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The phylogenetic placement of Phragmotrichum chailletii in Melanommataceae (Pleosporales) is re-evaluated and its phylogeographic structure is assessed based on a large number of specimens from Europe and North America together with a four-gene (ITS, LSU, RPB2, EF1-α) dataset. Morphologically, all collections produced identical conidiomata and conidia on the same substrate, which is spruce cones on litter. Their fruiting season was also similar as they originated from early spring soon after snow melts at a similar range of elevation. However, all isolates collected in Canada and associated with Picea species native to North America, clustered together in a clade separate from collections made in Europe and occurring on host species native to central-eastern Europe. A sibling species, Ph. thornhilliae, is introduced to accommodate this group of isolates using molecular and phylogeographic evidence. One specimen belonging to the novel species was also collected in Switzerland. Possible scenarios to explain this anomaly are provided and the most likely explanation is an accidental, human mediated introduction.

Jessica Peterson stared out the window of her family's white mini-van and sighed. Huge raindrops slid down the tinted glass. I'll never be able to work on my tree house [...]

ABSTRACTUsing the USA300, methicillin-resistant Staphylococcus aureusstrain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarAand sigBattenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB, and sarAmutants was increased protease production, with mutation of rothaving the least impact followed by mutation of codY, sigBand sarA, respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarAand sigBmutants, but not in the rotmutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarAand sigBmutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigBand rotmutants in a murine sepsis model but did not significantly impact the virulence of the codYmutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureusand that Rot, SigB and SarA play critical roles in this regard.

During the last decade, several analyses of prognostic factors for differentiated thyroid cancer (DTC) have been reported. Although these studies have established a framework for rational treatment planning, they have not fully answered questions regarding the prognostic significance of cervical lymph node metastases. An analysis of patients treated for DTC at our institution over a 34-year period has shown several factors to be significant by log-rank analysis, including the presence of cervical lymph node metastases, age greater than or equal to 50 years, a primary cancer size of greater than 3.O cm, and distant metastases. Further analysis has shown the node*negative and node-positive patient groups to be similar in regard to age, size of primary cancer, and the presence of distant metastases. This report compares our data with those of other studies that have investigated the association of cervical lymph node metastases and a poorer prognosis in patients with DTC. When considered as a group, these studies support the finding of the prognostic significance of cervical lymph node metastases, particularly that of palpable lymphadenopathy in older patients.

Mutation of purRwas previously shown to enhance the virulence of Staphylococcus aureusin a murine sepsis model, and this cannot be fully explained by increased expression of genes within the purine biosynthesis pathway. Rather, the increased production of specific S. aureusvirulence factors, including alpha toxin and the fibronectin-binding proteins, was shown to play an important role.

SUMMARYUsing DNA affinity chromatography we demonstrate that the S. aureusregulatory proteins MgrA, Rot, SarA, and SarS bind DNA baits derived from the promoter regions associated with the genes encoding aureolysin, ScpAB, SspABC, and SplA-F. Three of four baits also bound SarR and SarZ, the exception in both cases being the ScpAB-associated bait. Using the USA300, methicillin-resistant strain LAC and the USA200, methicillin-sensitive strain UAMS-1, we generated mutations in the genes encoding each of these proteins alone and in combination with sarAand examined the impact on protease production, the accumulation of high molecular weight proteins, and biofilm formation. These studies confirmed that multiple regulatory loci are involved in limiting protease production to a degree that impacts all of these phenotypes, but also demonstrate that sarAplays a predominant role in this regard. Using sarAmutants unable to produce individual proteases alone and in combination with each other, we also demonstrate that the increased production of aureolysin and ScpA is particularly important in defining the biofilm-deficient phenotype of LAC and UAMS-1 sarAmutants, while aureolysin alone plays a key role in defining the reduced accumulation of alpha toxin and overall cytotoxicity as assessed using both osteoblasts and osteoclasts.

ABSTRACTWe previously demonstrated that MgrA, SarA, SarR, SarS, SarZ, and Rot bind at least three of the four promoters associated with genes encoding primary extracellular proteases in Staphylococcus aureus(Aur, ScpA, SspA/SspB, SplA-F). We also showed that mutation of sarAresults in a greater increase in protease production, and decrease in biofilm formation, than mutation of the loci encoding any of these other proteins. However, these conclusions were based on in vitrostudies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteins in vivo. To this end, we compared the virulence of mgrA, sarA, sarR, sarS, sarZ, and rotmutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1, which was isolated directly from the bone of an osteomyelitis patient during surgical debridement. Mutation of mgrAand rotlimited virulence to a statistically significant extent in UAMS-1, but not in LAC, while the sarAmutant exhibited reduced virulence in both strains. The reduced virulence of the sarAmutant was correlated with reduced cytotoxicity for osteoblasts and osteoclasts, reduced biofilm formation, and reduced sensitivity to the antimicrobial peptide indolicidin, all of which were directly attributable to increased protease production in both LAC and UAMS-1. These results illustrate the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence and demonstrate the significance of SarA in limiting protease production in vivoin S. aureus.

The staphylococcal accessory regulator A (sarA) impacts the extracellular accumulation of Staphylococcus aureusvirulence factors at the level of intracellular production and extracellular protease-mediated degradation. We previously used a proteomics approach that measures protein abundance of all proteoforms to demonstrate that mutation of sarAresults in increased levels of extracellular proteases and assesses the impact of this on the accumulation of S. aureusexoproteins. Our previous approach was limited as it did not take into account that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, our goal was to use an expanded proteomics approach utilizing a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels to alleviate these false negatives and thereby provide for characterization of protease-dependent and -independent effects of sarAmutation on the S. aureusexoproteome. Proteins present in conditioned medium from overnight, stationary phase cultures of the USA300strain LAC, an isogenic sarAmutant, and a sarAmutant unable to produce any of the known extracellular proteases (sarA/protease) were resolved using one-dimensional gel electrophoresis. Quantitative proteomic comparisons of sarAversus sarA/proteasemutants identified proteins that were cleaved in a protease-dependent manner owing to mutation of sarA, and comparisons of sarA/proteasemutant versus the LAC parent strain identified proteins in which abundance was altered in a sarAmutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This expanded approach provided for a more comprehensive analysis of the impact of mutating sarAon the S. aureusexoproteome.

