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Evidence increasingly indicates that standardised, shorter regimens (SR) for multidrug-resistant TB (MDR-TB) is effective in treating this global disease, but there is little published data on associated adverse events. We report outcomes from a cohort treated with the SR in Port Moresby, Papua New Guinea (PNG). Among 26 patients treated with a TB SR from September 2017 to September 2018, 10 (39%) were successful treatments, 12 (46%) were failures, 2 died, and 2 were lost to follow-up. Of those whose treatment failed, most (Les preuves disponibles montrent de plus en plus que des protocoles standardisés, plus courts (SR) de la TB multirésistante (MDR-TB) traitent efficacement cette maladie mondiale, mais il y a peu de données publiées sur les effets secondaires. Nous rapportons les résultats d’une cohorte traitée par SR à Port Moresby, Papouasie Nouvelle-Guinée (PNG). Parmi 26 patients traités par SR de TB de septembre 2017 à septembre 2018 : 10 (39%) ont été traités avec succès, 12 (46%) ont échoué, 2 sont décédés et 2 ont été perdus de vue. Parmi les échecs, la majorité (

This chapter traces the rich literary, historical, artistic, architectural, and theatrical cross-pollination that took place in the nineteenth century as the French and English travelled back and forth across the English Channel (La Manche) to examine medieval cultural history and heritage and to share creative works of medievalism generated by such discoveries. France did not merely serve as a ‘source’ for Victorian medievalism, but rather as a locus for transnational representations of a European Middle Ages and its cultural productions. In order to better understand English medievalism in the nineteenth century, it is important to trace French developments from the same period.

The performance of stainless steel brackets, adhesively mounted with a soft polyurethane-based accelerated 1-component adhesive onto completely coated steel substrates, is investigated. The investigated joint connections on typical marine top coats, namely polyurethane, polysiloxane and epoxy, exhibited a high mechanical stability even after accelerated ageing (salt spray exposure, cyclic temperature variations, high relative humidity). Tensile strength and shear strength criteria for a designed loading capacity of 50 kg were achieved with a safety factor of >10. All failure strength values were lower than the adhesion strength of the coatings to the steel. The adhesive material maintained its high deformability after ageing. The torsional rigidity of the adhesive joint exceeded the required clamping torque of the designed bolt. A cyclical lifetime of >9·106 load cycles was estimated. Effects of cyclic ageing on the creep performance of the adhesive joint were found to be insignificant. All adhesive joint connections met the requirements for a safety factor for the design for marine applications. Under sealed conditions, the ingress of moisture and chloride was avoided, and deterioration mechanisms typical for unsealed joints in marine environments (e.g. moisture penetration, adhesive material plastification, interfacial delamination) did not take place. Effects of mechanical and structural parameters as well as surface parameters of the top coat materials on the performance of the joints were statistically insignificant.

A reference to insanity among New England Protestant missionary women serving in the Near East from a letter written during the early nineteenth century offers a provocative opportunity to reexamin...

Die Blickdiagnose von angeborenen Stoffwechselerkrankungen ist oft schwierig. Mehrere Hundert verschiedene Erkrankungen und ihre Symptome lassen sich von einem einzelnen Arzt kaum im Kopf halten. Ein Zeitraum von mehreren Jahren von Symptombeginn bis zur endgultigen Diagnose ist bei seltenen Erkrankungen nicht unublich. Auf der anderen Seite ist eine ganze Reihe dieser Erkrankungen behandelbar, und eine fruhe Blickdiagnose ermoglicht eine fruhe Therapie. Exemplarisch wird die Fruherkennung einiger behandelbarer Stoffwechselerkrankungen dargestellt, wie Homocystinurie, Progerie oder „Congenital-disorder-of-glycosylation“(CDG)-Ia-Erkrankung.

The selection and adjustment of an effective abrasive mass flow rate is one of the most important requirements for efficient blast-cleaning processes. Steel grit is one of the most widely u...

