Trapped lipopolysaccharide and LptD intermediates reveal lipopolysaccharide translocation steps across the Escherichia coli outer membrane

Lipopolysaccharide (LPS) is a main component of the outer membrane of Gram-negative bacteria, which is essential for the vitality of most Gram-negative bacteria and plays a critical role for drug resistance. LptD/E complex forms a N-terminal LPS transport slide, a hydrophobic intramembrane hole and the hydrophilic channel of the barrel, for LPS transport, lipid A insertion and core oligosaccharide and O-antigen polysaccharide translocation, respectively. However, there is no direct evidence to confirm that LptD/E transports LPS from the periplasm to the external leaflet of the outer membrane. By replacing LptD residues with an unnatural amino acid p-benzoyl-L-phenyalanine (pBPA) and UV-photo-cross-linking in E.coli, the translocon and LPS intermediates were obtained at the N-terminal domain, the intramembrane hole, the lumenal gate, the lumen of LptD channel, and the extracellular loop 1 and 4, providing the first direct evidence and “snapshots” to reveal LPS translocation steps across the outer membrane

Alloactivation of naïve CD4⁺ CD8⁻ CD25⁺T regulatory cells: Expression of CD8α identifies potent suppressor cells that can promote transplant tolerance induction

Therapy with alloantigen-specific CD4+ CD25+ T regulatory cells (Treg) for induction of transplant tolerance is desirable, as naïve thymic Treg (tTreg) are not alloantigen-specific and are weak suppressor cells. Naïve tTreg from DA rats cultured with fully allogeneic PVG stimulator cells in the presence of rIL-2 express IFN-gamma receptor (IFNGR) and IL-12 receptor beta2 (IL-12Rβ2) and are more potent alloantigen-specific regulators that we call Ts1 cells. This study examined additional markers that could identify the activated alloantigen-specific Treg as a subpopulation within the CD4+ CD25+ Foxp3+ Treg. After culture of naïve DA CD4+ CD8− CD25+ T cells with rIL-2 and PVG alloantigen, or rIL-2 without alloantigen, CD8α was expressed on 10–20% and CD8β on <5% of these cells. These cells expressed ifngr and Il12rb2. CD8α+ cells had increased Ifngr that characterizes Ts1 cells as well was Irf4, a transcription factor induced by TCR activation. Proliferation induced by re-culture with rIL-12 and alloantigen was greater with CD4+ CD8α+ CD25+ Treg consistent with the CD8α+ cells expressing IL-12R. In MLC, the CD8α+ fraction suppressed responses against allogeneic stimulators more than the mixed Ts1 population, whereas the CD4+ CD8− CD25+ T cells were less potent. In an adoptive transfer assay, rIL-2 and alloantigen activated Treg suppress rejection at a ratio of 1:10 with naïve effector cells, whereas alloantigen and rIL-2 activated tTreg depleted of the CD8α+ cells were much less effective. This study demonstrated that expression of CD8α by rIL-2 and alloantigen activation of CD4+ CD8− CD25+ Foxp3+ T cells was a marker of activated and potent Treg that included alloantigen-specific Treg

A common intronic variant of PARP1 confers melanoma risk and mediates melanocyte growth via regulation of MITF

Previous genome-wide association studies have identified a melanoma-associated locus at 1q42.1 that encompasses a ~100-kb region spanning the PARP1 gene. Expression quantitative trait locus (eQTL) analysis in multiple cell types of the melanocytic lineage consistently demonstrated that the 1q42.1 melanoma risk allele (rs3219090[G]) is correlated with higher PARP1 levels. In silico fine-mapping and functional validation identified a common intronic indel, rs144361550 (−/GGGCCC; r2 = 0.947 with rs3219090), as displaying allele-specific transcriptional activity. A proteomic screen identified RECQL as binding to rs144361550 in an allele-preferential manner. In human primary melanocytes, PARP1 promoted cell proliferation and rescued BRAFV600E-induced senescence phenotypes in a PARylation-independent manner. PARP1 also transformed TERT-immortalized melanocytes expressing BRAFV600E. PARP1-mediated senescence rescue was accompanied by transcriptional activation of the melanocyte-lineage survival oncogene MITF, highlighting a new role for PARP1 in melanomagenesis

