A secreted bacterial protein protects bacteria from cationic antimicrobial peptides by entrapment in phase-separated droplets.

Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly killing bacterial invaders; however, many pathogens have evolved resistance evasion mechanisms such as cell surface modification, CAP sequestration, degradation, or efflux. We have discovered that several pathogenic and commensal proteobacteria, including the urgent human threat Neisseria gonorrhoeae, secrete a protein (lactoferrin-binding protein B, LbpB) that contains a low-complexity anionic domain capable of inhibiting the antimicrobial activity of host CAPs. This study focuses on a cattle pathogen, Moraxella bovis, that expresses the largest anionic domain of the LbpB homologs. We used an exhaustive biophysical approach employing circular dichroism, biolayer interferometry, cross-linking mass spectrometry, microscopy, size-exclusion chromatography with multi-angle light scattering coupled to small-angle X-ray scattering (SEC-MALS-SAXS), and NMR to understand the mechanisms of LbpB-mediated protection against CAPs. We found that the anionic domain of this LbpB displays an α-helical secondary structure but lacks a rigid tertiary fold. The addition of antimicrobial peptides derived from lactoferrin (i.e. lactoferricin) to the anionic domain of LbpB or full-length LbpB results in the formation of phase-separated droplets of LbpB together with the antimicrobial peptides. The droplets displayed a low rate of diffusion, suggesting that CAPs become trapped inside and are no longer able to kill bacteria. Our data suggest that pathogens, like M. bovis, leverage anionic intrinsically disordered domains for the broad recognition and neutralization of antimicrobials via the formation of biomolecular condensates.

Knowledge of Sugars Consumption and the WHO Sugars Guideline among Canadian Dietitians and Other Health Professionals.

Purpose: The objective was to assess knowledge related to sugars consumption and World Health Organization (WHO) sugars guideline among Canadian dietitians and other health professionals. Methods: A multiple-choice style survey was administered at Dietitians of Canada and Canadian Diabetes Association conferences in 2014. Results: The study showed that only 12% of the surveyed respondents (n = 335) in 2014 were able to correctly identify the amount of added sugars consumed by Canadians, whereas two-thirds overestimated this amount. About 10% of the respondents knew that the 10% guideline by WHO for free sugars was based on evidence related to dental caries. Registered dietitians had relatively better knowledge of Canadian sugars consumption (P = 0.003), but not of the WHO free sugars guideline compared with other surveyed health professionals such as medical doctors or nurses. Conclusions: Knowledge gaps existed among surveyed Canadian health professionals on topics related to sugars consumption and the WHO sugars guideline. Future research should focus on tools to support better communication of sugars guideline and consistent use of sugars terminology.

A Quiescent Bcl11b High Stem Cell Population Is Required for Maintenance of the Mammary Gland.

Stem cells in many tissues sustain themselves by entering a quiescent state to avoid genomic insults and to prevent exhaustion caused by excessive proliferation. In the mammary gland, the identity and characteristics of quiescent epithelial stem cells are not clear. Here, we identify a quiescent mammary epithelial cell population expressing high levels of Bcl11b and located at the interface between luminal and basal cells. Bcl11bhigh cells are enriched for cells that can regenerate mammary glands in secondary transplants. Loss of Bcl11b leads to a Cdkn2a-dependent exhaustion of ductal epithelium and loss of epithelial cell regenerative capacity. Gain- and loss-of-function studies show that Bcl11b induces cells to enter the G0 phase of the cell cycle and become quiescent. Taken together, these results suggest that Bcl11b acts as a central intrinsic regulator of mammary epithelial stem cell quiescence and exhaustion and is necessary for long-term maintenance of the mammary gland.

A Drosophila LexA Enhancer-Trap Resource for Developmental Biology and Neuroendocrine Research.

Novel binary gene expression tools like the LexA-LexAop system could powerfully enhance studies of metabolism, development, and neurobiology in Drosophila However, specific LexA drivers for neuroendocrine cells and many other developmentally relevant systems remain limited. In a unique high school biology course, we generated a LexA-based enhancer trap collection by transposon mobilization. The initial collection provides a source of novel LexA-based elements that permit targeted gene expression in the corpora cardiaca, cells central for metabolic homeostasis, and other neuroendocrine cell types. The collection further contains specific LexA drivers for stem cells and other enteric cells in the gut, and other developmentally relevant tissue types. We provide detailed analysis of nearly 100 new LexA lines, including molecular mapping of insertions, description of enhancer-driven reporter expression in larval tissues, and adult neuroendocrine cells, comparison with established enhancer trap collections and tissue specific RNAseq. Generation of this open-resource LexA collection facilitates neuroendocrine and developmental biology investigations, and shows how empowering secondary school science can achieve research and educational goals.