The performance of a sealed stainless steel bracket system, adhesively bonded with two polyurethane-based adhesives (flexible adhesive, stiff adhesive) onto a marine polyurethane top coat, is investigated. The investigated joint connections on the coatings exhibited a high mechanical stability even after accelerated ageing (salt spray exposure, cyclic temperature variations, high relative humidity). The aged joints retained up to 81% of their tensile strength and up to 92% of their lap-shear strength. The torsional rigidity of the adhesive joint exceeded the required clamping torque of the designed bolt. A cyclical lifetime of >9·106 load cycles was estimated. Effects of cyclic ageing on the creep performance of the adhesive joint were found to be insignificant. Under tensile loads, the joints with stiff adhesive material exhibited a linear-elastic performance without the capability to deform prior to failure. The joints with the flexible adhesive material, in contrast, exhibited a behavior typically for ductile materials featuring a pronounced yield plateau prior to failure. Failure loads were higher, and displacement was notably lower, for the joints with stiff adhesive material. All joint connections met the requirements for a safety factor for the design for marine applications (11.6 for the flexible adhesive; 14.3 for the stiff adhesive). When the flexible adhesive is applied, the cohesive strength of the adhesive material is the limiting design factor. When the stiff adhesive is utilized, the load carrying capacity is higher, and the interface between priming coat and steel substrate is the limiting design area of the joints. The strength utilization of the adhesive materials depended on the particular structure of the entire joint configuration, particularly on the interface between steel substrate and the coating material. A number of assessment factors, namely stress limit factor, coating adhesion factor and safety factor, are introduced and discussed for tensile and shear loads.

Coating specifications for offshore wind energy devices (OWEAs) are still based on specifications for the offshore oil and gas industry. Recently, two standards, developed for OWEA in the German offshore section (North Sea and Baltic Sea), were issued. This article reviews current offshore standards and commercial specifications used in the German OWEA industry. Stresses are defined for different zones of the structures. Specified coating systems for different stresses, including testing methods, are discussed. A review of 34 commercial coating specifications for OWEA in the North Sea and the Baltic Sea is provided. Evaluation parameters include number of layers, dry film thickness, and binder types for different coats. Special coating requirements are discussed, namely impact resistance, abrasion resistance, icing/deicing performance, low friction, color and gloss stability, and low-temperature performance. Finally, trends for the utilization of thermal spray metals are reviewed.

The proteinase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), which forms part of the caspase recruitment domain-containing protein 11-B-cell lymphoma 10-MALT1 signalosome complex, plays a direct role in nuclear factor kappa B activation. Here, we describe the case of a female infant with severe immune dysregulation leading to recurrent systemic infections, failure to thrive and severe crises of ichthyosiform erythroderma with high levels of serum IgE. Hence, initial symptoms indicated Netherton syndrome or Omenn syndrome. Surprisingly, sequence analyses of SPINK5 and RAG1/RAG2, respectively, excluded these diseases. During the hospital stay the patient's health deteriorated, despite intensive care therapy, and she died. In order to delineate the diagnosis, whole-exome sequencing was performed. Two compound heterozygous mutations in MALT1 were found and verified by Sanger sequencing (exon 2 c.245T>C, exon 2 c.310dup), which led to a MALT1 deficiency at the protein level. Based on these results, an immunological analysis was performed, as was immunofluorescence staining of key skin proteins, to confirm a diagnosis of MALT1 deficiency. This case report provides a closer description of the clinical and histological skin phenotype of MALT1 deficiency, and we conclude that MALT1 deficiency must be considered a possible differential diagnosis of Netherton and Omenn syndromes. What's already known about this topic? Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) deficiency is a combined immunodeficiency. MALT1 is part of the caspase recruitment domain-containing protein 11-B-cell lymphoma 10-MALT1 signalosome complex, which is essential for nuclear factor kappa B activation. Current publications describe a phenotype of recurrent systemic infections; only in a few cases has an inflammatory involvement of the integument been described. What does this study add? A closer description of the cutaneous phenotype of MALT1 deficiency in a patient with two novel MALT1 mutations. Immune mapping of follicular epidermis shows lympho-epithelial Kazal-type-related inhibitor is reduced in MALT1 deficiency and absent on interfollicular staining. Clinically, MALT1 deficiency mimics Netherton syndrome and Omenn syndrome, and should be considered a differential diagnosis.