Nonlinear response of the vacuum Rabi resonance

On the level of single atoms and photons, the coupling between atoms and the electromagnetic field is typically very weak. By employing a cavity to confine the field, the strength of this interaction can be increased many orders of magnitude to a point where it dominates over any dissipative process. This strong-coupling regime of cavity quantum electrodynamics has been reached for real atoms in optical cavities, and for artificial atoms in circuit QED and quantum-dot systems. A signature of strong coupling is the splitting of the cavity transmission peak into a pair of resolvable peaks when a single resonant atom is placed inside the cavity - an effect known as vacuum Rabi splitting. The circuit QED architecture is ideally suited for going beyond this linear response effect. Here, we show that increasing the drive power results in two unique nonlinear features in the transmitted heterodyne signal: the supersplitting of each vacuum Rabi peak into a doublet, and the appearance of additional peaks with the characteristic sqrt(n) spacing of the Jaynes-Cummings ladder. These constitute direct evidence for the coupling between the quantized microwave field and the anharmonic spectrum of a superconducting qubit acting as an artificial atom.Comment: 6 pages, 4 figures. Supplementary Material and Supplementary Movies are available at http://www.eng.yale.edu/rslab/publications.htm

Induction of humoral immune response to multiple recombinant Rhipicephalus appendiculatus antigens and their effect on tick feeding success and pathogen transmission

BACKGROUND: Rhipicephalus appendiculatus is the primary vector of Theileria parva, the etiological agent of East Coast fever (ECF), a devastating disease of cattle in sub-Saharan Africa. We hypothesized that a vaccine targeting tick proteins that are involved in attachment and feeding might affect feeding success and possibly reduce tick-borne transmission of T. parva. Here we report the evaluation of a multivalent vaccine cocktail of tick antigens for their ability to reduce R. appendiculatus feeding success and possibly reduce tick-transmission of T. parva in a natural host-tick-parasite challenge model. METHODS: Cattle were inoculated with a multivalent antigen cocktail containing recombinant tick protective antigen subolesin as well as two additional R. appendiculatus saliva antigens: the cement protein TRP64, and three different histamine binding proteins. The cocktail also contained the T. parva sporozoite antigen p67C. The effect of vaccination on the feeding success of nymphal and adult R. appendiculatus ticks was evaluated together with the effect on transmission of T. parva using a tick challenge model. RESULTS: To our knowledge, this is the first evaluation of the anti-tick effects of these antigens in the natural host-tick-parasite combination. In spite of evidence of strong immune responses to all of the antigens in the cocktail, vaccination with this combination of tick and parasite antigens did not appear to effect tick feeding success or reduce transmission of T. parva. CONCLUSION: The results of this study highlight the importance of early evaluation of anti-tick vaccine candidates in biologically relevant challenge systems using the natural tick-host-parasite combination

Age-Related Attenuation of Dominant Hand Superiority

The decline of motor performance of the human hand-arm system with age is well-documented. While dominant hand performance is superior to that of the non-dominant hand in young individuals, little is known of possible age-related changes in hand dominance. We investigated age-related alterations of hand dominance in 20 to 90 year old subjects. All subjects were unambiguously right-handed according to the Edinburgh Handedness Inventory. In Experiment 1, motor performance for aiming, postural tremor, precision of arm-hand movement, speed of arm-hand movement, and wrist-finger speed tasks were tested. In Experiment 2, accelerometer-sensors were used to obtain objective records of hand use in everyday activities