ABSTRACTStaphylococcus aureuscauses acute and chronic forms of infection, the latter often associated with formation of a biofilm. It has previously been demonstrated that mutation of atl, codY, rot, sarA, and sigBlimits biofilm formation in the USA300 strain LAC while mutation of agr, fur, and mgrAhas the opposite effect. Here we used a murine sepsis model to assess the impact of these same loci in acute infection. Mutation of agr, atl, and furhad no impact on virulence, while mutation of mgrAand rotincreased virulence. In contrast, mutation of codY, sarA, and sigBsignificantly attenuated virulence. Mutation of sigBresulted in reduced accumulation of AgrA and SarA, while mutation of sarAresulted in reduced accumulation of AgrA, but this cannot account for the reduced virulence of sarAor sigBmutants because the isogenic agrmutant was not attenuated. Indeed, as assessed by accumulation of alpha toxin and protein A, all of the mutants we examined exhibited unique phenotypes by comparison to an agrmutant and to each other. Attenuation of the sarA, sigBand codYmutants was correlated with increased production of extracellular proteases and global changes in extracellular protein profiles. These results suggest that the inability to repress the production of extracellular proteases plays a key role in attenuating the virulence of S. aureusin acute as well as chronic, biofilm-associated infections, thus opening up the possibility that strategies aimed at the de-repression of protease production could be used to broad therapeutic advantage. They also suggest that the impact of codY, sarA, and sigBon protease production occurs via an agr-independent mechanism.

This study investigated the effects of injectable vitamin C (VC) at weaning and prior to transit on growth performance and immune function in early-weaned beef steers. On day 0, 91 Angus × Simmental steers (92 ± 4 kg) were weaned (65 ± 11 d of age), given vaccination boosters, blocked by age, and randomly assigned to weaning (WEAN) treatments: intramuscular injections (20 mL per steer) of VC (250-mg sodium ascorbate per mL; 5 g per steer) or saline (SAL). From days 0 to 48, steers were housed at the Dixon Springs Agricultural Center (Simpson, IL) in pens (six pens; N= 14 to 16 steers per pen) equipped with two to three Vytelle bunks to measure individual daily feed disappearance. On day 49, half of the steers in each WEAN treatment were randomly assigned to an additional injection treatment (20 mL per steer) of VC or SAL prior to transport (TRANS). After administering pretransit injections, all steers were loaded onto a commercial livestock trailer with equal representation of treatments across compartments. Steers were transported for 6 h (approximately 480 km) to the Illinois Beef and Sheep Field Laboratory (Urbana, IL). Upon arrival, steers were sorted into pens (six pens; N= 13 to 17 steers per pen) with 2 Vytelle bunks per pen. Steers were weighed on days 0, 1, 14, 48, 49, 64, 78, 106, and 107. Blood was collected (WEAN = 24 steers per treatment; TRANS = 12 steers per treatment) on days 0, 1, 2, 14, 49 (pre- and posttransit), 50, and 51. Data were analyzed using the MIXED procedure of SAS 9.4 with fixed effects of age block, WEAN, TRANS, and WEAN × TRANS. Plasma ascorbate concentrations were greater (WEAN × time P< 0.01) on days 1 and 2 for steers that received VC at weaning. Similarly, for steers that received VC on day 49 pretransit, ascorbate concentrations were greater (TRANS × time P= 0.04) on days 49 posttransit, 50, and 51. Treatments did not affect (P≥ 0.13) body weight, average daily gain, or gain to feed throughout the trial. Serum Bovine Viral Diarrhea Virus type 1 and 2 antibody titers on days 14 and 51 were not affected (P≥ 0.32) by treatment. Injectable VC administered to early-weaned beef steers at the time of weaning or pretransit increased plasma ascorbate concentrations but did not improve growth performance or antibody response to vaccination booster.Beef calves weaned at a younger age may encounter greater physiological or oxidative stress during weaning and transit. This research examined the ability of an injectable vitamin C to affect growth performance and immune function in early-weaned beef calves.Weaning and transit represent the primary stressors for beef calves in the United States and are responsible for increasing inflammation, suppressing the immune system, and decreasing antioxidant status. These adverse physiological responses to stressors may decrease growth and increase morbidity in beef calves. Vitamin C is the primary water-soluble antioxidant in plasma and when provided intramuscularly prior to the stress event, may be able to attenuate aspects of a stress response on growth and immune function. This study evaluated the effects of injectable vitamin C given to early-weaned beef calves prior to weaning on day 0 and a 6-h transit on day 49 after weaning. Basal levels of plasma ascorbate were lower than prior studies in older and larger animals. As expected, injectable vitamin C rapidly increased plasma ascorbate concentrations at 24 h, but concentrations also increased in control calves receiving a saline injection. Treatments did not affect overall growth performance or dry matter intake. Treatments also did not impact the immune response to a booster vaccination provided at weaning. While other research has indicated a positive effect of injectable vitamin C prior to transit, additional research is needed to refine the dosage and physiological need for exogenous antioxidants like vitamin C based on the severity and duration of a stress event in lightweight beef calves.

The objective was to determine the effects of injectable vitamin E (VE) before or after transit on feedlot cattle receiving performance, health, and blood parameters. Angus × Simmental steers (n= 196; body weight [BW] = 163 ± 29 kg) were utilized in a randomized complete block design. Steers were blocked by BW and randomly assigned to 1 of 3 treatments: intramuscular injections of saline pre- and post-transit (CON), intramuscular injections of VE (2,000 mg d-α-tocopherol) pre-transit and saline post-transit (PRE), or intramuscular injections of saline pre-transit and VE (2,000 mg d-α-tocopherol) post-transit (POST). Pre-transit injections were administered on day 0, and steers were transported on day 7 for approximately 4 h (348 km). After arrival, steers were fed a common corn silage-based diet in GrowSafe bunks. Final BW tended to be greater (P= 0.08) for CON steers compared with POST steers while PRE steers were intermediate. From days 7 to 63, treatment affected average daily gain (ADG) with PRE and CON steers exhibiting (P= 0.04) greater ADG compared with POST steers. Dry matter intake (DMI), water intake, and gain to feed from days 7 to 63 were not affected (P≥ 0.17) by treatment. Day 0 serum α-tocopherol concentrations were considered marginal (2.3 ± 0.2 mg/l). A treatment × day interaction (P< 0.01) was observed for serum α-tocopherol concentrations. Serum α-tocopherol concentrations were greatest for PRE steers on day 7 (prior to and post-transit), but greater for POST steers on dys 10 and 14. Plasma ferric-reducing antioxidant potential concentrations increased (P= 0.04) for POST steers compared with CON steers and PRE steers being intermediate. Plasma non-esterified fatty acids (NEFA) concentrations exhibited a treatment × day interaction (P= 0.04) with CON and POST steers being 16% and 14% greater than PRE steers on day 14, respectively. On day 21, NEFA concentrations were greatest for POST steers compared with PRE steers and CON steers being intermediate. There was no main effect (P≥ 0.14) of treatment on the number of bovine respiratory disease morbidity treatments. Hair cortisol concentrations were decreased (P< 0.01) 14 days after transit for PRE and POST steers compared with CON steers. Overall, injectable VE administered before or after transit increased serum tocopherol concentrations while reducing stress, but did not improve the growth performance of beef steers during the receiving phase.Transportation stressors can negatively impact lightweight calf performance and health. An injection of VE before or after the transit may reduce stress through improved antioxidant status during the receiving phase.Cattle are transported multiple times throughout their lifespan due to the geographic distribution of the United States beef industry. However, transportation can elicit a variety of stressors that jeopardize cattle growth performance and health. Lightweight feeder calves are at the greatest risk for stress-related morbidity and mortality during the feedlot receiving phase. This study evaluated the effects of injectable vitamin E (VE) before or after transit on feedlot receiving phase growth performance, health, and blood parameters of lightweight beef steers. Steers receiving an injection of VE before or after transit had increased serum α-tocopherol concentrations. However, treatment with VE did not improve growth performance and feed intake. Steers injected with VE before or after transit experienced a decrease in hair cortisol concentrations 14 d after transit while steers injected with VE after transit had improved antioxidant status 14 d after transit compared with control steers and those receiving VE before transit. These results indicate that an injection of VE around the time of transit had no effect on growth performance and intake but can improve antioxidant status during the receiving phase.