The paper deals with a systematic investigation into the flow of copper slag abrasive particles through a metering valve. The investigation involves abrasive mass flow rate measurements, and the results are statistically interpreted based on DoE (Design of Experiments) and ANOVA (Analysis of Variance). Three process parameters are varied, namely static air pressure, nozzle diameter and valve opening. Abrasive mass flow rate increases if air pressure, nozzle diameter or valve opening increase. All three main factors provide statistically significant effects, whereby the strongest effect occurs due to changes in valve opening. Although a non-standardized quadratic regression model delivers a satisfactory agreement with experimental results, it misjudges the statistical significance of the process parameters. A standardization of the regression function omits multicollinear effects and simplifies the regression function. A standardized quadratic regression model is found to be capable to statistically describe the relationships in the scope of the evaluation effort.

Herbicide options for management of volunteer corn in soybean include a variety of acetyl CoA carboxylase-inhibiting herbicides, yet often, applications of acetyl CoA carboxylase herbicides are delayed until the weed is visible above the soybean canopy. Volunteer corn growing above the soybean canopy is a highly competitive weed, and herbicides applied at this point can kill the weed, yet soybean yield loss is still a concern. Our objective was to compare the effect of controlling various densities of volunteer corn growing in soybean EARLY (≤ 30 cm) versus LATE (≈ 90 cm) on percent control and soybean yield. Seven volunteer corn densities (0, 0.5, 2, 4, 8, 12, and 16 plants m−2) were hand planted into 19-cm row soybean. Clethodim 79 g ai ha−1was tank-mixed with glyphosate at 840 g ae ha−1and applied to the volunteer corn EARLY and LATE. The EARLY application provided higher and less variable control of volunteer corn 14 d after treatment (DAT) compared to LATE applications at all volunteer corn densities. There was no difference in control at 28 DAT for both the EARLY and LATE applications. Soybean yield was not affected by either application timing. Although no yield reduction was seen with the LATE treatments, later-season applications of clethodim to control volunteer corn may offer more variable control and could allow for additional Bt selection pressure on targeted insect pests.

BACKGROUND Combining herbicide-resistant and Bacillus thuringiensis (Bt) traits in corn (Zea mays L.) hybrids may affect insect resistance management owing to volunteer corn. Some Bt toxins may be expressed at lower levels by nitrogen-deficient corn roots. Corn plants with sublethal levels of Bt expression could accelerate the evolution of Bt resistance in target insects. The present objective was to quantify the concentration of Bt (Cry3Bb1) in corn root tissue with varying tissue nitrogen concentrations. RESULTS Expression of Cry3Bb1 toxin in root tissue was highly variable, but there were no differences in the overall concentration of Cry3Bb1 expressed between roots taken from Cry3Bb1-positive volunteer and hybrid corn plants. The nitrogen rate did affect Cry3Bb1 expression in the greenhouse, less nitrogen resulted in decreased Cry3Bb1 expression, yet this result was not documented in the field. CONCLUSION A positive linear relationship of plant nitrogen status on Cry3Bb1 toxin expression was documented. Also, high variability in Cry3Bb1 expression is potentially problematic from an insect resistance management perspective. This variability could create a mosaic of toxin doses in the field, which does not fit into the high-dose refuge strategy and could alter predictions about the speed of evolution of resistance to Cry3Bb1 in western corn rootworm Diabrotica virgifera virgifera LeConte. © 2013 Society of Chemical Industry

