PD-1 blockade: a therapeutic option for treatment of metastatic Merkel cell carcinoma.

The immune system is extremely important in the development and progression of Merkel cell carcinoma (MCC). Immune checkpoint blockade has recently been shown to enable efficacious treatment of a variety of tumours. We report the use of an anti-programmed death receptor 1 (PD-1) antibody for treatment of a patient with metastatic MCC. An 80-year-old patient with metastatic MCC received off-label treatment with the anti-PD-1 antibody pembrolizumab after the disease had progressed during therapy with oral etoposide. A positron emission tomography (PET) computed tomography scan performed after three cycles of pembrolizumab revealed responses to therapy with reduced size of the adrenal gland metastases and less PET activity in the adrenal gland and lymph node metastases. Treatment was resumed owing to disease progression after a treatment-free interval of > 4 months. During subsequent months of treatment, the size of the metastases stabilized and uptake of nuclide by all tumour sites once again decreased. These results reveal the potential efficacy of an anti-PD-1 antibody for treatment of metastatic MCC. Thus, they contribute to currently limited data on the use of anti-PD-1 antibodies for the treatment of MCC. Moreover, this is the first report of successful resumption of treatment of metastatic MCC with an anti-PD-1 antibody. Results from ongoing trials will contribute to determination of the relevance of PD-1 blockade in metastatic MCC.

Development of a Greenhouse Screening Method for Adult Plant Response in Wheat to Stem Rust.

Screening for adult plant resistance in wheat to stem rust, caused by Puccinia graminis f. sp. tritici, is generally conducted in field plots. Although such evaluations are successful if managed properly, field ratings are time consuming, expensive, weather dependent, and open to inoculum of unwanted races or other confounding diseases. The objective of this study was to develop a dependable system of screening the response of adult plants to stem rust under greenhouse conditions. A comparison of inoculation methods and incubation environments showed that plants inoculated with urediniospores suspended in water, followed by a 24 h dew period in a plastic chamber constructed in a greenhouse, gave the most consistent results. Measurements of response type, stem rust severity, and frequency in follow-up experiments indicated that the most reliable infection was obtained when plants sprayed with 1.25 mg urediniospores per ml water were incubated in the plastic chamber. Using the optimized protocol, a Kariega × Avocet S doubled haploid population was inoculated with two P. graminis f. sp. tritici races. Depending on the race, composite interval mapping showed flag leaf infection type to be significantly influenced by regions on chromosomes 6A, 6D, and 7D. Stem rust severity and reaction type mapped to chromosomes 6D and/or 6A. The Lr34/Yr18/Sr57 gene derived from Kariega on chromosome 7D affected the rust response on flag leaves but not on stems of greenhouse-grown plants. This study showed that phenotyping and genetic analysis of especially major effect stem rust resistance in adult wheat plants is possible and reproducible under controlled conditions in a greenhouse.

Efficacy of different carrier gases for barrier discharge plasma generation compared to chlorhexidine on the survival of Pseudomonas aeruginosa embedded in biofilm in vitro.

Because of its antimicrobial properties, nonthermal plasma could serve as an alternative to chemical antisepsis in wound treatment. Therefore, this study investigated the inactivation of biofilm-embedded Pseudomonas aeruginosa SG81 by a surface barrier-discharged (SBD) plasma for 30, 60, 150 and 300 s. In order to optimize the efficacy of the plasma, different carrier gases (argon, argon admixed with 1% oxygen, and argon with increased humidity up to approx. 80%) were tested and compared against 0.1% chlorhexidine digluconate (CHG) exposure for 600 s. The antimicrobial efficacy was determined by calculating the difference between the numbers of colony-forming units (CFU) of treated and untreated biofilms. Living bacteria were distinguished from dead by fluorescent staining and confocal laser scanning microscopy. Both SBD plasmas and CHG showed significant antimicrobial effects compared to the untreated control. However, plasma treatment led to a higher antimicrobial reduction (argon plasma 4.9 log10 CFU/cm(2), argon with admixed oxygen 3 log10 CFU/cm(2), and with increased gas humidity 2.7 log10 CFU/cm(2) after 300 s) compared to CHG. In conclusion, SBD plasma is suitable as an alternative to CHG for inactivation of Pseudomonas aeruginosa embedded in biofilm. Further development of SBD plasma sources and research on the role of carrier gases and humidity may allow their clinical application for wound management in the future.

