Evolved Resistance to a Novel Cationic Peptide Antibiotic Requires High Mutation Supply

Background and Objectives A key strategy for resolving the antibiotic resistance crisis is the development of new drugs with antimicrobial properties. The engineered cationic antimicrobial peptide WLBU2 (also known as PLG0206) is a promising broad-spectrum antimicrobial compound that has completed Phase I clinical studies. It has activity against Gram-negative and Gram-positive bacteria including infections associated with biofilm. No definitive mechanisms of resistance to WLBU2 have been identified. Methodology Here, we used experimental evolution under different levels of mutation supply and whole genome sequencing (WGS) to detect the genetic pathways and probable mechanisms of resistance to this peptide. We propagated populations of wild-type and hypermutator Pseudomonas aeruginosa in the presence of WLBU2 and performed WGS of evolved populations and clones. Results Populations that survived WLBU2 treatment acquired a minimum of two mutations, making the acquisition of resistance more difficult than for most antibiotics, which can be tolerated by mutation of a single target. Major targets of resistance to WLBU2 included the orfN and pmrB genes, previously described to confer resistance to other cationic peptides. More surprisingly, mutations that increase aggregation such as the wsp pathway were also selected despite the ability of WLBU2 to kill cells growing in a biofilm. Conclusions and implications The results show how experimental evolution and WGS can identify genetic targets and actions of new antimicrobial compounds and predict pathways to resistance of new antibiotics in clinical practice

Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage, A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. the mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. the variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. the new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (K-i 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase, the variant LDTI-10T binds to thrombin but does not inhibit it, the relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V), the data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions. (C) 1999 Federation of European Biochemical Societies.UNIFESP, Dept Bioquim, EPM, BR-04044020 São Paulo, BrazilUniv Munich, Klinikum Innenstadt, Chirurg Klin & Poliklin, Klin Chem & Klin Biochem Abt, D-8000 Munich, GermanyUNIFESP, Dept Med, Disciplina Hematol, EPM, BR-04044020 São Paulo, BrazilUNIFESP, Dept Bioquim, EPM, BR-04044020 São Paulo, BrazilUNIFESP, Dept Med, Disciplina Hematol, EPM, BR-04044020 São Paulo, BrazilWeb of Scienc

Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo- mouse model of FKRP-deficient muscular dystrophy.

BACKGROUND: Fukutin-related protein (FKRP) mutations are the most common cause of dystroglycanopathies known to cause both limb girdle and congenital muscular dystrophy. The P448Lneo- mouse model has a knock-in mutation in the FKRP gene and develops skeletal, respiratory, and cardiac muscle disease. METHODS: We studied the natural history of the P448Lneo- mouse model over 9 months and the effects of twice weekly treadmill running. Forelimb and hindlimb grip strength (Columbus Instruments) and overall activity (Omnitech Electronics) assessed skeletal muscle function. Echocardiography was performed using VisualSonics Vevo 770 (FujiFilm VisualSonics). Plethysmography was performed using whole body system (ADInstruments). Histological evaluations included quantification of inflammation, fibrosis, central nucleation, and fiber size variation. RESULTS: P448Lneo- mice had significantly increased normalized tissue weights compared to controls at 9 months of age for the heart, gastrocnemius, soleus, tibialis anterior, quadriceps, and triceps. There were no significant differences seen in forelimb or hindlimb grip strength or activity monitoring in P448Lneo- mice with or without exercise compared to controls. Skeletal muscles demonstrated increased inflammation, fibrosis, central nucleation, and variation in fiber size compared to controls (p \u3c 0.05) and worsened with exercise. Plethysmography showed significant differences in respiratory rates and decreased tidal and minute volumes in P448Lneo- mice (p \u3c 0.01). There was increased fibrosis in the diaphragm compared to controls (p \u3c 0.01). Echocardiography demonstrated decreased systolic function in 9-month-old mutant mice (p \u3c 0.01). There was increased myocardial wall thickness and mass (p \u3c 0.001) with increased fibrosis in 9-month-old P448Lneo- mice compared to controls (p \u3c 0.05). mRNA expression for natriuretic peptide type A (Nppa) was significantly increased in P448Lneo- mice compared to controls at 6 months (p \u3c 0.05) and for natriuretic peptide type B (Nppb) at 6 and 9 months of age (p \u3c 0.05). CONCLUSIONS: FKRP-deficient P448Lneo- mice demonstrate significant deficits in cardiac and respiratory functions compared to control mice, and this is associated with increased inflammation and fibrosis. This study provides new functional outcome measures for preclinical trials of FKRP-related muscular dystrophies