Transgenic volunteer corn is a competitive weed in soybean that decreases soybean yield at densities as low as 0.5 plants m−2, yet the competitive effects of volunteer corn in corn have yet to be quantified in the peer-reviewed literature. In order to quantify competition between volunteer corn and hybrid corn, seed was harvested from transgenic hybrid corn. The seed was then hand-planted at two locations (Lafayette, IN and Wanatah, IN) into 3 by 9 m plots of hybrid corn at five densities: 0 (control), 0.5, 2, 4, and 8 plants m−2. Volunteer corn competition reduced leaf area and biomass of hybrid corn plants. Hybrid corn grain yield at Lafayette, IN, was reduced by 23 and 22% due to competition with volunteer corn growing in densities of 8 plants m−2in 2010 and 2011, respectively, but when volunteer corn grain yield was combined with the hybrid corn grain yield, there was no reduction in total grain yield. This study demonstrates that the competitive effects on the grain yield of the hybrid corn will be offset by the grain yield of the volunteer plants. However, because the unpredictable locations and densities of volunteer corn plants present challenges to machine harvesting, future studies should examine what proportion of the volunteer crop is actually harvestable.

Volunteer corn (VC) in hybrid corn has become more prevalent in recent years and can reduce grain yield. Nitrogen (N) management can influence VC interference in corn. Field experiments were established to determine the effects of N fertilizer management and VC interference on hybrid corn growth and grain yield. Treatments consisted of three VC densities (control, 0 plants m−2; low density, 1 plant m−2; high density, 4 plants m−2) and six N fertilizer treatments (0 kg N ha−1, 67 kg N ha−1 at planting, 67 kg N ha−1 at planting + 133 kg N ha−1 at V5 corn growth stage, 67 kg N ha−1 at planting + 133 kg N ha−1 at V10 corn growth stage, 200 kg N ha−1 at V5 corn growth stage, and 200 kg N ha−1 at V10 corn growth stage). The effect of VC on hybrid corn was dependent on N rate. When 200 kg N ha−1 was applied, regardless of application timing, hybrid corn dry weight, hybrid corn N content, and hybrid corn grain yield were reduced by the high VC density. However, when VC grain yield was added to hybrid corn grain yield, VC density did not affect total grain yield. When 0 and 67 kg N ha−1 were applied, neither hybrid corn dry weight nor hybrid corn N content was affected by either VC density, but the high VC density reduced hybrid corn grain yield for both N rates by 19% and total grain yield by 9 and 10%, respectively. Application timing of N fertilizer had no effect on hybrid corn dry weight, N content, or grain yield. However, late N fertilizer applications (200 kg N ha−1 at V10 and 67 kg N ha−1 at planting +133 kg N ha−1 at V10) resulted in greater VC N content, VC grain yield, and total yield. Assuming the harvestability of VC, the ability of a late N treatment (V10) to maximize total grain yield allows growers to use a late N application to reduce the competitive effects of VC in hybrid corn.

Glyphosate-resistant (GR) volunteer corn has emerged as a problematic weed in corn:soybean rotational systems, partly because of the rapid increase in adoption of corn hybrids that contain traits for both glyphosate and insect resistance. Volunteer GR corn can decrease soybean yields. The objectives of this study were to quantify the impact of volunteer corn on soybean growth and yield and determine how volunteer corn densities affect western corn rootworm (WCR) emergence. Volunteer corn seed was hand-planted at targeted densities of 0.5, 2, 4, 8, 12, and 16 seeds m−2at soybean planting and 21 d after planting to evaluate both early- and late-emerging cohorts. WCR emergence was assessed with the use of field emergence traps placed over individual corn plants in the 0.5- and 16-plants-m−2plots in 2008 and 2009. In 2010, WCR emergence traps were also placed over individual and clumped volunteer corn plants at densities of two and eight plants m−2. Soybean yield reductions ranged from 10 to 41% where early-emerging volunteer corn densities ranged from 0.5 to 16 plants m−2. No soybean yield loss occurred with the late-emerging cohort of volunteer corn. Twice as many adult WCRs emerged from a single volunteer corn plant growing at densities of 8 and 16 plants m−2, compared with plots containing 0.5 and 2 plants m−2. These results demonstrate that controlling volunteer corn will not only prevent soybean yield loss, but also may reduce the risk of WCR larval survival after exposure to Bt (Bacillus thuringiensisBerliner derived) corn.