Identification of adult plant resistance to stripe rust in the wheat cultivar Cappelle-Desprez.

Following the appearance of stripe rust in South Africa in 1996, efforts have been made to identify new sources of durable resistance. The French cultivar Cappelle-Desprez has long been considered a source of durable, adult plant resistance (APR) to stripe rust. As Cappelle-Desprez contains the seedling resistance genes Yr3a and Yr4a, wheat lines were developed from which Yr3a and Yr4a had been removed, while selecting for Cappelle-Desprez derived APR effective against South African pathotypes of the stripe rust fungus, Puccinia striiformis f. sp. tritici. Line Yr16DH70, adapted to South African wheat growing conditions, was selected and crossed to the stripe rust susceptible cultivar Palmiet to develop a segregating recombinant inbred line mapping population. A major effect QTL, QYr.ufs-2A was identified on the short arm of chromosome 2A derived from Cappelle-Desprez, along with three QTL of smaller effect, QYr.ufs-2D, QYr.ufs-5B and QYr.ufs-6D. QYr.ufs-2D was located within a region on the short arm of chromosome 2D believed to be the location of the stripe rust resistance gene Yr16. An additional minor effect QTL, QYr.ufs-4B, was identified in the cv. Palmiet. An examination of individual RILs carrying single or combinations of each QTL indicated significant resistance effects when QYr.ufs-2A was combined with the three minor QTL from Cappelle-Desprez, and between QYr.ufs-2D and QYr.ufs-5B.

Clinical use of the antiseptic polihexanide for genital tract infections.

BACKGROUND: In clinical practice, treatment of genital tract infections is based on administration of either antibiotics or antiseptics. While antibiotics may be applied systemically or topically, antiseptics may be applied only topically. In case of bacterial vaginosis (BV), antibiotic therapy may often be limited and side effects due to systemic administration may develop. Polihexanide (PHMB) is a promising option for the topical treatment of genital tract infections, in particular BV and vaginitis. METHOD: A systematic search for publications on the use of PHMB for the treatment of genital infections in two electronic databases was performed. Titles, abstracts and citations were imported into a reference database. Duplicates were removed and two reviewers assessed each identified publication separately. RESULTS: Among a total of 204 references, 3 prospective randomized trials were identified. Two trials treated BV infections with PHMB in comparison to clindamycin as antibiotic standard therapy with no significant differences either in safety or in efficacy. The third controlled trial investigated the clinical efficacy of PHMB compared to placebo in the treatment of human papilloma virus. Patients treated with PHMB daily for up to 16-weeks showed significantly higher (52%) clearance of genital warts as compared to patients treated with placebo (4%). CONCLUSION: PHMB may be a clinically effective alternative for the treatment of BV and human papilloma virus. Although PHMB-based antiseptics are available since the late 90s, controlled trials to investigate its clinical potential for antiseptic treatment are scant. Clinical use of antiseptics for the treatment of infectious diseases should be explored and supported further.

First Report of a New Wheat Leaf Rust (Puccinia triticina) Race with Virulence for Lr12, 13, and 37 in South Africa.

