Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

Polarization of naïve CD4 T cells into T helper type 2 (TH2) cells is characterized by expression of IL‐4 and silencing of IFN‐γ. Here we show that during TH2 polarization, the DNA methyltransferase Dnmt3a is recruited to the IFN‐γ promoter and correspondingly the promoter undergoes progressive de novo methylation. Notably, the CpG located at the −53 position becomes methylated rapidly and this methylation inhibits ATF2/c‐Jun and CREB transcription factor binding in vitro. In vivo, the same factors bind to the unmethylated IFN‐γ promoter in T helper type 1 (TH1) cells but not the methylated IFN‐γ promoter in TH2 cells. Furthermore, methylation at the −53 CpG alone is sufficient to inhibit the IFN‐γ promoter‐driven reporter gene expression in a TH1 cell line. These findings suggest that rapid methylation of the evolutionarily conserved −53 CpG by Dnmt3a may suppress IFN‐γ transcription in developing TH2 cells by directly inhibiting transcription factor binding.

To study competition between naïve and memory T cells, we examined proliferation of adoptively transferred naïve CD8 ؉ T cells in lymphopenic recipients or recipients containing a clonal population of CD8 ؉ T cells. We find a hierarchy in the extent of T cell proliferation that appears to correlate with the strength of T cell receptor (TCR)-self-peptide-MHC (pepMHC) interactions. CD8 ؉ T cells also proliferate in recipients containing a full complement of CD8 ؉ cells with a different TCR if the transferred T cells experience stronger TCR-self-pepMHC interactions than the resident T cells. Furthermore, CD8 ؉ T cells proliferate in recipients that contain memory CD8 ؉ cells with a different TCR, but in this case the relative strengths of TCR-self-pepMHC interactions are not as critical. In contrast, CD8 ؉ T cells do not proliferate significantly in recipients harboring naïve or memory CD8 ؉ cells that bear the same TCR as the transferred cells. These results suggest that, among naïve T cells and between naïve and memory T cells, CD8 ؉ cells having the same TCR compete for both self-pepMHC and cytokines, whereas TCR-different CD8 ؉ cells compete for cytokines. These competitive relationships probably help maintain the size and TCR diversity of naïve and memory T cell populations required for optimal immune responses.I n a normal adult individual, peripheral T cells include both naïve cells that have not yet encountered antigen and antigenexperienced memory T cells. Both the naïve and memory cell compartments are composed of large numbers of T cells that express diverse antigen-specific T cell receptors (TCRs). Naïve T cells are critical for initiating immune responses to novel antigens, whereas memory T cells mount more rapid and vigorous responses on reencountering the same antigens. Because the total number of peripheral T cells is maintained at a relatively constant level (homeostasis) (1, 2), a proper size and sufficient diversity of naïve and memory T cell compartments are critical for the immune system's ability to respond to unpredictable encounters with an enormous number of different antigens (3). The molecular mechanisms that regulate the size and diversity of naïve and memory T cell compartments are under active investigation but are still not well understood.The number of naïve T cells in the periphery is determined by their production in the thymus and by their survival, proliferation, and differentiation in peripheral lymphoid organs. Studies have shown that the number of T cells remains constant irrespective of whether the pool is oversupplied or undersupplied (4), indicating mechanisms of regulation in the periphery. Indeed, survival of naïve CD4 ϩ and CD8 ϩ T cells requires both soluble factors, such as IL-7 (5, 6), and interactions between T cells' TCR and self-peptide-MHC complexes (pepMHC) (MHC class I for CD8 ϩ T cells and MHC class II for CD4 ϩ T cells) (7-11). The numbers of memory T cells are similarly regulated by their generation, survival, proliferation, and differentiation. Unlike naïve ...

A protracted outbreak of New Delhi metallo-β-lactamase (NDM)–producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB–type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

Microbial inactivation by cold atmospheric plasmas has been a subject of tremendous research interest in recent years, in part, due to the ambiguity concerning the plasma factors responsible for bacterial inactivation. This work investigated the efficacy of an atmospheric‐pressure plasma jet ignited in either helium or helium/oxygen mixtures in inactivating Escherichia coli on agar. The correlation of data obtained from inactivation experiments and a 2D model describing the gas dynamics and afterglow chemistry showed that the inactivation mechanisms differed qualitatively between the two gas compositions. This work also provides insight into the reaction pathways that lead to the production and destruction of the key active species and illustrates the importance in these processes of admixing ambient air.

A protracted outbreak of New Delhi metallo–β–lactamase (NDM)–producing carbapenem–resistant Klebsiella pneumoniae, started in Tuscany, Italy, in November 2018 and has continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug–resistant, NDM–producing K. pneumoniae isolates cultured over a 20–month period from 76 patients at several health care facilities in South–East Tuscany. All isolates belonged to high–risk clone ST–147 and were typically non–susceptible to all first line antibiotics. Albeit sporadic, resistances to colistin, tigecycline and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST–147 isolates circulating in Tuscany were monophyletic, highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates) and shared a recent ancestor with isolates collected from the Middle East. While the blaNDM–1 gene was carried by an IncFIB–type plasmid, our investigations revealed that the ST–147 lineage from Italy also acquired a hybrid IncH–type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST–11 and ST–307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

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