Evolutionary distances in the twilight zone -- a rational kernel approach

Phylogenetic tree reconstruction is traditionally based on multiple sequence alignments (MSAs) and heavily depends on the validity of this information bottleneck. With increasing sequence divergence, the quality of MSAs decays quickly. Alignment-free methods, on the other hand, are based on abstract string comparisons and avoid potential alignment problems. However, in general they are not biologically motivated and ignore our knowledge about the evolution of sequences. Thus, it is still a major open question how to define an evolutionary distance metric between divergent sequences that makes use of indel information and known substitution models without the need for a multiple alignment. Here we propose a new evolutionary distance metric to close this gap. It uses finite-state transducers to create a biologically motivated similarity score which models substitutions and indels, and does not depend on a multiple sequence alignment. The sequence similarity score is defined in analogy to pairwise alignments and additionally has the positive semi-definite property. We describe its derivation and show in simulation studies and real-world examples that it is more accurate in reconstructing phylogenies than competing methods. The result is a new and accurate way of determining evolutionary distances in and beyond the twilight zone of sequence alignments that is suitable for large datasets.Comment: to appear in PLoS ON

Development and Evaluation of a Palliative Medicine Curriculum for Third-Year Medical Students

Abstract Objective: To assess the impact, retention, and magnitude of effect of a required didactic and experiential palliative care curriculum on third-year medical students' knowledge, confidence, and concerns about end-of-life care, over time and in comparison to benchmark data from a national study of internal medicine residents and faculty. Design: Prospective study of third-year medical students prior to and immediately after course completion, with a follow-up assessment in the fourth year, and in comparison to benchmark data from a large national study. Setting: Internal Medicine Clerkship in a public accredited medical school. Participants: Five hundred ninety-three third-year medical students, from July 2002 to December 2007. Main outcome measures: Pre- and postinstruction performance on: knowledge, confidence (self-assessed competence), and concerns (attitudes) about end-of-life care measures, validated in a national study of internal medicine residents and faculty. Medical student's reflective written comments were qualitatively assessed. Intervention: Required 32-hour didactic and experiential curriculum, including home hospice visits and inpatient hospice care, with content drawn from the AMA-sponsored Education for Physicians on End-of-life Care (EPEC) Project. Results: Analysis of 487 paired t tests shows significant improvements, with 23% improvement in knowledge (F1,486=881, p<0.001), 56% improvement in self-reported competence (F1,486=2,804, p<0.001), and 29% decrease in self-reported concern (F1,486=208, p<0.001). Retesting medical students in the fourth year showed a further 5% increase in confidence (p<0.0002), 13% increase in allaying concerns (p<0.0001), but a 6% drop in knowledge. The curriculum's effect size on M3 students' knowledge (0.56) exceeded that of a national cross-sectional study comparing residents at progressive training levels (0.18) Themes identified in students' reflective comments included perceived relevance, humanism, and effectiveness of methods used to teach and assess palliative care education. Conclusions: We conclude that required structured didactic and experiential palliative care during the clinical clerkship year of medical student education shows significant and largely sustained effects indicating students are better prepared than a national sample of residents and attending physicians.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98455/1/jpm%2E2010%2E0502.pd

HFE genotype modifies the influence of heme iron intake on iron status

Background: Public health policy to prevent iron deficiency through food fortification or other measures may be disadvantageous to people with hereditary hemochromatosis. Methods: From a cohort of U.K. women, 2531 women were typed for C282Y and H63D mutations in the hemochromatosis gene. These women completed food frequency questionnaires and provided blood for iron status. Results: C282Y homozygotes (n = 31) had serum ferritin concentrations 2.4 times higher (95% confidence interval = 1.9–3.1) than wild types (n = 1774), but heterozygotes (n = 726) were not different from wild types. H63D genotype had no effect on its own. The effect of heme iron intake (from meat, fish, and poultry) was 2.0 times greater (1.2–3.2) on C282Y homozygotes than other groups. Nonheme iron had little effect. Conclusions: There may be scope for dietary intervention in women homozygous for the C282Y mutation. C282Y heterozygotes and H63D homozygotes and heterozygotes have similar serum ferritin concentrations to wild type and need not reduce their meat intake other than as part of a normal healthy diet