ABSTRACTWe used in vitroand in vivomodels of catheter-associated biofilm formation to compare the relative activity of antibiotics effective against methicillin-resistant Staphylococcus aureus(MRSA) in the specific context of an established biofilm. The results demonstrated that, under in vitroconditions, daptomycin and ceftaroline exhibited comparable activity relative to each other and greater activity than vancomycin, telavancin, oritavancin, dalbavancin, or tigecycline. This was true when assessed using established biofilms formed by the USA300 methicillin-resistant strain LAC and the USA200 methicillin-sensitive strain UAMS-1. Oxacillin exhibited greater activity against UAMS-1 than LAC, as would be expected, since LAC is an MRSA strain. However, the activity of oxacillin was less than that of daptomycin and ceftaroline even against UAMS-1. Among the lipoglycopeptides, telavancin exhibited the greatest overall activity. Specifically, telavancin exhibited greater activity than oritavancin or dalbavancin when tested against biofilms formed by LAC and was the only lipoglycopeptide capable of reducing the number of viable bacteria below the limit of detection. With biofilms formed by UAMS-1, telavancin and dalbavancin exhibited comparable activity relative to each other and greater activity than oritavancin. Importantly, ceftaroline was the only antibiotic that exhibited greater activity than vancomycin when tested in vivoin a murine model of catheter-associated biofilm formation. These results emphasize the need to consider antibiotics other than vancomycin, most notably, ceftaroline, for the treatment of biofilm-associated S. aureusinfections, including by the matrix-based antibiotic delivery methods often employed for local antibiotic delivery in the treatment of these infections.

ABSTRACTWe used a murine model of acute, posttraumatic osteomyelitis to evaluate the virulence of two divergent Staphylococcus aureusclinical isolates (the USA300 strain LAC and the USA200 strain UAMS-1) and their isogenic sarAmutants. The results confirmed that both strains caused comparable degrees of osteolysis and reactive new bone formation in the acute phase of osteomyelitis. Conditioned medium (CM) from stationary-phase cultures of both strains was cytotoxic to cells of established cell lines (MC3TC-E1 and RAW 264.7 cells), primary murine calvarial osteoblasts, and bone marrow-derived osteoclasts. Both the cytotoxicity of CM and the reactive changes in bone were significantly reduced in the isogenic sarAmutants. These results confirm that sarAis required for the production and/or accumulation of extracellular virulence factors that limit osteoblast and osteoclast viability and that thereby promote bone destruction and reactive bone formation during the acute phase of S. aureusosteomyelitis. Proteomic analysis confirmed the reduced accumulation of multiple extracellular proteins in the LAC and UAMS-1 sarAmutants. Included among these were the alpha class of phenol-soluble modulins (PSMs), which were previously implicated as important determinants of osteoblast cytotoxicity and bone destruction and repair processes in osteomyelitis. Mutation of the corresponding operon reduced the cytotoxicity of CM from both UAMS-1 and LAC cultures for osteoblasts and osteoclasts. It also significantly reduced both reactive bone formation and cortical bone destruction by CM from LAC cultures. However, this was not true for CM from cultures of a UAMS-1 psmαmutant, thereby suggesting the involvement of additional virulence factors in such strains that remain to be identified.

ABSTRACTWe previously determined the extent to which mutations of different Staphylococcus aureusregulatory loci impact biofilm formation as assessed under in vitroconditions. Here we extend these studies to determine the extent to which those regulatory loci that had the greatest effect on biofilm formation also impact antibiotic susceptibility. The experiments were done under in vitroand in vivoconditions using two clinical isolates of S. aureus(LAC and UAMS-1) and two functionally diverse antibiotics (daptomycin and ceftaroline). Mutation of the staphylococcal accessory regulator (sarA) or sigBwas found to significantly increase susceptibilities to both antibiotics and in both strains in a manner that could not be explained by changes in the MICs. The impact of a mutation in sarAwas comparable to that of a mutation in sigBand greater than the impact observed with any other mutant. These results suggest that therapeutic strategies targeting sarAand/or sigBhave the greatest potential to facilitate the ability to overcome the intrinsic antibiotic resistance that defines S. aureusbiofilm-associated infections.

ABSTRACTWe demonstrate that mutation of xerC, which reportedly encodes a homologue of an Escherichia colirecombinase, limits biofilm formation in the methicillin-resistant Staphylococcus aureusstrain LAC and the methicillin-sensitive strain UAMS-1. This was not due to the decreased production of the polysaccharide intracellular adhesin (PIA) in either strain because the amount of PIA was increased in a UAMS-1 xerCmutant and undetectable in both LAC and its isogenic xerCmutant. Mutation of xerCalso resulted in the increased production of extracellular proteases and nucleases in both LAC and UAMS-1, and limiting the production of either class of enzymes increased biofilm formation in the isogenic xerCmutants. More importantly, the limited capacity to form a biofilm was correlated with increased antibiotic susceptibility in both strains in the context of an established biofilm in vivo. Mutation of xerCalso attenuated virulence in a murine bacteremia model, as assessed on the basis of the bacterial loads in internal organs and overall lethality. It also resulted in the decreased accumulation of alpha toxin and the increased accumulation of protein A. These findings suggest that xerCmay impact the functional status of agr. This was confirmed by demonstrating the reduced accumulation of RNAIII and AgrA in LAC and UAMS-1 xerCmutants. However, this cannot account for the biofilm-deficient phenotype of xerCmutants because mutation of agrdid not limit biofilm formation in either strain. These results demonstrate that xerCcontributes to biofilm-associated infections and acute bacteremia and that this is likely due to agr-independent and -dependent pathways, respectively.