Folate, vitamin B12, and vitamin B6 status of a group of high socioeconomic status women in the Alberta Pregnancy Outcomes and Nutrition (APrON) cohort.

Folic acid supplementation and food fortification policies have improved folate status in North American women of child bearing age. Recent studies have reported the possible inadequacy of vitamin B12 and B6 in the etiology of neural tube defects in folate-fortified populations. The aims of this study were to describe folate status and its relationship to supplementation and to assess vitamin B12 and B6 status in a cohort of pregnant women. Supplement intake data were collected in each trimester from the first cohort (n = 599) of the Alberta Pregnancy Outcomes and Nutrition (APrON) study. Red blood cell folate (RBCF) and plasma folate, holotranscobalamin, and pyridoxal 5-phosphate were measured. Overt folate deficiency was rare (3%) but 24% of women in their first trimester had suboptimal RBCF concentration (<906 nmol·L(-1)). The proportion of the cohort in this category declined substantially in second (9%) and third (7%) trimesters. High RBCF (>1360 nmol·L(-1)) was observed in approximately half of the women during each pregnancy trimester. Vitamin B12 and B6 deficiencies were rare (<1% of the cohort). Women consuming folic acid supplements above the upper level had significantly higher RBCF and plasma folate concentrations. In conclusion, the prevalence of vitamin B12 and B6 deficiency was very low. A quarter of the women had suboptimal folate status in the first trimester of pregnancy and over half the women had abnormally high RBCF, suggesting that supplementation during pregnancy is not appropriate in a cohort of women considered to be healthy and a low risk for nutritional deficiencies.

Effects of filtering visual short wavelengths during nocturnal shiftwork on sleep and performance.

Circadian phase resetting is sensitive to visual short wavelengths (450-480 nm). Selectively filtering this range of wavelengths may reduce circadian misalignment and sleep impairment during irregular light-dark schedules associated with shiftwork. We examined the effects of filtering short wavelengths (<480 nm) during night shifts on sleep and performance in nine nurses (five females and four males; mean age ± SD: 31.3 ± 4.6 yrs). Participants were randomized to receive filtered light (intervention) or standard indoor light (baseline) on night shifts. Nighttime sleep after two night shifts and daytime sleep in between two night shifts was assessed by polysomnography (PSG). In addition, salivary melatonin levels and alertness were assessed every 2 h on the first night shift of each study period and on the middle night of a run of three night shifts in each study period. Sleep and performance under baseline and intervention conditions were compared with daytime performance on the seventh day shift, and nighttime sleep following the seventh daytime shift (comparator). On the baseline night PSG, total sleep time (TST) (p < 0.01) and sleep efficiency (p = 0.01) were significantly decreased and intervening wake times (wake after sleep onset [WASO]) (p = 0.04) were significantly increased in relation to the comparator night sleep. In contrast, under intervention, TST was increased by a mean of 40 min compared with baseline, WASO was reduced and sleep efficiency was increased to levels similar to the comparator night. Daytime sleep was significantly impaired under both baseline and intervention conditions. Salivary melatonin levels were significantly higher on the first (p < 0.05) and middle (p < 0.01) night shifts under intervention compared with baseline. Subjective sleepiness increased throughout the night under both conditions (p < 0.01). However, reaction time and throughput on vigilance tests were similar to daytime performance under intervention but impaired under baseline on the first night shift. By the middle night shift, the difference in performance was no longer significant between day shift and either of the two night shift conditions, suggesting some adaptation to the night shift had occurred under baseline conditions. These results suggest that both daytime and nighttime sleep are adversely affected in rotating-shift workers and that filtering short wavelengths may be an approach to reduce sleep disruption and improve performance in rotating-shift workers.

Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1.

Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization.

Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.

BACKGROUND: High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. CONCLUSIONS: We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

Granulocyte-macrophage-colony-stimulating factor added to a multipeptide vaccine for resected Stage II melanoma.