Rapid adoption of glyphosate-resistant (GR) corn hybrids has led to the reemergence of volunteer corn as a problematic weed in soybean and has made controlling the initial stand of corn in a replant situation more difficult. If volunteer corn in soybean or the initial corn stand in a replant situation is not controlled, yield loss can occur. Clethodim and glufosinate are often used to control GR corn in corn replant situations and in soybean. The objectives of this research were to evaluate the response of two hybrid corn varieties and their F2progeny to clethodim and glufosinate and to evaluate the effect of plant nitrogen (N) concentration on clethodim and glufosinate efficacy. First, a dose-response study was conducted with clethodim and glufosinate on DeKalb 60-18 and 60-18F2, and DeKalb 63-42 and 63-42F2to compare the response of the hybrids and their F2progeny to the herbicides. DeKalb 63-42 was more tolerant to clethodim than 60-18 and 60-18F2. No differences were found between the hybrids and their respective F2progeny in the response to clethodim or glufosinate. In a second dose-response study assessing the effect of N conditions on herbicide efficacy, both clethodim and glufosinate were less injurious to plants growing in low N than in high N availability.

Understanding of the mating and dispersal behavior of the western corn rootworm, Diabrotica virgifera virgifera (LeConte), is essential to predicting potential resistance to Bt corn technologies recently deployed to combat this pest. To quantify movement of male beetles, field studies were conducted during 2006 and 2007 in commercial Bt cornfields that included the Environmental Protection Agency-mandated 20% refuge acreage. Wing traps containing a single virgin female beetle were placed along transects throughout these fields. Male beetles were collected from the sticky bottoms of traps to compare the mean number of beetles captured at the different transect distances. Gut contents of each captured male were assayed with protein test strips to determine if the Bt protein (Cry3Bb1) was present. This provided an estimate of the distances traveled by males to reach virgin females. These data indicated that the mean number of males captured approximately 200 m from refuge corn was not significantly different than the mean number of males captured close to the refuge (< 22 m). Complementary field observations were conducted to determine how far newly emerged female beetles moved before mating and the time of day mating occurs. Results indicated that females do not move far from the site of emergence before mating and do not mate within the first 4 h of adult life. The implications of these data for movement and mating patterns of adult rootworms in Bt/refuge environments are discussed.

Congenital disorders of glycosylation (CDG, formerly named carbohydrate-deficient glycoprotein syndromes) are a rapidly growing family of inherited disorders affecting the assembly or processing of glycans on glycoconjugates. The clinical spectrum of the different types of CDG discovered so far is variable, ranging from severe multisystemic disorders to disorders restricted to specific organs. This review deals with clinical, diagnostic, and biochemical aspects of all characterized CDGs, including a disorder affecting the N-glycosylation of erythrocytes, congenital dyserythropoietic anemia type II (CDA II/HEMPAS), and the first disorders affecting O-glycosylation. Since the clinical spectrum of symptoms in CDG is variable and may be unspecific, a generous selective screening for the presence of CDG is recommended.

We describe the successful treatment of a neonate with Kasabach-Merritt syndrome who received local irradiation and interferon alpha therapy after failure of corticosteroid treatment. A male neonate, born after an uneventful pregnancy, had a huge haemangioma involving the upper right cervical region as well as severe thrombocytopenia. He was treated with corticosteroids, interferon alpha and radiotherapy. Prednisolone therapy (5 mg kg(-1) day(-1)) was started at 41 days of age. No therapeutic effect was observed after 2 weeks. At this time the tumour size had increased dramatically, platelet counts had decreased progressively and coagulation abnormalities had developed. Because corticosteroid therapy had been ineffective and the child was in a life-threatening condition, irradiation was delivered up to a total dose of 9.5 Gy in five fractions. Simultaneously, prednisolone therapy was slowly decreased and interferon alpha therapy (3 million U m(-2) day(-1)) was started and continued for 6 weeks. After irradiation with 9.5 Gy and beginning interferon alpha therapy, the tumour decreased in size and coagulation parameters normalized within 4 weeks. 6 months later, platelet counts and coagulation parameters were still normal. The tumour had further decreased in size. No acute severe side effects were observed. Radiation therapy combined with interferon alpha treatment is an alternative treatment modality when high dose corticoid steroid therapy has been ineffective in patients with Kasabach-Merritt syndrome, despite the risks of growth delay and secondary malignancy. In children showing no response to corticosteroids, radiotherapy and/or interferon alpha should be considered in Kasabach-Merritt syndrome.