Comparisons of dissolved organic matter and its optical characteristics in small low and high Arctic catchments

Climate change is affecting the rate of carbon cycling, particularly in the Arctic. Permafrost degradation through deeper thaw and physical disturbances results in the release of carbon dioxide and methane to the atmosphere and to an increase in lateral dissolved organic matter (DOM) fluxes. Whereas riverine DOM fluxes of the large Arctic rivers are well assessed, knowledge is limited with regard to small catchments that cover more than 40 % of the Arctic drainage basin. Here, we use absorption measurements to characterize changes in DOM quantity and quality in a low Arctic (Herschel Island, Yukon, Canada) and a high Arctic (Cape Bounty, Melville Island, Nunavut, Canada) setting with regard to geographical differences, impacts of permafrost degradation, and rainfall events. We find that DOM quantity and quality is controlled by differences in vegetation cover and soil organic carbon content (SOCC). The low Arctic site has higher SOCC and greater abundance of plant material resulting in higher chromophoric dissolved organic matter (cDOM) and dissolved organic carbon (DOC) than in the high Arctic. DOC concentration and cDOM in surface waters at both sites show strong linear relationships similar to the one for the great Arctic rivers. We used the optical characteristics of DOM such as cDOM absorption, specific ultraviolet absorbance (SUVA), ultraviolet (UV) spectral slopes (S275–295), and slope ratio (SR) for assessing quality changes downstream, at base flow and storm flow conditions, and in relation to permafrost disturbance. DOM in streams at both sites demonstrated optical signatures indicative of photodegradation downstream processes, even over short distances of 2000 m. Flow pathways and the connected hydrological residence time control DOM quality. Deeper flow pathways allow the export of permafrost-derived DOM (i.e. from deeper in the active layer), whereas shallow pathways with shorter residence times lead to the export of fresh surface- and near-surface-derived DOM. Compared to the large Arctic rivers, DOM quality exported from the small catchments studied here is much fresher and therefore prone to degradation. Assessing optical properties of DOM and linking them to catchment properties will be a useful tool for understanding changing DOM fluxes and quality at a pan-Arctic scale

Skunk River Review Fall 2001, vol 13

https://openspace.dmacc.edu/skunkriver/1022/thumbnail.jp

Colorectal and other cancer risks for carriers and noncarriers from families with a DNA mismatch repair gene mutation: A Prospective Cohort Study

To determine whether cancer risks for carriers and noncarriers from families with a mismatch repair (MMR) gene mutation are increased above the risks of the general population. We prospectively followed a cohort of 446 unaffected carriers of an MMR gene mutation (MLH1, n = 161; MSH2, n = 222; MSH6, n = 47; and PMS2, n = 16) and 1,029 their unaffected relatives who did not carry a mutation every 5 years at recruitment centers of the Colon Cancer Family Registry. For comparison of cancer risk with the general population, we estimated country-, age-, and sex-specific standardized incidence ratios (SIRs) of cancer for carriers and noncarriers. Over a median follow-up of 5 years, mutation carriers had an increased risk of colorectal cancer (CRC; SIR, 20.48; 95% CI, 11.71 to 33.27; P < .001), endometrial cancer (SIR, 30.62; 95% CI, 11.24 to 66.64; P < .001), ovarian cancer (SIR, 18.81; 95% CI, 3.88 to 54.95; P < .001), renal cancer (SIR, 11.22; 95% CI, 2.31 to 32.79; P < .001), pancreatic cancer (SIR, 10.68; 95% CI, 2.68 to 47.70; P = .001), gastric cancer (SIR, 9.78; 95% CI, 1.18 to 35.30; P = .009), urinary bladder cancer (SIR, 9.51; 95% CI, 1.15 to 34.37; P = .009), and female breast cancer (SIR, 3.95; 95% CI, 1.59 to 8.13; P = .001). We found no evidence of their noncarrier relatives having an increased risk of any cancer, including CRC (SIR, 1.02; 95% CI, 0.33 to 2.39; P = .97). We confirmed that carriers of an MMR gene mutation were at increased risk of a wide variety of cancers, including some cancers not previously recognized as being a result of MMR mutations, and found no evidence of an increased risk of cancer for their noncarrier relatives