A new race of Puccinia triticina was collected from common wheat (Triticum aestivum) in the Eastern and Western Cape provinces during the annual rust survey in 2009. Six single-pustule isolates from a field collection, which were shown to be a new race in preliminary analyses, were inoculated onto seedlings of 16 Thatcher (Tc) near-isogenic differential lines (1) and other tester lines with known Lr genes. Standard procedures for inoculation, incubation, and rust evaluation were followed (4) and all infection studies were repeated. The low infection type of Lr18 was confirmed at 18°C. All six isolates were avirulent (infection types [ITs] 0; to 2) to Lr1, 2a, 2c, 9, 11, 16, 18, and 24 and virulent (ITs 3 to 4) to Lr3, 3ka, 10, 14a, 17, 26, 30, B, and Tc (control). The new race, named 3SA145 according to the ARC-Small Grain Institute notation, corresponds to race CCPS in the North American system (1). On the basis of seedling ITs of the extended Lr gene set, 3SA145 was avirulent (ITs 0; to 22+) to Lr2b, 19, 21, 23, 25, 28, 29, 32, 36 (E84081), 38, 45, 47 (KS90H450), 50 (KS96WGRC36), 51 (R05), and 52 and virulent to Lr3bg, 15, 20 (Thew), 27+31 (Gatcher), and 33. Lines containing the adult plant resistance (APR) genes Lr12 (RL6011, IT 3++), Lr13 (CT263, IT 3), Lr22b (Tc, IT 4), and Lr37 (RL6081, IT 3) were susceptible in the adult stage to 3SA145, whereas lines with the APR genes Lr22a (RL6044, IT ;1), Lr34 (RL6058, IT Z1), and Lr35 (RL6082, IT ;1) were resistant in controlled infection studies in a greenhouse. A control, the common race (3SA133), was virulent only on Tc adult plants. In seedlings, 3SA133 was avirulent to Lr15, 17, 26, and 27+31, but unlike 3SA145, it was virulent to Lr1, 2c, 11, 18, 24, and 28. Races 3SA133 and 3SA145 did not differ in their virulence to the remaining seedling genes. Virulence to Lr37 has been reported in several countries, including Australia, Canada, Uruguay, and the United States (1,2). Prior to the detection of 3SA145, adult plants of RL6081 were resistant to all wheat leaf rust races in South Africa. In 2009, however, RL6081 showed severity levels of up to 30S at certain Western Cape trap plot sites. Of 124 South African bread wheat cultivars and advanced breeding lines tested at the seedling stage, 3SA145 was virulent to 48, whereas 3SA133 was virulent to 36 entries. A further six entries were heterogeneous in their reaction to 3SA145. In adult plant infection studies of 48 South African spring wheats in a greenhouse, 19 were susceptible (flag leaf IT ≥3) and 22 were resistant to 3SA145. Seven entries showed a Z3 flag leaf IT indicating adult plant resistance. According to a simple sequence repeat (SSR) study using 17 primer-pair combinations described by Szabo and Kolmer (3), 3SA145 showed 30% homology with the dominant South African races. Although virulence to Lr12 and Lr13 has been known in different leaf rust races in South Africa, to our knowledge, this is the first report of combined virulence to Lr12, 13, and 37. The SSR data and unique avirulence/virulence profile suggest that 3SA145 may be an exotic introduction to South Africa. References: (1) J. A. Kolmer et al. Plant Dis. 89:1201, 2005. (2) B. McCallum and P. Seto-Goh. Can. J. Plant Pathol. 31:80, 2009. (3) L. Szabo and J. Kolmer. Mol. Ecol. Notes 7:708, 2007. (4) T. Terefe et al. S. Afr. J. Plant Soil 26:51, 2009.

Plant pathogen forensics: capabilities, needs, and recommendations.

A biological attack on U.S. crops, rangelands, or forests could reduce yield and quality, erode consumer confidence, affect economic health and the environment, and possibly impact human nutrition and international relations. Preparedness for a crop bioterror event requires a strong national security plan that includes steps for microbial forensics and criminal attribution. However, U.S. crop producers, consultants, and agricultural scientists have traditionally focused primarily on strategies for prevention and management of diseases introduced naturally or unintentionally rather than on responding appropriately to an intentional pathogen introduction. We assess currently available information, technologies, and resources that were developed originally to ensure plant health but also could be utilized for postintroduction plant pathogen forensics. Recommendations for prioritization of efforts and resource expenditures needed to enhance our plant pathogen forensics capabilities are presented.