»Congenital disorders of glycosylation« (CDG; angeborene Glykosylierungskrankheiten) umfassen eine Gruppe von Stoffwechselkrankheiten, die durch eine fehlerhafte Glykosylierung von Proteinen oder Lipiden entstehen.

A 2-year-old boy who developed hypophosphatemic rickets without signs of muscular weakness or neurological disturbances is presented. Biochemical findings included hypophosphatemia, metabolic acidosis, hypouricemia, hyperphosphaturia, severe glucosuria, generalized hyperaminoaciduria, hypercalciuria, proteinuria with elevated excretion of IgG, transferrin, albumin and high levels of alpha-1-microglobulin. Urine concentration capacity and creatinine clearance were normal. Lactaturia without elevated levels of plasma lactate and a high urinary excretion of beta-hydroxybutyrate were suggestive for mitochondriopathy. Partial deficiency of cytochrome c oxidase (complex IV of the respiratory chain) was found in skeletal muscle. A renal biopsy specimen demonstrated enlarged mitochondria with abnormal arborization and disorientation of the cristae in the proximal tubular cells. Reduced activity of mitochondrial cytochrome c oxidase in tubular cells could be demonstrated by ultracytochemistry. In conclusion, rickets due to the renal Fanconi syndrome can be the first clinical sign of mitochondrial cytopathies without extra-renal symptoms. Elevated excretion of lactate and ketone bodies in urine may serve as a diagnostic marker.

Previous studies in our laboratory have shown that chemical treatments may induce increases in proliferin gene family mRNA accumulation in cultured murine embryonic cells. Proliferin inductions are highly correlated with subsequent promotional outcomes during two-stage focus-formation assays in C3H/10T1/2 cell cultures. In work reported here, the strong affiliation between these two responses was further validated after treating cells with di-n-butyltin dichloride which is a polyvinyl chloride (PVC) plastic additive that often contaminates food and water. Increased proliferin expression and promotion of morphological transformation occurred at similar concentrations. Promotion of transformation was detected at di-n-butyltin dichloride concentrations of 80 n m (24 ng/ml) and above, if added to initiated cultures before confluent monolayers had formed. Proliferin induction and morphological transformation were both reduced in confluent cultures treated with di-n-butyltin dichloride, as compared to subconfluent cultures. Proliferin expression measured in near-confluent cultures was induced up to 10-fold during the 36-hr period following di-n-butyltin dichloride exposure and was accompanied by increased accumulation of transcripts from many genes regulated by oxidative stresses, growth-inducing agents, and/or other promoting agents (asbestos, superoxide radicals ). Di-n-butyltin dichloride-induced mRNA species included members of the fos and jun proto-oncogene families, c-myc, egr1, ribonucleotide reductase (R2 subunit), odc, macrophage chemotactic protein/je, hsp70, metallothionine IIA, c-sod and mn-sod. The observed patterns of RNA accumulation suggested that a small subset of mRNA species, including proliferin, exhibit regulatory behaviour as a response to dissimilar agents or conditions that promote focus-formation in C3H/10T1/2 cultures. Plausible predictions of promotional effects in two-stage morphological transformation assays can be made from gene-expression responses to test agents.

Genetically-modifi ed (GM) corn (Zea mays L.) and soybean [Glycine max (L.) Merr.] dominate the North American agricultural landscape and are becoming increasingly important as biofuels. However, as herbicide-tolerance and insecticidal traits are oft en simultaneously expressed by individual plants, glyphosate [N-(phosphonomethyl) glycine]-resistant (GR) volunteer corn is becoming a widespread problem as a weed in corn-soybean rotational systems. We show that these volunteer corn plants not only have herbicide-tolerance genes but also express insecticidal “Bt” protein. We also report high levels of damage to these plants from larvae of the target pest, the western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte). Th is suggests that volunteer herbicide-tolerant Bt corn has the potential to present problems both for weed management and insect resistance management, as it may facilitate more rapid evolution of Bt resistance in corn rootworm populations.