Purpose:Congenital hypogonadotropic hypogonadism (CHH) and split hand/foot malformation (SHFM) are two rare genetic conditions. Here we report a clinical entity comprising the two.Methods:We identified patients with CHH and SHFM through international collaboration. Probands and available family members underwent phenotyping and screening for FGFR1 mutations. The impact of identified mutations was assessed by sequence- and structure-based predictions and/or functional assays.Results:We identified eight probands with CHH with (n = 3; Kallmann syndrome) or without anosmia (n = 5) and SHFM, seven of whom (88%) harbor FGFR1 mutations. Of these seven, one individual is homozygous for p.V429E and six individuals are heterozygous for p.G348R, p.G485R, p.Q594*, p.E670A, p.V688L, or p.L712P. All mutations were predicted by in silico analysis to cause loss of function. Probands with FGFR1 mutations have severe gonadotropin-releasing hormone deficiency (absent puberty and/or cryptorchidism and/or micropenis). SHFM in both hands and feet was observed only in the patient with the homozygous p.V429E mutation; V429 maps to the fibroblast growth factor receptor substrate 2a binding domain of FGFR1, and functional studies of the p.V429E mutation demonstrated that it decreased recruitment and phosphorylation of fibroblast growth factor receptor substrate 2a to FGFR1, thereby resulting in reduced mitogen-activated protein kinase signaling.Conclusion:FGFR1 should be prioritized for genetic testing in patients with CHH and SHFM because the likelihood of a mutation increases from 10% in the general CHH population to 88% in these patients.Genet Med 17 8, 651–659.

Porphyrin molecules are ubiquitous and play an important role in chemical and biological processes. Their wide panoply of functions may be controlled by modifying their peripheral substituents. Electron injection from the tip of a scanning tunneling microscope into single 5,10,15,20-tetrakis(4-bromophenyl)-porphyrin-cobalt molecules adsorbed to Ag(111) was used to controllably remove Br atoms from the molecule periphery. Spectroscopy of the differential conductance, together with density functional calculations, assign spectral features to the spectroscopic signatures of molecular frontier orbitals. Different molecular orbitals are visible in the spectra of intact and dehalogenated molecules. These findings combine single-molecule chemistry with a characterization of each product’s electronic structure.

A 144-day study assessed the effects of dietary energy and implant potency to determine optimum strategies for managing feed conversion (F:G) and marbling. Fifty-four Angus steers (327 ± 8 kg) from Iowa State University’s herd genetically selected for enhanced marbling were stratified by initial bodyweight (BW), ultrasound intramuscular fat, and age to a 2 × 3 factorial. Dietary treatments included: low energy (1.30 Mcal NEg/kg DM, 18% roughage level; LE) or high energy finishing ration (1.39 Mcal NEg/kg DM, 8% roughage; HE). Implant treatments (IMP; Merck) included: no implant (NOIMP), Revalor-IS (RIS), or Revalor-200 (R200) on d 0 and 74. Steers were fed via bunks capturing daily individual feed disappearance (n = 9 steers/treatment). Steers were weighed on d 0, 74, and 144 and harvested on d 145. Data were analyzed in Proc Mixed of SAS with fixed effects of diet, IMP, and interaction. No interactions were observed for feedlot performance (P >0.17). Steers fed HE had greater average daily gain (ADG) and final BW than LE steers (P < 0.01) while LE steers had greater F:G (P = 0.04). Final BW and ADG were greatest for R200, intermediate for RIS, and lowest for NOIMP (P < 0.01). F:G was greatest for NOIMP, intermediate for RIS, and lowest for R200 (P < 0.01). Steers fed HE had increased ribeye area (P < 0.01) and tended to have greater marbling score (P = 0.06; 809) than LE steers (769). While ribeye area increased in response to implant potency (P < 0.01), marbling score was not impacted (P = 0.21) by IMP (815, 771, 782, for NOIMP, RIS, R200, respectively). Overall, steers graded 100% Choice or higher and 55% Prime. These data suggest implants, when used appropriately, improve growth performance and efficiency in beef steers without compromising carcass quality.

We report on the solution‐based synthesis and characterization of three‐dimensional networks of ZnO nanoparticles where the formation of structures is achieved by covalently linking the nanocrystals with bifunctional organic ligands. The colloidal synthesis will be presented with application of two ligands that vary in size and binding sites. Furthermore we report on structural characterization of dried powders and thin films by means of X‐ray diffraction and electron microscopy in order to examine the regularity of the structures. We also present first investigations of the optical properties and electrical conductance behavior in lateral direction of the differently linked hybrid ZnO networks.

ABSTRACTUsing a rabbit model of postsurgical osteomyelitis, we demonstrate that incorporation of xylitol into polymethylmethacrylate (PMMA) bone cement enhances the elution of daptomycin under in vivoconditions. We also demonstrate that this can be correlated with an improved therapeutic outcome in the treatment of a chronic bone infection following surgical debridement.

Dasyspora gregaria, the single species of the allegedly monotypic rust genus Dasyspora(Basidiomycota, Pucciniales), was investigated by light microscopy and DNA sequencing (ITS1–5.8S–ITS2 region, partial LSU and SSU of the nuclear rDNA, mt cytochrome oxidase subunit 3). Both methods indicated that D. gregariais not a single species but can be split in 11 distinct taxa, each of which appear confined to a single Xylopiaspecies (Annonaceae) host. Herein nine of these are described as new. Both the phylogenetic analyses and morphology show that the species are grouped into two main clades designated Dasyspora gregariaand D. winteri. The first comprises D. gregaria, the type species of the genus, which is restricted to X. cayennensis,two new species on X. aromatica, D. segregariafrom northern South America and D. echinatafrom Brazil. The second clade is formed by D. winteri, recombined from Puccinia winterion X. sericea,and the new species D. amazonicaon X. amazonica, D. emarginataeon X. emarginata, D. frutescentison X. frutescens, D. ferrugineaeon X. frutescensvar. ferruginea, D. guianensison X. benthamii, D. mesoamericanaon X. frutescens, and D. nitidaeon X. nitida. Dasyspora frutescentisand D. mesoamericanawere not clearly distinguishable by their morphology and host associations but differed from another in their sequences and geographic distributions. They are considered cryptic species. An identification key and the distributions are given for all recognized species. Along with molecular data we discuss the systematic position of Dasysporain the Pucciniales.

