SUMMARY Primordial germ cells (PGCs) undergo dramatic rearrangements to their methylome during embryo-genesis, including initial genome-wide DNA demethylation that establishes the germline epigenetic ground state. The role of the 5-methylcytosine (5mC) dioxygenases Tet1 and Tet2 in the initial genome-wide DNA demethylation process has not been examined directly. Using PGCs differentiated from either control or Tet2−/−; Tet1 knockdown embryonic stem cells (ESCs), we show that in vitro PGC (iPGC) formation and genome-wide DNA demethylation are unaffected by the absence of Tet1 and Tet2, and thus 5-hydroxymethylcytosine (5hmC). However, numerous promoters and gene bodies were hypermethylated in mutant iPGCs, which is consistent with a role for 5hmC as an intermediate in locus-specific demethylation. Altogether, our results support a revised model of PGC DNA demethylation in which the first phase of comprehensive 5mC loss does not involve 5hmC. Instead, Tet1 and Tet2 have a locus-specific role in shaping the PGC epige-nome during subsequent development.

No abstract

Background Previous studies suggest that immediate reconstruction following mastectomy produces superior results over delayed procedures. However, for medical or oncological reasons, some patients may be poor candidates for immediate reconstruction. We compared complications and patient-reported outcomes between immediate and delayed breast reconstructions in a prospective, multicenter study. Methods 1957 patients (1806 immediate, 151 delayed) met eligibility criteria. Demographic data, major complications, infections, and reconstructive failure rates were evaluated. Patient-reported outcomes were assessed with BREAST-Q, PROMIS, and EORTC QLQ-BR23 surveys, pre- and two years postoperatively. Subscale scores were compared across cohorts using mixed-effects regression models, controlling for patient characteristics and hospitals. Findings Complete data were available in 1639 immediate and 147 delayed reconstruction patients. There were significant baseline differences between immediate and delayed cohorts in age, BMI, prevalence of diabetes, lymph node management, use of radiation, and chemotherapy. Controlling for clinical covariates, the delayed group had lower odds of any (OR 0.38, p < 0.001) and major (OR 0.52, p = 0.016) complications, compared with immediate patients. Furthermore, delayed reconstruction was associated with a significantly lower failure rates (6% vs. 1.3%, p = 0.032). However, multivariate analyses found no significant differences in patient satisfaction or in psychosocial, sexual, or physical well-being at two years. Conclusions Compared with immediate techniques, delayed reconstruction following mastectomy was associated with lower rates of overall and major complication, while providing equivalent patient satisfaction and quality of life benefits. Although immediate reconstruction is still the preferred choice of most patients and surgeons, delaying reconstruction does not appear to compromise clinical or patient-reported outcomes.

is an important pathogen, causing life-threatening infections such as endocarditis and severe sepsis in immunocompromised patients. The β-lactam antibiotics are the usual therapy of choice for this organism, but their effectiveness is threatened by the frequent emergence of resistance. The lipopeptide daptomycin (DAP) has been suggested for therapy against such resistant strains due to its bactericidal activity and demonstrated efficacy against other Gram-positive pathogens. Unlike other bacteria, however, has the unique ability to rapidly develop stable, high-level resistance to DAP upon exposure to the drug both and Using isogenic DAP-susceptible and DAP-resistant strain pairs, we describe a mechanism of resistance to both DAP and cationic antimicrobial peptides that involves loss-of-function mutations in (encoding a phosphatidate cytidylyltransferase). CdsA catalyzes the synthesis of cytidine diphosphate-diacylglycerol, an essential phospholipid intermediate for the production of membrane phosphatidylglycerol and cardiolipin. DAP-resistant strains demonstrated a total disappearance of phosphatidylglycerol, cardiolipin, and anionic phospholipid microdomains from membranes. In addition, these strains exhibited cross-resistance to cationic antimicrobial peptides from human neutrophils (i.e., hNP-1). Interestingly, CdsA-mediated changes in phospholipid metabolism were associated with DAP hyperaccumulation in a small subset of the bacterial population, without any binding by the remaining larger population. Our results indicate that CdsA is the major mediator of high-level DAP resistance in and suggest a novel mechanism of bacterial survival against attack by antimicrobial peptides of both innate and exogenous origins.

Summary Three pathogenic forms, or formae speciales, of Fusarium oxysporum infect the roots of Arabidopsis thaliana belowground, instigating symptoms of wilt disease in leaves aboveground. In prior reports, Arabidopsis mutants that are deficient in the biosynthesis of abscisic acid or salicylic acid or insensitive to ethylene or jasmonates exhibit more or less wilt disease than wild type, implicating the involvement of hormones in the normal host response to F. oxysporum. Our analysis of hormone-related mutants finds no evidence that endogenous hormones contribute to infection in roots. Mutants that are deficient in abscisic acid and insensitive to ethylene have no less infection than wild type, though they exhibit less disease. Whether a mutant that is insensitive to jasmonates affects infection depends on which forma specialis is infecting roots. Insensitivity to jasmonates suppresses infection by F. oxysporum forma specialis conglutinans and F. oxysporum forma specialis matthioli, which produce isoleucine- and leucine-conjugated jasmonate (JA-Ile/Leu) in culture filtrates; whereas, insensitivity to jasmonates has no effect on infection by F. oxysporum forma specialis raphani, which produces no detectable JA-Ile/Leu. Furthermore, insensitivity to jasmonates has no effect on wilt disease of tomato, and the tomato pathogen F. oxysporum forma specialis lycopersici produces no detectable jasmonates. Thus, some but not all F. oxysporum pathogens appear to utilize jasmonates as effectors, promoting infection in roots and/or development of symptoms in shoots. Only when infection of roots is promoted by jasmonates is wilt disease enhanced in a mutant deficient in salicylic acid biosynthesis.