A concave pyridine 1c has been attached to dendrimers of the Frechet type by an ethylenoxide linker on the convex outside of the catalyst. Two generations of dendrons [G-1]-OH, [G-2]-OH,[G-1]-Brand [G-2]-Br and of dendrimers with 4,4′-dihydroxybiphenyl (3) and 1,1,1-tris(4-hydroxyphenyl)ethane (4) as di- and trivalent cores [G-1]2–[C2], [G-1]3–[C3] and [G-2]3–[C3] have been synthesized. The resulting molecules possess up to twelve concave catalytic pyridine centers on the outside of the molecules, and can be used as selective catalysts in the base catalyzed addition of alcohols to diphenylketene.

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.

Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol is required for targeting of membrane proteins to the Golgi complex. We used insect cells for our studies because they are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. We found that two well-characterized mammalian Golgi proteins were targeted to the Golgi region of Aedes albopictus cells, both in the presence and absence of cellular cholesterol. Our results imply that a cholesterol gradient through the secretory pathway is not required for membrane protein targeting to the Golgi complex, at least in insect cells.

Combining herbicide-resistant and Bacillus thuringiensis (Bt) traits in corn (Zea mays L.) hybrids may affect insect resistance management owing to volunteer corn. Some Bt toxins may be expressed at lower levels by nitrogen-deficient corn roots. Corn plants with sublethal levels of Bt expression could accelerate the evolution of Bt resistance in target insects. The present objective was to quantify the concentration of Bt (Cry3Bb1) in corn root tissue with varying tissue nitrogen concentrations.Expression of Cry3Bb1 toxin in root tissue was highly variable, but there were no differences in the overall concentration of Cry3Bb1 expressed between roots taken from Cry3Bb1-positive volunteer and hybrid corn plants. The nitrogen rate did affect Cry3Bb1 expression in the greenhouse, less nitrogen resulted in decreased Cry3Bb1 expression, yet this result was not documented in the field.A positive linear relationship of plant nitrogen status on Cry3Bb1 toxin expression was documented. Also, high variability in Cry3Bb1 expression is potentially problematic from an insect resistance management perspective. This variability could create a mosaic of toxin doses in the field, which does not fit into the high-dose refuge strategy and could alter predictions about the speed of evolution of resistance to Cry3Bb1 in western corn rootworm Diabrotica virgifera virgifera LeConte.

Semliki Forest virus (SFV), an enveloped alphavirus, infects cells by endocytosis followed by low pH-triggered fusion of the virus and endocytic vesicle membranes. Progeny virus is released by budding from the cell plasma membrane.In vitro,SFV fusion with artificial liposomes is triggered by low pH and is dependent on the presence of cholesterol and sphingolipid in the target liposome membrane. In tissue culture, both SFV fusion and virus exit are strongly cholesterol-dependent when assayed in cholesterol-depleted insect cells. We here describe the preparation of insect cells that while not containing detectable amounts of cholesterol, have adapted to sterol-depleted conditions, resulting in a more permissive phenotype for SFV infection. Although still less efficient at supporting SFV infection than control cholesterol-containing cells, the adapted cells show a 45-fold increase in primary infection by SFV, increased release of progeny virus, and enhanced virus growth kinetics compared to nonadapted cholesterol-depleted cells. The adapted cells are also about 85-fold more permissive for low pH-induced fusion of SFV with the plasma membrane, suggesting that adaptation correlates with a change in the cell membrane.

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur cholesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the viral spike proteins were synthesized and transported to the site of p62 cleavage with normal kinetics. Surprisingly, the subsequent exit of virus particles was dramatically slowed compared to cholesterol-containing cells. The inhibition of virus production could be reversed by the addition of cholesterol to depleted cells. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatitis virus or a cholesterol-independent SFV fusion mutant. Thus, cholesterol was only critical in the exit pathway of viruses that also require cholesterol for fusion. These results demonstrate a specific and unexpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budding of SFV.