ABSTRACTRecent studies suggest that extracellular DNA promotes biofilm formation in Staphylococcus aureusand, conversely, that extracellular nucleases limit the ability to form a biofilm. S. aureusproduces at least two extracellular nucleases, and in the study described in this report, we examined the impact of each of these nucleases on biofilm formation under both in vitroand in vivoconditions. Our results demonstrate that both nucleases impact biofilm formation in the clinical isolate UAMS-1. Under certain in vitroconditions, this impact is negative, with mutation of either or both of the nuclease genes (nuc1and nuc2) resulting in an enhanced capacity to form a biofilm. However, this effect was not apparent in vivoin a murine model of catheter-associated biofilm formation. Rather, mutation of either or both nuclease genes appeared to limit biofilm formation to a degree that could be correlated with increased susceptibility to daptomycin.

Recent studies suggest that extracellular DNA promotes biofilm formation in Staphylococcus aureus and, conversely, that extracellular nucleases limit the ability to form a biofilm. S. aureus produces at least two extracellular nucleases, and in the study described in this report, we examined the impact of each of these nucleases on biofilm formation under both in vitro and in vivo conditions. Our results demonstrate that both nucleases impact biofilm formation in the clinical isolate UAMS-1. Under certain in vitro conditions, this impact is negative, with mutation of either or both of the nuclease genes (nuc1 and nuc2) resulting in an enhanced capacity to form a biofilm. However, this effect was not apparent in vivo in a murine model of catheter-associated biofilm formation. Rather, mutation of either or both nuclease genes appeared to limit biofilm formation to a degree that could be correlated with increased susceptibility to daptomycin.

Selbstständige Versicherungsvermittler haben eine außerordentlich hohe Bedeutung für die Versicherungsbranche, da sie über 90% des Versicherungsgeschäfts vermitteln und die Kunden betreuen. über die Wettbewerbsstruktur dieses Teilmarktes ist dennoch wenig bekannt. Im vorliegenden Beitrag wird die Wettbewerbsstruktur auf dem deutschen Versicherungsvermittlungsmarkt erstmals umfassend untersucht. Diese Studie betrachtet den Versicherungsvermittlungsmarkt als eigenständigen Markt und deren Akteure nicht nur als reine Absatzorgane von Versicherungsunternehmen. Basierend auf Porters Branchenstrukturmodell wurden Versicherungsvermittler zu ihrer Einschätzung des Wettbewerbs innerhalb des Versicherungsvermittlungsmarktes und zu ihrem Umsatz befragt. Mittels eines Regressionsmodells wird der Zusammenhang zwischen Branchenstruktur und Umsatz analysiert. Die Ergebnisse belegen, dass die Wettbewerbsstruktur einen deutlichen Einfluss auf den Umsatzerfolg der Versicherungsvermittler besitzt. Insurance agents (including independent insurance brokers) have an extraordinarily high importance for the German insurance industry since they mediate over 90% of insurance companies’ sales volume and provide additional services for customers and policyholders. Nevertheless little is known about the market structure within the insurance agents’ market. Insurance agents are mostly known as a distribution entity of insurance companies. Therefore only limited research has focused on the market of German insurance agents as an independent market. The following article focuses on the market structure of the German insurance agents market which is comprehensively examined for the first time. This study considers the insurance agent market as an independent market and their participants not only as pure distribution entities of insurance companies. Based on PortersFive Forces of Competition Framework exclusive insurance agents and independent insurance brokers were asked to evaluate the competition and market structure within the German insurance agents’ market. We examine the effect of PortersFive Forces on agents’ performance to measure the relation between market structure and performance. The results show that the market structure has a strong influence on total revenues of insurance agents. We find different results for exclusive agents and independent brokers and show that the competitive environment and the market structure affect insurance agents’ performance in different ways.

Seven species of the rust genus Sphaerophragmiumoccur on members of the tropical plant family Annonaceae. Uropyxis gerstneriis recombined to S. gerstneri. A new species, S. xylopiae, is described from Xylopia acutiflora. The host plant of S. boanenseis identified as Mitrellasp. Sphaerophragmium pulchrumis transferred to Dicheirinia. The anatomy of telia with teliospores and parasitizing mycelium is described and illustrated in detail. A new type of M-haustorium, which emanates laterally from intracellular hypha, is detected in S. monodorae. An identification key is given.

The article discusses the role of business gurus. Topics covered include the high number of chief executive officers (CEO) and business managers from various industries who seek help from professional consultants or business coaches, ActionCOACH Business Coaching owner Monte Wyatt citing the benefits of utilizing the help of a consultant such as revenue growth and additional employees, and the Business Resources program of Des Moines Area Community College (DMACC) in Iowa.

We have previously shown that both, polymer conformation and dynamics are crucial for the exciton transport in conjugated polymers. Thereby we found that the usual Förstertype hopping transfer model – even if one applies the linedipole approximation – falls short in one crucial aspect: the nature of the sites the excitons are transferred between is still unclear. We found that the simple model of spectroscopic units defined as segments of the polymer chains separated by structural defects breaking the πconjugation is only justified for chemical defects like hydrogenated double bonds, or extreme gauche 90° torsions between the monomers. Both defects are far too rare in a wellprepared conjugated polymer to explain the mean spectroscopicunit length of typically 6–7 monomers. Meanwhile, also the concept of dynamical formation of the spectroscopic units, we had previously suggested, has also failed. Thus the question of a paradigma change concerning the exciton transport in conjugated polymers appears on the agenda.

ABSTRACTWe used a murine model of catheter-associated biofilm formation to determine whether the mutation of the staphylococcal accessory regulator (sarA) has an impact on the susceptibility of established Staphylococcus aureusbiofilms to treatment with daptomycin in vivo. The experiments were done with two clinical isolates, one of which (UAMS-1) was obtained from the bone of a patient suffering from osteomyelitis, while the other (UAMS-1625) is an isolate of the USA300 clonal lineage of community-acquired methicillin (meticillin)-resistant S. aureus. UAMS-1625 had a reduced capacity to form a biofilm in vivo compared to that of UAMS-1 (P= 0.0015), but in both cases the mutation of sarAlimited biofilm formation compared to that of the corresponding parent strain (P≤ 0.001). The mutation of sarAdid not affect the daptomycin MIC for either strain, but it did result in increased susceptibility in vivo in the context of an established biofilm. Specifically, daptomycin treatment resulted in the clearance of detectable bacteria from <10% of the catheters colonized with the parent strains, while treatment with an equivalent daptomycin concentration resulted in the clearance of 46.4% of the catheters colonized with the UAMS-1 sarAmutant and 69.1% of the catheters colonized with the UAMS-1625 sarAmutant. In the absence of daptomycin treatment, mice with catheters colonized with the UAMS-1625 parent strain also developed skin lesions in the region adjacent to the implanted catheter. No such lesions were observed in any other experimental group, including untreated mice containing catheters colonized with the UAMS-1625 sarAmutant.