Efficacy of chlorhexidine, polihexanide and tissue-tolerable plasma against Pseudomonas aeruginosa biofilms grown on polystyrene and silicone materials.

BACKGROUND: The formation of biofilms is crucial in the pathogenesis of many acute and subacute microbial infections, including chronic wounds and foreign-body-related infections. Topical antimicrobial therapy with chemical antiseptics or physical treatment with tissue-tolerable plasma (TTP) may be promising to control bacterial infection. METHODS: We assessed the efficacy of 0.1% chlorhexidine digluconate (CHX), 0.02 and 0.04% polihexanide (polyhexamethylene biguanide, PHMB) and of TTP against Pseudomonas aeruginosa SG81 biofilm grown in microtitre plates (polystyrene) and on silicone materials in an artificial wound fluid. RESULTS: Overall, PHMB was as effective as CHX in reducing the total amount of biofilm (gentian violet assay) and in reducing the bacterial metabolism in biofilms (XTT assay). TTP also led to a significant reduction in colony-forming units. CONCLUSION: The antimicrobial activity of PHMB in biofilms is comparable to that of CHX. TTP could become an interesting physical alternative to chemical antisepsis in the future.

Antiseptic efficacy and tolerance of tissue-tolerable plasma compared with two wound antiseptics on artificially bacterially contaminated eyes from commercially slaughtered pigs.

AIM: To compare the tissue tolerance and efficacy of two wound antiseptics with tissue-tolerable plasma (TTP) on enucleated contaminated eyes from slaughtered pigs in order to draw consequences for the use of TTP on wounds. METHOD: The corneas of extracted eyes were contaminated with Staphylococcus aureus or Pseudomonas aeruginosa. One and 10 min after application of 10% povidone (PVP)-iodine and 0.04% polyhexanide, respectively, the eyes were rinsed with inactivating solution. To test TTP, the plasma pen meandered over the eyes at a speed of 30 mm/s and a distance of 5 mm; the eyes were then rinsed with balanced salt solution. The reduction factor was calculated by the difference between the logarithm of colony-forming units in the rinse before and after antisepsis or TTP application. RESULTS: The efficacy of TTP (reduction factor 2.4-2.9) was significantly higher (p < 0.001) than that of PVP-iodine and polyhexanide (reduction factor 1.7-2.1). CONCLUSION: TTP is more effective than the tested wound antiseptics. The lack of histological damage to the eyes of slaughtered pigs would seem to make its use as a wound antiseptic a viable alternative. In contrast to antiseptics, it supplies additional energy in the form of heat, electric fields and radicals by TTP.

First Report of Virulence for the Wheat Leaf Rust (Puccinia triticina) Resistance Gene Lr32 in South Africa.

