospitalized with suspected foodborne botulism, precipitating an investigation by state and local public health departments in California. METHODS: We interviewed suspected botulism patients and their families, inspected the suspect establishment, and collected suspect food. We tested patient sera, stool, and gastric aspirates using mouse bioassay for BoNT and/or culture for Clostridium botulinum. We tested suspect food and environmental samples for BoNT and confirmed presumptive positives using direct mouse bioassay and culture. We performed whole-genome sequencing on food and clinical isolates. RESULTS: From April 2017 through May 2017, 10 patients in the Sacramento area were hospitalized with laboratory-confirmed botulism; 7 required mechanical ventilation, and 1 died. Of 9 patients with information, all had visited Gas Station X before illness onset, where 8 reported consuming a commercial cheese sauce. BoNT/A and/or BoNT/A-producing C. botulinum were detected from each patient and from leftover cheese sauce. Clostridium botulinum isolates from 4 patients were closely related to cheese sauce isolates by whole-genome high-quality single-nucleotide polymorphism analysis. No other botulism cases associated with this cheese sauce were reported elsewhere in the United States. CONCLUSIONS: This large foodborne botulism outbreak in California was caused by consumption of commercial cheese sauce dispensed at a gas station market. The epidemiologic and laboratory evidence confirmed the cheese sauce as the outbreak source. The cheese sauce was likely locally contaminated, although the mechanism is unclear.

Mechanisms of establishment and functional significance of DNA demethylation during erythroid differentiation.

Erythroid differentiation is associated with global DNA demethylation, but a complete methylome was lacking in the erythroid lineage. We have generated allele-specific base resolution methylomes of primary basophilic erythroblasts (BasoEs) and compared these with 8 other cell types. We found that DNA demethylation during differentiation from hematopoietic stem/progenitor cells (HSPCs) to BasoEs occurred predominantly in intergenic sequences and in inactive gene bodies causing the formation of partially methylated domains (PMDs) in 74% of the BasoE methylome. Moreover, differentially methylated regions (DMRs) between HSPCs and BasoEs occurred mostly in putative enhancer regions and were most often associated with GATA, EKLF, and AP1 binding motifs. Surprisingly, promoters silent in both HSPCs and BasoEs exhibited much more dramatic chromatin changes during differentiation than activated promoters. Unmethylated silent promoters were often associated with active chromatin states in highly methylated domains (HMDs) but with polycomb-repression in PMDs, indicating that silent promoters are generally regulated differently in HMDs and PMDs. We show that long PMDs replicate late, but that short PMDs replicate early and therefore that the partial methylation of DNA after replication during erythroid expansion occurs throughout S phase of the cell cycle. We propose that baseline maintenance methylation following replication decreases during erythroid differentiation resulting in PMD formation and that the presence of HMDs in the BasoE methylome results from transcription-associated DNA methylation of gene bodies. We detected ∼700 large allele-specific DMRs that were enriched in single-nucleotide polymorphisms, suggesting that primary DNA sequence might be a determinant of DNA methylation levels within PMDs.

Identification of a BET family bromodomain/casein kinase II/TAF-containing complex as a regulator of mitotic condensin function.

Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02), casein kinase II (CKII), and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation.

p16(INK4a) -mediated suppression of telomerase in normal and malignant human breast cells.

The cyclin-dependent kinase inhibitor p16(INK4a) (CDKN2A) is an important tumor suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal.

BORIS (CTCFL) is not expressed in most human breast cell lines and high grade breast carcinomas.

BORIS (CTCFL) is the only known paralog of the versatile regulatory protein CTCF, a multifunctional DNA binding protein that mediates distinct gene regulatory functions involved in cell growth, differentiation, and apoptosis. Unlike CTCF, the expression of BORIS is normally restricted to specific cells in testes (the only cells where CTCF is not expressed), where it may play a role in reprogramming the methylation pattern of male germ line DNA. Frequent amplification of the 20q13.2 region, which contains the BORIS gene, and expression of BORIS transcripts in diverse human tumors and cell lines have led to the hypothesis that aberrant expression of BORIS may play a role in tumorigenesis by interfering with CTCF functions. However, recent studies using more quantitative methods indicate low frequency of BORIS expression in melanoma, ovarian, prostate, and bladder carcinomas. To investigate the relationship between chromosome 20q13 amplification and BORIS mRNA levels within breast cancer cell lines and tissues, we developed a quantitative RT-PCR assay to measure the levels of BORIS mRNA. Endpoint RT-PCR assays were also used to investigate the possible expression of alternatively spliced variants. Using multiple primer sets and controls, we found that neither mature BORIS transcripts nor spliced variants are commonly expressed at detectable levels in malignant breast cells or tissues, although endogenous BORIS transcripts can be induced in MCF-7 cells following 5-aza-2'-deoxycytidine treatment. In conclusion, in most breast cancer cells, endogenous BORIS is unlikely to be expressed at sufficient levels to interfere with CTCF functions. Thus it is improbable that aberrant BORIS expression plays a role in most human breast cancers.

