In mammals, Bone Morphogenetic Protein (BMP) pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. ...A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.
Cross-reactivity within flavivirus antibody assays, produced by shared epitopes in the envelope proteins, can complicate the serological diagnosis of Zika virus (ZIKAV) infection. We assessed the ...utility of the plaque reduction neutralization test (PRNT) to confirm recent ZIKAV infections and rule out misleading positive immunoglobulin M (IgM) results in areas with various levels of past dengue virus (DENV) infection incidence. We reviewed PRNT results of sera collected for diagnosis of ZIKAV infection from 1 January through 31 August 2016 with positive ZIKAV IgM results, and ZIKAV and DENV PRNTs were performed. PRNT result interpretations included ZIKAV, unspecified flavivirus, DENV infection, or negative. For this analysis, ZIKAV IgM was considered false positive for samples interpreted as a DENV infection or negative. In U.S. states, 208 (27%) of 759 IgM-positive results were confirmed to be ZIKAV compared to 11 (21%) of 52 in the U.S. Virgin Islands (USVI), 15 (15%) of 103 in American Samoa, and 13 (11%) of 123 in Puerto Rico. In American Samoa and Puerto Rico, more than 80% of IgM-positive results were unspecified flavivirus infections. The false-positivity rate was 27% in U.S. states, 18% in the USVI, 2% in American Samoa, and 6% in Puerto Rico. In U.S. states, the PRNT provided a virus-specific diagnosis or ruled out infection in the majority of IgM-positive samples. Almost a third of ZIKAV IgM-positive results were not confirmed; therefore, providers and patients must understand that IgM results are preliminary. In territories with historically higher rates of DENV transmission, the PRNT usually could not differentiate between ZIKAV and DENV infections.
We examined the role of community face experience on 6- and 8-month-old Caucasian infants' scanning of own- and other-race face scanning. We measured infants' proportional fixation time and scan path ...amplitudes as indices of face processing. Proportional fixation time to informationally rich face regions varied as a function of age and face race for infants living in a racially homogeneous community, whereas scan path amplitudes varied as a function of age and face race for infants living in a racially diverse community. In both communities 6-month-old infants did not show different responding to own- and other-race faces, whereas 8-month-old infants responded differently to own- and other-race faces. However, 8-month-old infants from the two communities showed different patterns of cross-race face scanning. Therefore, experience in the community beyond the home appears to contribute to the development of differential scanning of own- versus other-race faces between 6 and 8 months of age.
Peanut allergy (PA) is a complex disease with both environmental and genetic risk factors. Previously, PA loci were identified in filaggrin (FLG) and HLA in candidate gene studies, and loci in HLA ...were identified in a genome-wide association study and meta-analysis.
We sought to investigate genetic susceptibility to PA.
Eight hundred fifty cases and 926 hyper-control subjects and more than 7.8 million genotyped and imputed single nucleotide polymorphisms (SNPs) were analyzed in a genome-wide association study to identify susceptibility variants for PA in the Canadian population. A meta-analysis of 2 phenotypes (PA and food allergy) was conducted by using 7 studies from the Canadian, American (n = 2), Australian, German, and Dutch (n = 2) populations.
An SNP near integrin α6 (ITGA6) reached genome-wide significance with PA (P = 1.80 × 10−8), whereas SNPs associated with Src kinase–associated phosphoprotein 1 (SKAP1), matrix metallopeptidase 12 (MMP12)/MMP13, catenin α3 (CTNNA3), rho GTPase–activating protein 24 (ARHGAP24), angiopoietin 4 (ANGPT4), chromosome 11 open reading frame (C11orf30/EMSY), and exocyst complex component 4 (EXOC4) reached a threshold suggestive of association (P ≤ 1.49 × 10−6). In the meta-analysis of PA, loci in or near ITGA6, ANGPT4, MMP12/MMP13, C11orf30, and EXOC4 were significant (P ≤ 1.49 × 10−6). When a phenotype of any food allergy was used for meta-analysis, the C11orf30 locus reached genome-wide significance (P = 7.50 × 10−11), whereas SNPs associated with ITGA6, ANGPT4, MMP12/MMP13, and EXOC4 and additional C11orf30 SNPs were suggestive (P ≤ 1.49 × 10−6). Functional annotation indicated that SKAP1 regulates expression of CBX1, which colocalizes with the EMSY protein coded by C11orf30.