The objective of these experiments was to assess the effects of food and water deprivation and transit duration on the behavior of beef feedlot steers. In Experiment 1, 36 Angus-cross steers (353 ± 10 kg) were stratified to 6 pens and assigned one of three treatments (n= 12 steers per treatment): control (CON; stayed in home pens with ad libitum access to feed and water), deprived (DEPR; stayed in home pens but deprived of feed and water for 18 h), or transported (TRANS; subjected to 18-h transit event and returned to home pens). In Experiment 2, 60 Angus-cross steers (398 ± 5 kg; 6 steers per pen) were transported either 8 (8H) or 18 (18H) h. Four 8H pens (n= 24 steers) and six 18H pens (n= 36 steers) were used for behavioral analysis. In both experiments, the time to eat, drink, and lay down was recorded for each steer upon return to home pens. Total pen displacements from the feed bunk were also assessed for the 2 h following feed access in both experiments. Data were analyzed using Proc Mixed of SAS 9.4, with treatment as a fixed effect. Steer was the experimental unit for behavioral activities, while pen was the experimental unit for bunk displacements. Displacements were analyzed as repeated measures with the repeated variable of time. In Experiment 1, the time to eat and drink was similar across treatments (P≥ 0.17). However, TRANS laid down in 16.5 min while DEPR did not lay down until 70.5 min post-arrival to pen (P< 0.01). Deprived steers had greater bunk displacements in the first 70 min post-feed access than CON or TRANS, though displacements among treatments from 100 to 120 min post-feed access were similar (treatment × time: P= 0.02). In Experiment 2, both 8H and 18H steers laid down approximately 25 min post-home pen arrival (P= 0.14). There was no effect of transit duration or duration by time on bunk displacements (P≥ 0.20), though displacements were greater from 0 to 20 min than from 20 to 30 min post-feed access (time: P= 0.04). Steers that were deprived of feed and water were highly motivated to access those resources, while transported steers prioritized laying down. Producers should consider these priorities when preparing to receive cattle from a long transit event.Because of the segmentation of the cattle industry, cattle are transported at least once during their lives. The objective of these two studies was to determine if transportation, feed and water deprivation, and/or transit duration changed the behavior of feedlot steers. The first study found steers transported for 18 h preferred to lay down instead of competing for food, unlike steers that were deprived of food and water for 18 h. Bunk displacements were also increased in steers deprived of food and water, indicating increased aggression. In the second study examining effects of transit duration (8 vs. 18 h), steers from both treatments laid down within 25 min of arrival back to the home pens. There were no differences in the frequency of bunk displacements between treatments. Producers should consider the increased motivation for cattle to lay down after transportation and the increased aggression at the feed bunk in food-deprived cattle when developing post-arrival management strategies.After transportation, steers prefer to lay down instead of competing for food at the bunk. However, steers deprived of food and water for 18 h have increased bunk displacements during feed reintroduction.

The near-surface electronic structure of the room-temperature ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide has been investigated with ultraviolet and X-ray photoelectron spectroscopy as well as metastable induced electron spectroscopy. The results have been compared with density functional theory calculations. The good agreement between the experimental and theoretical data provides detailed insight into the origin of the observed spectral features. In particular, we found that a simple composition of the spectra of the isolated ions does not suffice to fit to the experimental results, but interionic interactions have to be considered.

AbstractIt is a remarkable age in molecular biology when one can argue that our current understanding of a process is influenced as much by structural studies as it is by genetic and physiological manipulations. This statement is particularly poignant with membrane proteins for which structural knowledge has been long impeded by the inability to easily obtain crystal structures in a lipid matrix. Thus, several highresolution structures of the components comprising tripartite multidrug efflux pumps from Escherichia coliand Pseudomonas aeruginosaare now available and were received with much acclaim over ever-evolving crystal structures of soluble, aqueous proteins. These structures, in conjunction with functional mutagenesis studies, have provided insight into substrate capture and binding domains and redefined the potential interactions between individual pump constituents. However, correct assembly of the components is still a matter of debate as is the functional contribution of each to the translocation of drug substrates over long distances spanning the Gram-negative cell envelope.

Ectomycorrhizae (ECM) of Lactarius aff. omphaliformisRomagn., Russula alnijorullensis(Sing.) Sing. and Cortinarius tucumanensisMos. on Andean alder (Alnus acuminataKunth) were characterized and identified. The identification of the fungal symbionts was achieved by morpho-anatomical observations of mycorrhizae and by comparison of ITS-RFLP patterns obtained from ECM and fruitbodies. L. aff. omphaliformisECM differed in some morphological details such as ramification and mantle type from ECM of the same species on A. glutinosa. L. aff. omphaliformisECM show an orange to ochre mantle containing latex cells, which stain with sulpho-vanillin, emanating hyphae without clamps. R. alnijorullensisECM represent a typical Russula-type-ECM, light yellow to pinkish, the outer mantle being composed of triangular latex-filled cells staining with sulpho-vanillin, emanating hyphae without clamps. C. tucumanensisECM exhibit a white (silvery) to yellowish brown mantle covered with soil particles, emanating hyphae with clamps.

Controlled ensemble formation of protein-surfactant systems provides a fundamental concept for the realization of nanoscale devices with self-organizing capability. In this context, spectroscopic monitoring of pigment-containing proteins yields detailed structural information. Here we have studied the association behavior of the bacterial light-harvesting protein LH2 from Rhodobacter spheroides in an n,n-dimethyldodecylamine-n-oxide/water environment. Time-resolved studies of the excitation annihilation yielded information about aggregate sizes and packing of the protein complexes therein. The results are compared to transmission electron microscopy images of instantaneously frozen samples. Our data indicate the manifestation of different phases, which are discussed with respect to the thermodynamic equilibrium in ternary protein-surfactant-water systems. Accordingly, by varying the concentration the formation of different types of aggregates can be controlled. Conditions for the appearance of isolated LH2 complexes are defined.

Retinoids that activate the nuclear retinoid X receptors (RXRs) display potential for chemoprevention of breast cancer. We previously reported that 9cUAB30 (1) is an RXR-selective retinoid. To explore its in vivo chemopreventive activity, multigram quantities of 1 were needed. Here, we describe a modified synthesis that yields up to 100 g of 1. We further demonstrate that 1 is very effective in the prevention of N-methyl-N-nitrosourea induced mammary cancers in rats without signs of toxicity.