The wheat leaf rust resistance gene Lr32 was transferred from Aegilops tauschii Coss. to bread wheat (Triticum aestivum L.) (1). Despite virulence for Lr32 in some isolates from Bulgaria, Israel, and Turkey, the gene has been reported to be effective in Australia, Mexico, the United States, and South Africa (1,2). A leaf rust isolate that differed in its avirulence/virulence profile from previously recorded races of Puccinia triticina Eriks. in South Africa was collected from triticale (× Triticosecale) in the Western Cape in 2005. According to the South African leaf rust differential set (3), this isolate (UVPt19) was avirulent for Lr3a, 3bg, 3ka, 10, 11, 16, 20, 26, and 30 and virulent for Lr1, 2a, 2b, 2c, 14a, 15, 17, 24, and Thatcher (Tc, control). Except for Lr20 in cv. Thew, all differentials are Tc near-isogenic lines. In comparison with known South African races (3), it differed from race 3SA132 at the Lr10 locus. Using standard rust pathology protocols (3), an expanded set of Lr gene lines (non Tc lines indicated) showed that UVPt19 is avirulent on wheat seedlings containing Lr9, 19, 21, 25, 27+31 (Gatcher), 29, 36 (ER84018), 37, 41 (KS91WGRC10), 44, 45, 47 (KS90H450), 50 (KS96WGRC36), 51 (R05), and 52, and virulent for Lr12, 22a, 23, 28, 32, 33, and 35. In the seedling stage, UVPt19 was virulent for the temperature sensitive genes Lr13, 18, and 34 at 25°C, but produced lower infection types (ITs) on Lr18 and 34 at 14 to 18°C. Seedlings of Pavon 76 (Lr46) were resistant (IT ;1=) to UVPt19. The susceptible response of lines carrying Lr32 was confirmed by high ITs (3++4) on RL5713/2*Mq, RL6086 (TcLr32), and RL5713/2*Mq//6*Palmiet. A control isolate (UVPt9) produced ITs ;1+, ;1+, and ;;1= on these lines, respectively. UVPt19 was virulent on line RL6092 (TcLr20) but avirulent on Thew. When tested on adult plants of lines RL6011 (TcLr12), CT263 (TcLr13), RL6044 (TcLr22a), RL6058 (TcLr34), RL6082 (TcLr35), RL6081 (TcLr37), and Tc (control), UVPt19 was only virulent (IT 3+) on CT263 and Tc. Flag leaves of RL6011 (IT ;1), RL6044 (IT 1), RL6058 (IT Z3-), RL6082 (IT 0;), and RL6081 (IT ;1) were resistant. UVPt19 was virulent on seedlings of 11 of 13 triticale cultivars and lines tested as opposed to UVPt9, which was virulent to only one entry. From a collection of 105 South African bread wheat cultivars and elite breeding lines, UVPt19 was virulent on 13 and five were mixed in their response to this isolate. All IT experiments were repeated. Although virulence has emerged for Lr32 in South Africa, the gene has not been used in local cultivars. Previously, McIntosh et al. (1) also reported that Lr32 has not been exploited in wheat production. On the basis of current evidence, UVPt19 appears to be potentially more damaging to triticale than bread wheat. Furthermore, the race seems rare because it was not collected in a recent wheat leaf rust survey in South Africa (3). References: (1) R. A. McIntosh et al. The Wheat Rusts: An Atlas of Resistance Genes, CSIRO-Kluwer, Dordrecht, the Netherlands, 1995. (2) Z. A. Pretorius. Phytophylactica 21:195, 1989. (3) T. Tarekegn et al. S. Afr. J. Plant Soil 26:51, 2009.

First Report of a Puccinia graminis f. sp. tritici Race Virulent to the Sr24 and Sr31 Wheat Stem Rust Resistance Genes in South Africa.