This study identifies multiple novel loci as risk factors for PA and food allergy and establishes C11orf30 as a risk locus for both PA and food allergy. Multiple genes (C11orf30/EMSY, SKAP1, and CTNNA3) identified by this study are involved in epigenetic regulation of gene expression.
Upon irradiation with visible light, the photosensitizer-peptide conjugate eosin-(KLAKLAK)2 kills a broad spectrum of bacteria without damaging human cells. Eosin-(KLAKLAK)2 therefore represents an ...interesting lead compound for the treatment of local infection by photodynamic bacterial inactivation. The mechanisms of cellular killing by eosin-(KLAKLAK)2, however, remain unclear and this lack of knowledge hampers the development of optimized therapeutic agents. Herein, we investigate the localization of eosin-(KLAKLAK)2 in bacteria prior to light treatment and examine the molecular basis for the photodynamic activity of this conjugate.
By employing photooxidation of 3,3-diaminobenzidine (DAB), (scanning) transmission electron microscopy ((S)TEM), and energy dispersive X-ray spectroscopy (EDS) methodologies, eosin-(KLAKLAK)2 is visualized at the surface of E. coli and S. aureus prior to photodynamic irradiation. Subsequent irradiation leads to severe membrane damage. Consistent with these observations, eosin-(KLAKLAK)2 binds to liposomes of bacterial lipid composition and causes liposomal leakage upon irradiation. The eosin moiety of the conjugate mediates bacterial killing and lipid bilayer leakage by generating the reactive oxygen species singlet oxygen and superoxide. In contrast, the (KLAKLAK)2 moiety targets the photosensitizer to bacterial lipid bilayers. In addition, while (KLAKLAK)2 does not disrupt intact liposomes, the peptide accelerates the leakage of photo-oxidized liposomes.
Together, our results suggest that (KLAKLAK)2 promotes the binding of eosin Y to bacteria cell walls and lipid bilayers. Subsequent light irradiation results in membrane damage from the production of both Type I & II photodynamic products. Membrane damage by oxidation is then further aggravated by the (KLAKLAK)2 moiety and membrane lysis is accelerated by the peptide. These results therefore establish how photosensitizer and peptide act in synergy to achieve bacterial photo-inactivation. Learning how to exploit and optimize this synergy should lead to the development of future bacterial photoinactivation agents that are effective at low concentrations and at low light doses.
Quetol 651, a low viscosity epoxy resin, is miscible with alcohols, acetone, and water. It is versatile and can be used as a single epoxide or mixed with other epoxides and anhydrides. The most ...important characteristic is that the addition of Quetol 651 to a formulation results in a lower viscosity embedding medium and allows for good detection of antigenic activity. Properly formulated and mixed resins containing Quetol 651 have excellent sectioning properties and good beam stability. The decrease in viscosity lends to lower specific gravity of the embedding medium and less interfering electron density between specimen elements resulting in better spatial resolution. New formulations and viscosity data are presented and compared to long used, embedding formulations and the extensive uses of Quetol 651 are reviewed.
We used eye tracking to examine 4.5‐ to 12.5‐month‐old infants’ (N = 92) eye movements during 3‐s presentations of upright and inverted faces. Scanning of inverted faces was statistically ...indistinguishable at 4.5, 6.5, 8, and 12.5 months of age; at each of these ages, infants disproportionately scanned the region containing the eyes. Scanning of upright faces changed over this age range. When viewing upright faces, 4.5‐month‐old and 6.5‐month‐old infants focused disproportionately on the region containing the eyes, whereas 12.5‐month‐old and 8‐month‐old infants distributed looking more broadly, scanning more of the internal area of the faces. These results are consistent with other observed developmental differences in face processing and provide insight into how moment‐to‐moment face processing changes during infancy.