Mutation of sarA resulted in a reduced capacity to form a biofilm in six of the eight Staphylococcus aureus strains we tested (UAMS-1, UAMS-601, SA113, SC-01, S6C, and DB). The exceptions were Newman, which formed a poor biofilm under all conditions, and RN6390, which consistently formed a biofilm only after mutation of agr. Mutation of agr in other strains had little impact on biofilm formation. In every strain other than Newman, including RN6390, simultaneous mutation of sarA and agr resulted in a phenotype like that observed with the sarA mutants. Complementation studies using a sarA clone confirmed that the defect in biofilm formation was due to the sarA mutation.

ABSTRACTMutation of sarAresulted in a reduced capacity to form a biofilm in six of the eight Staphylococcus aureusstrains we tested (UAMS-1, UAMS-601, SA113, SC-01, S6C, and DB). The exceptions were Newman, which formed a poor biofilm under all conditions, and RN6390, which consistently formed a biofilm only after mutation of agr. Mutation of agrin other strains had little impact on biofilm formation. In every strain other than Newman, including RN6390, simultaneous mutation of sarAand agrresulted in a phenotype like that observed with the sarAmutants. Complementation studies using a sarAclone confirmed that the defect in biofilm formation was due to the sarAmutation.

In recent years there has been a dramatic increase in the use of breast conservation therapy for patients with cancer. There are several factors associated with local recurrence but none are considered absolute contraindication for breast conserving therapy except multifocal carcinoma. This single-institution series investigates the effects of multiple factors on local relapse-free survival after breast-conserving therapy for women with invasive cancer. One-hundred and ninety-two patients (193 cancers) with invasive carcinoma underwent breast-conserving therapy (surgery, radiation therapy and chemotherapy if indicated) at University of Alabama at Birmingham Hospital from 1986 through 1995. The Kaplan-Meier method was used to calculate curves for local recurrence. The log rank statistic test was used for statistical comparison between curves. The Cox proportional hazards model was used for multivariate analysis. Significance was defined as p<0.05. Mean patient age was 56±12 years (range 25–83 years). Nineteen patients (9.8%) were 40 years age or younger. With a median time follow-up of 67 months the local recurrence rate for all patients was 6.7%. Ten patients developed local recurrence and three had combined local and distant recurrence. The mean local relapse-free interval was 71 months (range 10–148). The 5-year actuarial local relapse-free survival for all patients was 96%. Comparison of patients younger and older than 40 years age revealed a significantly lower risk of recurrence with increasing age (86 vs. 97% respectively, p=0.013). Patients 40 years age or younger with poorly differentiated tumors had a 5-year local relapse-free survival of only 40%. In multivariate analysis, only young age and poor tumor differentiation were found statistically significant in predicting local recurrence. Age and tumor differentiation are independent risk factors in our serie. These two factors should be taken into consideration when counseling breast cancer patients. En años recientes ha habido un dramático incremento en la cirugía conservadora de mama para el tratamiento de pacientes con cáncer. Existen varios factores reportados que incrementan el riesgo de recurrencia local pero ninguno es considerado contraindicación absoluta para terapia conservadora, excepto el carcinoma multifocal. Esta serie de una sola institución investiga los efectos de múltiples factores sobre la sobrevida libre de recurrencia local después de tratamiento conservador para mujeres con carcinoma invasor de mama. Ciento noventa y dos pacientes (193 cánceres) con carcinoma de mama invasivo fueron sometidas a tratamiento conservador (lumpectomía, radioterapia y quimioterapia si se encontraba indicada) en el Hospital de la Universidad de Alabama en Birmingham, USA de 1986 a 1995. Se utilizó el método de Kaplan-Meier para calcular las curvas de sobrevida y recurrencia local y la prueba de logrank para comparación estadística entre las curvas. Se utilizó el método de Cox para el análisis multivariado. Se definió significancia con una p<0.05. La edad promedio de las pacientes fue 56±12 años (rango 25–83 años). Diecinueve pacientes (9,8%) eran menores de 40 años de edad. Con una mediana de seguimiento de 67 meses, la tasa de recurrencia local para todas las pacientes fue 6.7%. Diez pacientes desarrollaron recurrencia local aislada y tres pacientes la combinación de recurrencia local y a distancia. El intervalo promedio libre de recurrencia local en estas pacientes fue de 71 bre de recurrencia local en estas pacientes fue de 71 meses (rango 10–148 meses). La sobrevida actuarial a 5 años libre de recurrencia local fue de 96% para todo el grupo de pacientes. La comparación de pacientes menores y mayores de 40 años demostró un riesgo significativamente menor de recurrencia local con mayor edad (86 frente al 97% respectivamente, p=0,013). Las pacientes menores de 40 años de edad con tumores poco diferenciados tuvieron una sobrevida actuarial libre de recurrencia local a 5 años de solo 40%. En el análisis mutivariado sólo la edad menor a 40 años y la pobre differenciación del tumor fueron encontrados predictores significativos de recurrencial local. La edad y la diferenciación del tumor fueron encontrados predictores independientes. Estos factores deben ser tomados en consideración al plantear el tratamiento para las pacientes con carcinoma de mama invasivo.

We recently demonstrated that mutation of sarA in clinical isolates of Staphylococcus aureus results in a phenotype that is distinct by comparison to sarA mutants generated in the laboratory strain RN6390 (J. S. Blevins, K. E. Beenken, M. O. Elasri, B. K. Hurlburt, and M. S. Smeltzer, Infect. Immun. 70:470-480, 2002). This raises the possibility that studies demonstrating that RN6390 sarA mutants are attenuated do not accurately reflect the role of sarA in the pathogenesis of staphylococcal disease. To test this hypothesis, we used a murine model of musculoskeletal infection to assess the virulence of sarA and agr mutants generated in a clinical isolate of S. aureus (UAMS-1). By using this model, we confirmed that mutation of sarA and/or agr results in a reduced capacity to cause both septic arthritis and osteomyelitis.

ABSTRACTWe recently demonstrated that mutation of sarAin clinical isolates of Staphylococcus aureusresults in a phenotype that is distinct by comparison to sarAmutants generated in the laboratory strain RN6390 (J. S. Blevins, K. E. Beenken, M. O. Elasri, B. K. Hurlburt, and M. S. Smeltzer, Infect. Immun. 70:470-480, 2002). This raises the possibility that studies demonstrating that RN6390 sarAmutants are attenuated do not accurately reflect the role of sarAin the pathogenesis of staphylococcal disease. To test this hypothesis, we used a murine model of musculoskeletal infection to assess the virulence of sarAand agrmutants generated in a clinical isolate of S. aureus(UAMS-1). By using this model, we confirmed that mutation of sarAand/or agrresults in a reduced capacity to cause both septic arthritis and osteomyelitis.