Isolates of Puccinia graminis f. sp. tritici belonging to the Ug99 race group are virulent to a broad spectrum of resistance genes, rendering most of the world's wheat germplasm susceptible to stem rust (3). Following the initial detection of Ug99 (TTKSK, North American [NA] race notation) in Uganda, virulence to the widely used Sr31 resistance gene has been reported from Kenya, Ethiopia, Sudan, and Iran (2,3). In November 2009, a wheat genotype suspected to carry Sr31 showed a susceptible response to stem rust in a disease nursery (29°08'05.02''S, 30°38'29.18''E), inoculated with race TTKSP, near Greytown in KwaZulu-Natal, South Africa. Inoculation of urediniospores of the field collection (isolate UVPgt60) onto seedlings of line Federation4*/Kavkaz confirmed virulence for Sr31. In three independent, replicated, and comparative seedling tests, eight single-pustule isolates of UVPgt60 all typed to race PTKST following the NA race nomenclature. These isolates produced compatible infection types (ITs) (3+ to 4) on the Sr31 testers Gamtoos, Sr31/6*LMPG, Federation4*/Kavkaz, Kavkaz, and Clement, whereas isolate UVPgt59 (TTKSP) was avirulent (ITs ;1 to 1) on these genotypes. In addition to Sr31 virulence, the new race differed from TTKSP by producing a lower IT (2 to 2++) on Cns_T.mono_ deriv., the accepted entry for Sr21 in the NA differential set. The UVPgt60 isolates were clearly avirulent on Einkorn (Sr21) (IT ;1=), a response that also differed from those produced by BPGSC, TTKSF, and TTKSP (IT 2). With the exception of Sr21, UVPgt60 isolates had a virulence pattern similar to race TTKST (1), notably the virulence combination for Sr24 and Sr31. Isolate UVPgt60.6 was randomly selected for testing on additional Sr genes and South African wheat cultivars and breeding lines. Similar to the race identification experiments seedling tests were duplicated and compared with reactions produced by TTKSP and other races. Greenhouse temperatures for all seedling tests ranged between 18 and 25°C. On the basis of primary leaf responses, PTKST is avirulent (ITs 0; to 2++) for Sr13, 14, 21, 22, 25, 26, 27, 29, 32, 33, 35, 36, 37, 39, 42, 43, 44, Em, Tmp, and Satu and virulent (ITs 3 to 4) for Sr5, 6, 7b, 8a, 8b, 9a, 9b, 9d, 9e, 9g, 10, 11, 16, 17, 24, 30, 31, 34, 38, 41, and McN. From 103 South African wheat cultivars and lines tested as seedlings, 59 and 47 were susceptible (IT ≥ 3) to races PTKST and TTKSP, respectively. Simple-sequence repeat analysis (4) with selected primer pairs showed that PTKST clusters with isolates belonging to the Ug99 lineage. Subsequent to the collection made at Greytown, stem rust sampled in December 2009 from naturally infected breeders' lines at Cedara (29°32'19.59''S, 30°16'03.50''E), KwaZulu-Natal, revealed five isolates with a virulence profile similar to PTKST. On the basis of current evidence it appears that PTKST may be an introduction to South Africa rather than a single-step mutation from local stem rust races. References: (1) Y. Jin et al. Plant Dis. 92:923, 2008. (2) K. Nazari et al. Plant Dis. 93:317, 2009. (3) R. P. Singh et al. Adv. Agron. 98:271, 2008. (4) B. Visser et al. Mol. Plant Pathol. 10:213, 2009.

Living with death: the evolution of the mitochondrial pathway of apoptosis in animals.

The mitochondrial pathway of cell death, in which apoptosis proceeds following mitochondrial outer membrane permeabilization, release of cytochrome c, and APAF-1 apoptosome-mediated caspase activation, represents the major pathway of physiological apoptosis in vertebrates. However, the well-characterized apoptotic pathways of the invertebrates C. elegans and D. melanogaster indicate that this apoptotic pathway is not universally conserved among animals. This review will compare the role of the mitochondria in the apoptotic programs of mammals, nematodes, and flies, and will survey our knowledge of the apoptotic pathways of other, less familiar model organisms in an effort to explore the evolutionary origins of the mitochondrial pathway of apoptosis.

Potential Energy Surface Including Electron Correlation for F + H2 rarr FH + H: Refined Linear Surface.

A priori quantum mechanical calculations have been carried out at about 150 linear geometries for the fluorine plus hydrogen molecule system. An extended basis set of Gaussian functions was used, and electron correlation was treated explicitly by configuration interaction. Comparison with the experimental activation energy and exothermicity suggests that the theoretical potential surface is quite realistic.

Predicting the evolution of human influenza A.