Atm1p is an ABC transporter localized in the mitochondrial inner membrane; it functions to export an unknown species into the cytosol and is involved in cellular iron metabolism. Depletion or ...deletion of Atm1p causes Fe accumulation in mitochondria and a defect in cytosolic Fe/S cluster assembly but reportedly not a defect in mitochondrial Fe/S cluster assembly. In this study the nature of the accumulated Fe was examined using Mössbauer spectroscopy, EPR, electronic absorption spectroscopy, X-ray absorption spectroscopy, and electron microscopy. The Fe that accumulated in aerobically grown cells was in the form of iron(III) phosphate nanoparticles similar to that which accumulates in yeast frataxin Yfh1p-deleted or yeast ferredoxin Yah1p-depleted cells. Relative to WT mitochondria, Fe/S cluster and heme levels in Atm1p-depleted mitochondria from aerobic cells were significantly diminished. Atm1p depletion also caused a buildup of nonheme Fe(II) ions in the mitochondria and an increase in oxidative damage. Atm1p-depleted mitochondria isolated from anaerobically grown cells exhibited WT levels of Fe/S clusters and hemes, and they did not hyperaccumulate Fe. Atm1p-depleted cells lacked Leu1p activity, regardless of whether they were grown aerobically or anaerobically. These results indicate that Atm1p does not participate in mitochondrial Fe/S cluster assembly and that the species exported by Atm1p is required for cytosolic Fe/S cluster assembly. The Fe/S cluster defect and the Fe-accumulation phenotype, resulting from the depletion of Atm1p in aerobic cells (but not in anaerobic cells), may be secondary effects that are observed only when cells are exposed to oxygen during growth. Reactive oxygen species generated under these conditions might degrade iron−sulfur clusters and lower heme levels in the organelle.
Irradiation penetrates food tissues and effectively reduces the number of food microorganisms in fresh produce, but the efficacy of the process against internalized bacteria is unknown. The objective ...of this study was to understand the mechanisms of pathogen colonization of plants relative to lettuce leaf structures so that radiation treatment of fresh leafy vegetables can be optimized. Leaves of iceberg, Boston, green leaf, and red leaf lettuces were cut into pieces, submerged in a cocktail mixture of two isolates of
Escherichia coli (Rifampicin resistant), and subjected to a vacuum perfusion process to force the bacterial cells into the intercellular spaces in the leaves. Sixty bags containing 20
g of lettuce each were tested. The inoculated leaves were gamma irradiated (Lanthanum-140, 0.16
kGy/h) at 0.25–1.0-kGy (surface dose values), with increments of 0.25
kGy at 15
°C. Microbial analysis was performed right after irradiation, including non-irradiated leaf pieces (controls). A dose uniformity ratio (max/min dose) of 2.8 was set to confirm the effect of non-uniform dose distribution. Calculated
D
10-values varied between 48 and 62% based on the dose distribution from the entrance dose. However, despite the subtle differences in composition and structure among the four lettuce varieties, the
D
10-values were not significantly different. Irradiation up to 1.0-kGy resulted in 3–4-log reduction of internalized
E. coli on the lettuce leaves. The SEM images suggest that the contamination sites of pathogens in leafy vegetables are mainly localized on crevices and into the stomata. This study shows that irradiation effectively reduces viable
E. coli cells internalized in lettuce, and decontamination is not influenced by lettuce variety. Ionizing irradiation effectively reduced the population of internalized pathogen in a dose-dependent manner and could be used as an effective killing step to mitigate the risk of foodborne disease outbreaks.
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