LSST: from Science Drivers to Reference Design and Anticipated Data Products

(Abridged) We describe here the most ambitious survey currently planned in the optical, the Large Synoptic Survey Telescope (LSST). A vast array of science will be enabled by a single wide-deep-fast sky survey, and LSST will have unique survey capability in the faint time domain. The LSST design is driven by four main science themes: probing dark energy and dark matter, taking an inventory of the Solar System, exploring the transient optical sky, and mapping the Milky Way. LSST will be a wide-field ground-based system sited at Cerro Pach\'{o}n in northern Chile. The telescope will have an 8.4 m (6.5 m effective) primary mirror, a 9.6 deg$^2$ field of view, and a 3.2 Gigapixel camera. The standard observing sequence will consist of pairs of 15-second exposures in a given field, with two such visits in each pointing in a given night. With these repeats, the LSST system is capable of imaging about 10,000 square degrees of sky in a single filter in three nights. The typical 5$\sigma$ point-source depth in a single visit in $r$ will be $\sim 24.5$ (AB). The project is in the construction phase and will begin regular survey operations by 2022. The survey area will be contained within 30,000 deg$^2$ with $\delta<+34.5^\circ$, and will be imaged multiple times in six bands, $ugrizy$, covering the wavelength range 320--1050 nm. About 90\% of the observing time will be devoted to a deep-wide-fast survey mode which will uniformly observe a 18,000 deg$^2$ region about 800 times (summed over all six bands) during the anticipated 10 years of operations, and yield a coadded map to $r\sim27.5$. The remaining 10\% of the observing time will be allocated to projects such as a Very Deep and Fast time domain survey. The goal is to make LSST data products, including a relational database of about 32 trillion observations of 40 billion objects, available to the public and scientists around the world.Comment: 57 pages, 32 color figures, version with high-resolution figures available from https://www.lsst.org/overvie

The Eco-Epidemiology of Pacific Coast Tick Fever in California

Rickettsia philipii (type strain “Rickettsia 364D”), the etiologic agent of Pacific Coast tick fever (PCTF), is transmitted to people by the Pacific Coast tick, Dermacentor occidentalis. Following the first confirmed human case of PCTF in 2008, 13 additional human cases have been reported in California, more than half of which were pediatric cases. The most common features of PCTF are the presence of at least one necrotic lesion known as an eschar (100%), fever (85%), and headache (79%); four case-patients required hospitalization and four had multiple eschars. Findings presented here implicate the nymphal or larval stages of D. occidentalis as the primary vectors of R. philipii to people. Peak transmission risk from ticks to people occurs in late summer. Rickettsia philipii DNA was detected in D. occidentalis ticks from 15 of 37 California counties. Similarly, non-pathogenic Rickettsia rhipicephali DNA was detected in D. occidentalis in 29 of 38 counties with an average prevalence of 12.0% in adult ticks. In total, 5,601 ticks tested from 2009 through 2015 yielded an overall R. philipii infection prevalence of 2.1% in adults, 0.9% in nymphs and a minimum infection prevalence of 0.4% in larval pools. Although most human cases of PCTF have been reported from northern California, acarological surveillance suggests that R. philipii may occur throughout the distribution range of D. occidentalis