Eighteen codons in the HA1 domain of the hemagglutinin genes of human influenza A subtype H3 appear to be under positive selection to change the amino acid they encode. Retrospective tests show that viral lineages undergoing the greatest number of mutations in the positively selected codons were the progenitors of future H3 lineages in 9 of 11 recent influenza seasons. Codons under positive selection were associated with antibody combining site A or B or the sialic acid receptor binding site. However, not all codons in these sites had predictive value. Monitoring new H3 isolates for additional changes in positively selected codons might help identify the most fit extant viral strains that arise during antigenic drift.

Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness.

An avian H5N1 influenza A virus (A/Hong Kong/156/97) was isolated from a tracheal aspirate obtained from a 3-year-old child in Hong Kong with a fatal illness consistent with influenza. Serologic analysis indicated the presence of an H5 hemagglutinin. All eight RNA segments were derived from an avian influenza A virus. The hemagglutinin contained multiple basic amino acids adjacent to the cleavage site, a feature characteristic of highly pathogenic avian influenza A viruses. The virus caused 87.5 to 100 percent mortality in experimentally inoculated White Plymouth Rock and White Leghorn chickens. These results may have implications for global influenza surveillance and planning for pandemic influenza.

Pseudomonas syringae phytotoxins: mode of action, regulation, and biosynthesis by peptide and polyketide synthetases.

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents.

Prior stress promotes the generalization of contextual fear memories: Involvement of the gabaergic signaling within the basolateral amygdala complex.

Fear generalization occurs when a response, previously acquired with a threatening stimulus, is transferred to a similar one. However, it could be maladaptive when stimuli that do not represent a real threat are appraised as dangerous, which is a hallmark of several anxiety disorders. Stress exposure is a major risk factor for the occurrence of anxiety disorders and it is well established that it influences different phases of fear memory; nevertheless, its impact on the generalization of contextual fear memories has been less studied. In the present work, we have characterized the impact of acute restraint stress prior to contextual fear conditioning on the generalization of this fear memory, and the role of the GABAergic signaling within the basolateral amygdala complex (BLA) on the stress modulatory effects. We have found that a single stress exposure promoted the generalization of this memory trace to a different context that was well discriminated in unstressed conditioned animals. Moreover, this effect was dependent on the formation of a contextual associative memory and on the testing order (i.e., conditioning context first vs generalization context first). Furthermore, we observed that increasing GABA-A signaling by intra-BLA midazolam administration prior to the stressful session exposure prevented the generalization of fear memory, whereas intra-BLA administration of the GABA-A antagonist (Bicuculline), prior to fear conditioning, induced the generalization of fear memory in unstressed rats. We concluded that stress exposure, prior to contextual fear conditioning, promotes the generalization of fear memory and that the GABAergic transmission within the BLA has a critical role in this phenomenon.

Roles of cell division and gene transcription in the methylation of CpG islands.

De novo methylation of CpG islands within the promoters of eukaryotic genes is often associated with their transcriptional repression, yet the methylation of CpG islands located downstream of promoters does not block transcription. We investigated the kinetics of mRNA induction, demethylation, and remethylation of the p16 promoter and second-exon CpG islands in T24 cells after 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment to explore the relationship between CpG island methylation and gene transcription. The rates of remethylation of both CpG islands were associated with time but not with the rate of cell division, and remethylation of the p16 exon 2 CpG island occurred at a higher rate than that of the p16 promoter. We also examined the relationship between the remethylation of coding sequence CpG islands and gene transcription. The kinetics of remethylation of the p16 exon 2, PAX-6 exon 5, c-ABL exon 11, and MYF-3 exon 3 loci were examined following 5-Aza-CdR treatment because these genes contain exonic CpG islands which are hypermethylated in T24 cells. Remethylation occurred most rapidly in the p16, PAX-6, and c-ABL genes, shown to be transcribed prior to drug treatment. These regions also exhibited higher levels of remethylation in single-cell clones and subclones derived from 5-Aza-CdR-treated T24 cells. Our data suggest that de novo methylation is not restricted to the S phase of the cell cycle and that transcription through CpG islands does not inhibit their remethylation.