BACKGROUND: Forty-eight patients with resected Stages IIA and IIB melanoma were immunized with two tumor antigen epitope peptides derived from gp100(209-217) (210M) (IMDQVPSFV) and tyrosinase(368-376) (370D) (YMDGTMSQV) emulsified with incomplete Freund's adjuvant (IFA). Patients were assigned randomly to receive either peptides/IFA alone or with 250 microm of granulocyte-macrophage-colony-stimulating factor (GM-CSF) subcutaneously daily for 5 days to evaluate the toxicities and immune responses in either arm. Time to recurrence and survival were secondary end points. METHODS: Immunizations were administered every 2 weeks x 4, then every 4 weeks x 3, and once 8 weeks later. A leukapheresis to obtain peripheral blood mononuclear cells for immune analyses and skin testing with peptides and recall reagents was performed before and after eight vaccinations. RESULTS: Local pain and granuloma formation, fever, and lethargy of Grade 1 or 2 were observed. Transient vaccine-related Grade III and no Grade IV toxicity was observed. Seventeen of the 40 patients for whom posttreatment skin tests were performed developed a positive skin test response to the gp100 peptide, but only 1 of the 40 patients developed a positive skin test response to tyrosinase. Immune responses were measured by release of interferon-gamma (IFN-gamma) in an enzyme-linked immunosorbent assay (ELISA) by effector cells in the presence of peptide-pulsed antigen-presenting cells, by cytokine release of IFN-gamma, GM-CSF, and tumor necrosis factor-alpha in a Luminex assay, or by an antigen-specific tetramer flow cytometry assay. Thirty-four of the 39 patients for whom the ELISA data were performed demonstrated an immune response after vaccination, as did 37 of 42 patients by tetramer assay. Enzyme-linked immunosorbent assay, Luminex, and tetramer responses in the GM-CSF/peptide/IFA group were higher than in the peptide/IFA group. Epitope spreading to the MART-1/MelanA 27-35 and 26-35 (27L) epitopes was detected by tetramer assay in 10 patients. Seven of 48 patients experienced disease recurrence with a median of 24 months of follow-up and 2 patients in this intermediate to high risk group have died. CONCLUSION: These data suggest a significant number of patients with resected melanoma mount an antigen-specific immune response against a peptide vaccine. There is a trend for GM-CSF to modestly increase the immune response and support further development of GM-CSF as a vaccine adjuvant.

Totally endoscopic atrial septal defect repair with robotic assistance.

BACKGROUND: The development of minimally invasive cardiac surgery has been characterized by the performance of increasingly complex operations through progressively smaller incisions. Computer (robotic) enhancement has emerged as a potential facilitator of these procedures, initially by providing enhanced endoscopic camera control and, more recently, by allowing the manipulation of surgical instruments through limited thoracic incisions. This report describes the next step in this progression, namely the performance of an atrial septal defect (ASD) repair entirely through thoracoscopic port incisions. This represents the first U.S. application of robotic technology for totally endoscopic open-heart surgery. MATERIALS AND METHODS: A 33-year-old woman with a secundum atrial septal defect underwent totally endoscopic repair through four port incisions by means of the Da Vinci (Intuitive Surgical, Mountain View, CA) robotic surgical system. Cardiopulmonary bypass was achieved peripherally (femoral Estech endoaortic balloon cannula; femoral and right internal jugular venous Bio-medicus cannulae). The myocardium was protected with antegrade cold blood cardioplegia delivered through the distal port of the arterial cannula. After port insertion, the entire operation, including pericardiotomy, bicaval occlusion, atriotomy, atrial septopexy, and atrial closure, was performed by computer-aided control of a camera and two instrument arms manipulated by a surgeon seated 15 feet away. The fourth port was used for suction and suture passage by the patient-side assistant. The aortic cross-clamp time was 43 minutes, and the postoperative transesophageal echocardiogram demonstrated normal ventricular function and the absence of interatrial shunting. The patient was extubated on the night of surgery, was ambulatory within 15 hours, and was discharged on the morning of postoperative day 3, 63 hours after the procedure. At 30-day follow-up, the patient was well and without complaints, and transthoracic echocardiogram confirmed the continued absence of interatrial shunting. CONCLUSIONS: Computer-aided robotic surgical technology can be used to perform open-heart procedures with a totally endoscopic approach. The benefits of this approach may include decreased perioperative pain, decreased recovery times, and improved cosmesis and patient acceptance. Clinical trials currently in progress will demonstrate whether this technology will be of reproducible value in the management of patients with intracardiac disease on a larger scale.