Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length that can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides the foundation for nanopore sequencing of long, complex, natural DNA strands.

Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.nanopore DNA sequencing | DNA methylation | DNA hydroxymethylation | nanotechnology | next generation sequencing

Group B streptococci (GBS) are Gram-positive bacteria that cause infections in utero and in newborns. We recently showed that the GBS pigment is hemolytic and increased pigment production promotes bacterial penetration of human placenta. However, mechanisms utilized by the hemolytic pigment to induce host cell lysis and the consequence on fetal injury are not known. Here, we show that the GBS pigment induces membrane permeability in artificial lipid bilayers and host cells. Membrane defects induced by the GBS pigment trigger K+ efflux leading to osmotic lysis of red blood cells or pyroptosis in human macrophages. Macrophages lacking the NLRP3 inflammasome recovered from pigment-induced cell damage. In a murine model of in utero infection, hyperpigmented GBS strains induced fetal injury in both an NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent manner. These results demonstrate that the dual mechanism of action of the bacterial pigment/lipid toxin leading to hemolysis or pyroptosis exacerbates fetal injury and suggest that preventing both activities of the hemolytic lipid is likely critical to reduce GBS fetal injury and preterm birth.

Inflammasomes are mediators of inflammation, and constitutively activated NLRP3 inflammasomes have been linked to IL-1β-mediated tumorigenesis in human melanoma. Whereas NLRP3 regulation of caspase-1 activation requires the adaptor protein ASC, caspase-1 activation by another danger-signaling sensor NLRP1 does not require ASC because NLRP1 contains a C-terminal CARD domain that facilitates direct caspase-1 activation via CARD-CARD interaction. We hypothesized that NLRP1 has additional biological activities besides IL-1β maturation and investigated its role in melanoma tumorigenesis. NLRP1 expression in melanoma was confirmed by analysis of 216 melanoma tumors and 13 human melanoma cell lines. Unlike monocytic THP-1 cells with prominent nuclear localization of NLRP1, melanoma cells expressed NLRP1 mainly in the cytoplasm. Knocking down NLRP1 revealed a tumor promoting property of NLRP1 both in vitro and in vivo. Mechanistic studies showed that caspase-1 activity, IL-1β production, IL-1β secretion, and NF-kB activity were reduced by knocking down of NLRP1 in human metastatic melanoma cell lines 1205Lu and HS294T, indicating that NLRP1 inflammasomes are active in metastatic melanoma. However, unlike previous reports showing that NLRP1 enhances pyroptosis in macrophages, NLRP1 in melanoma behaved differently in the context of cell death. Knocking down NLRP1 increased caspase-2, -9, and -3/7 activities and promoted apoptosis in human melanoma cells. Immunoprecipitation revealed interaction of NLRP1 with CARD-containing caspase-2 and -9, whereas NLRP3 lacking a CARD motif did not interact with the caspases. Consistent with these findings, NLRP1 activation but not NLRP3 activation reduced caspase-2, -9, and -3/7 activities and provided protection against apoptosis in human melanoma cells, suggesting a suppressive role of NLRP1 in caspase-3/7 activation and apoptosis via interaction with caspase-2 and -9. In summary, we showed that NLRP1 promotes melanoma growth by enhancing inflammasome activation and suppressing apoptotic pathways. Our study demonstrates a tumor-promoting role of NLRP1 in cancer cells.

Cancers are comprised of heterogeneous subpopulations with various tumor-initiating capacities, yet key stem cell genes associated with enhanced tumor initiating capacities and their regulatory mechanisms remain elusive. Here we analyzed patient-derived xenografts from melanoma, colon, and pancreatic cancer tissues and identified enrichment of tumor-initiating cells in MHC class I-hi cells, where CDK1, a master regulator of the cell cycle, was upregulated. Overexpression of CDK1, but not its kinase-dead variant, in melanoma cells increased their spheroid forming ability, tumorigenic potential, and tumor-initiating capacity; inhibition of CDK1 with pharmacological agents reduced these characteristics, which was unexplained by the role of CDK1 in regulating the cell cycle. Proteomic analysis revealed an interaction between CDK1 and the pluripotent stem cell transcription factor Sox2. Blockade or knockdown of CDK1 resulted in reduced phosphorylation, nuclear localization, and transcriptional activity of Sox2. Knockout of Sox2 in CDK1-overexpressing cells reduced CDK1-driven tumor-initiating capacity substantially. Furthermore, GSEA analysis of CDK1-hi tumor cells identified a pathway signature common in all three cancer types, including E2F, G2M, MYC, and spermatogenesis, confirming a stem-like nature of CDK1-hi tumor cells. These findings reveal a previously unrecognized role for CDK1 in regulating tumor-initiating capacity in melanoma and suggest a novel treatment strategy in cancer via interruption of CDK1 function and its protein-protein interactions.