The accessory gene regulator (agr) and the staphylococcal accessory regulator (sar) are central regulatory elements that control the production of Staphylococcus aureus virulence factors. To date, the functions of these loci have been defined almost exclusively using RN6390, which is representative of the laboratory strain 8325-4. However, RN6390 was recently shown to have a mutation in rsbU that results in a phenotype resembling that of a sigB mutant (I. Kullik et al., J. Bacteriol. 180:4814-4820, 1998). For that reason, it remains unclear whether the regulatory events defined in RN6390 are representative of the events that take place in clinical isolates of S. aureus. To address this issue, we generated mutations in the sarA and agr loci of three laboratory strains (RN6390, Newman, and S6C) and four clinical isolates (UAMS-1, UAMS-601, DB, and SC-1). Mutation of sarA in the cna-positive strains UAMS-1 and UAMS-601 resulted in an increased capacity to bind collagen, while mutation of agr had little impact. Northern blot analysis confirmed that the increase in collagen binding was due to increased cna transcription. Without exception, mutation of sarA resulted in increased production of proteases and a decreased capacity to bind fibronectin. Mutation of agr had the opposite effect. Although mutation of sarA resulted in a slight reduction in fnbA transcription, changes in the ability to bind fibronectin appeared to be more directly correlated with changes in protease activity. Lipase production was reduced in both sarA and agr mutants. While mutation of sarA in RN6390 resulted in reduced hemolytic activity, it had the opposite effect in all other strains. There appeared to be reduced levels of the sarC transcript in RN6390, but there was no difference in the overall pattern of sar transcription or the production of SarA. Although mutation of sarA resulted in decreased RNAIII transcription, this effect was not evident under all growth conditions. Taken together, these results suggest that studies defining the regulatory roles of sarA and agr by using RN6390 are not always representative of the events that occur in clinical isolates of S. aureus.

ABSTRACTThe accessory gene regulator (agr) and the staphylococcal accessory regulator (sar) are central regulatory elements that control the production of Staphylococcus aureusvirulence factors. To date, the functions of these loci have been defined almost exclusively using RN6390, which is representative of the laboratory strain 8325-4. However, RN6390 was recently shown to have a mutation in rsbUthat results in a phenotype resembling that of a sigBmutant (I. Kullik et al., J. Bacteriol. 180:4814–4820, 1998). For that reason, it remains unclear whether the regulatory events defined in RN6390 are representative of the events that take place in clinical isolates of S. aureus. To address this issue, we generated mutations in the sarAand agrloci of three laboratory strains (RN6390, Newman, and S6C) and four clinical isolates (UAMS-1, UAMS-601, DB, and SC-1). Mutation of sarAin the cna-positive strains UAMS-1 and UAMS-601 resulted in an increased capacity to bind collagen, while mutation of agrhad little impact. Northern blot analysis confirmed that the increase in collagen binding was due to increased cnatranscription. Without exception, mutation of sarAresulted in increased production of proteases and a decreased capacity to bind fibronectin. Mutation of agrhad the opposite effect. Although mutation of sarAresulted in a slight reduction in fnbAtranscription, changes in the ability to bind fibronectin appeared to be more directly correlated with changes in protease activity. Lipase production was reduced in both sarAand agrmutants. While mutation of sarAin RN6390 resulted in reduced hemolytic activity, it had the opposite effect in all other strains. There appeared to be reduced levels of the sarCtranscript in RN6390, but there was no difference in the overall pattern of sartranscription or the production of SarA. Although mutation of sarAresulted in decreased RNAIII transcription, this effect was not evident under all growth conditions. Taken together, these results suggest that studies defining the regulatory roles of sarAand agrby using RN6390 are not always representative of the events that occur in clinical isolates of S. aureus.

Manifestation and extent of excitonic interactions in the red Chl-absorption region (Qy band) of trimeric LHC-II were investigated using two complementary nonlinear laser-spectroscopic techniques. Nonlinear absorption of 120-fs pulses indicates an increased absorption cross section in the red wing of the Qy band as compared to monomeric Chl a in organic solution. Additionally, the dependence of a nonlinear polarization response on the pump-field intensity was investigated. This approach reveals that one emitting spectral form, characterized by a 2.3(±0.8)-fold larger dipole strength than monomeric Chl a, dominates the fluorescence spectrum of LHC-II. Considering available structural and spectroscopic data, these results can be consistently explained assuming the existence of an excitonically coupled dimer located at Chl-bindings sites a2 and b2 (referring to the original notation of W. Kühlbrandt, D. N. Wang, and Y. Fujiyoshi, Nature, 1994, 367:614–621), which must not necessarily correspond to Chls a and b). This fluorescent dimer, terminating the excitation energy-transfer chain of the LHC-II monomeric subunit, is discussed with respect to its relevance for intra- and inter-antenna excitation energy transfer.

Studies of alkylation-induced mutations in Escherichia coliFX-11 revealed that both N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) produced tRNA suppressor mutations (G:C to A:T) but only ENU produced a significant number of backmutations (A:T to G:C, A:T to T:A and A:T to C:G). Further, the ENU-induced transversions were absent in a UmuC-defective strain. This suggested that transition mutations could result from alkylation of guanine or thymine at the O6- and O4-positions, respectively, but that transversions might result from alkylation of thymine at the O2-position. To test this idea, the gene encoding O6-alkylguanine-DNA methyltransferase (ogt) was recombined into a plasmid to overexpress the cellular levels of this enzyme. Ogt protein can de-alkylate O6-alkylguanine and O4-alkylthymine, but not O2-alkylthymine. Cells harboring the plasmid (or a control plasmid lacking the ogtgene) were exposed to different concentrations of MNU or ENU and the resulting mutations were analyzed. With either MNU or ENU, the frequency of GlnVosuppressors was reduced about 70-fold in the Ogt-overexpressing cells, suggesting that Ogt eliminated O6-alkylguanine. Similarly, GlnUosuppressor frequencies were substantially reduced. In contrast, the reduction in frequency for the backmutations was slight, only about 2.5-fold with MNU and less than two-fold for ENU. However, DNA sequence analysis of the backmutations showed that only A:T to G:C transitions were affected by overexpression of Ogt, suggesting repair of O4-alkylthymine. The frequency of transversions, in comparison, was essentially unaltered. These results implicate O2-alkylthymine as a likely candidate for transversion mutagenesis induced by ENU.