Promotion of variant human mammary epithelial cell outgrowth by ionizing radiation: an agent-based model supported by in vitro studies.

INTRODUCTION: Most human mammary epithelial cells (HMEC) cultured from histologically normal breast tissues enter a senescent state termed stasis after 5 to 20 population doublings. These senescent cells display increased size, contain senescence associated beta-galactosidase activity, and express cyclin-dependent kinase inhibitor, p16INK4A (CDKN2A; p16). However, HMEC grown in a serum-free medium, spontaneously yield, at low frequency, variant (v) HMEC that are capable of long-term growth and are susceptible to genomic instability. We investigated whether ionizing radiation, which increases breast cancer risk in women, affects the rate of vHMEC outgrowth. METHODS: Pre-stasis HMEC cultures were exposed to 5 to 200 cGy of sparsely (X- or gamma-rays) or densely (1 GeV/amu 56Fe) ionizing radiation. Proliferation (bromodeoxyuridine incorporation), senescence (senescence-associated beta-galactosidase activity), and p16 expression were assayed in subcultured irradiated or unirradiated populations four to six weeks following radiation exposure, when patches of vHMEC became apparent. Long-term growth potential and p16 promoter methylation in subsequent passages were also monitored. Agent-based modeling, incorporating a simple set of rules and underlying assumptions, was used to simulate vHMEC outgrowth and evaluate mechanistic hypotheses. RESULTS: Cultures derived from irradiated cells contained significantly more vHMEC, lacking senescence associated beta-galactosidase or p16 expression, than cultures derived from unirradiated cells. As expected, post-stasis vHMEC cultures derived from both unirradiated and irradiated cells exhibited more extensive methylation of the p16 gene than pre-stasis HMEC cultures. However, the extent of methylation of individual CpG sites in vHMEC samples did not correlate with passage number or treatment. Exposure to sparsely or densely ionizing radiation elicited similar increases in the numbers of vHMEC compared to unirradiated controls. Agent-based modeling indicated that radiation-induced premature senescence of normal HMEC most likely accelerated vHMEC outgrowth through alleviation of spatial constraints. Subsequent experiments using defined co-cultures of vHMEC and senescent cells supported this mechanism. CONCLUSIONS: Our studies indicate that ionizing radiation can promote the outgrowth of epigenetically altered cells with pre-malignant potential.

Telomerase activation by c-Myc in human mammary epithelial cells requires additional genomic changes.

A central question in breast cancer biology is how cancer cells acquire telomerase activity required for unlimited proliferation. According to one model, proliferation of telomerase(-) pre-malignant cells leads to telomere dysfunction and increased genomic instability. Such instability leads in rare cases to reactivation of telomerase and immortalization. The mechanism of telomerase reactivation remains unknown. We have studied immortalization of cultured human mammary epithelial cells by c-Myc, a positive transcriptional regulator of the hTERT gene encoding the catalytic subunit of telomerase. Retrovirally introduced c-Myc cDNA resulted in immortalization of human mammary epithelial cells in which the cyclin dependent kinase inhibitor, p16(INK4A), was inactivated by an shRNA-encoding retrovirus. However, while c-Myc introduction immediately resulted in increased activity of transiently transfected hTERT promoter reporter constructs, endogenous hTERT mRNA levels did not change until about 60 population doublings after c-Myc introduction. Increased endogenous hTERT transcripts and stabilization of telomeric DNA in cells expressing exogenous c-Myc coincided with telomere dysfunction-associated senescence in control cultures. Genome copy number analyses of immortalized cells indicated amplifications of some or all of chromosome 5, where hTERT genes are located. hTERT gene copy number, however, was not increased in one case. The results are consistent with the hypothesis that changes in chromosome 5, while not necessarily increasing hTERT gene copy number, resulted in removal of repressive chromatin structures around hTERT loci, allowing induction of hTERT transcription. These in vitro results model one possible sequence of events leading to immortalization of breast epithelial cells during cancer progression.

CTCF interacts with and recruits the largest subunit of RNA polymerase II to CTCF target sites genome-wide.

CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Serial" chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the beta-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

Poly(ADP-ribosyl)ation regulates CTCF-dependent chromatin insulation.

Chromatin insulators demarcate expression domains by blocking the cis effects of enhancers or silencers in a position-dependent manner. We show that the chromatin insulator protein CTCF carries a post-translational modification: poly(ADP-ribosyl)ation. Chromatin immunoprecipitation analysis showed that a poly(ADP-ribosyl)ation mark, which exclusively segregates with the maternal allele of the insulator domain in the H19 imprinting control region, requires the bases that are essential for interaction with CTCF. Chromatin immunoprecipitation-on-chip analysis documented that the link between CTCF and poly(ADP-ribosyl)ation extended to more than 140 mouse CTCF target sites. An insulator trap assay showed that the insulator function of most of these CTCF target sites is sensitive to 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase activity. We suggest that poly(ADP-ribosyl)ation imparts chromatin insulator properties to CTCF at both imprinted and nonimprinted loci, which has implications for the regulation of expression domains and their demise in pathological lesions.