Distribution of Three Verticillium dahliae Races in Coastal California and Evaluation of Resistance in Lettuce.

Verticillium wilt, caused by Verticillium dahliae, is one of the most devastating soilborne diseases of lettuce (Lactuca sativa L.). There are three races of V. dahliae, and each race has been characterized by markers representing race-specific effectors. Race 1 is differentiated by the presence of the functional secretory Ave1 effector. Similarly, races 2 and 3 are differentiated by effectors VdR2e and VdR3e, respectively. Although the presence of race 1 in coastal California was well established, the presence of effector-based races 2 and 3 was uncertain. This study therefore focused on characterizing 727 isolates collected from 142 ranches of symptomatic lettuce and other crops from coastal California. Based on this evaluation, 523 isolates were designated as race 1, 20 isolates as race 2, 23 isolates as race 3, and 17 as race undefined. Isolates representing other Verticillium species totaled 110, and 34 were non-Verticillium fungal species. Because the use of resistant cultivars is a key strategy to manage this disease, we evaluated 48 lettuce germplasm lines and 1 endive (Cichorium endivia L.) line, comprising commercial cultivars and breeding lines, including the race 1-resistant heirloom cultivar La Brillante and the susceptible cultivar Salinas as controls. Resistance against races 1, 2, and 3 along with VdLs17, a virulent isolate of V. dahliae from lettuce that is currently not assigned to a race, was evaluated in replicated greenhouse experiments. Two crisphead lettuce lines, HL28 and HL29, exhibited resistance against race 1 and a partial resistance against race 2, whereas all other lines were highly susceptible to races 1 and 2 and VdLs17. The majority of lines exhibited higher resistance to race 3 relative to the other two races. This study documents the current distribution of the different races in coastal California. In addition, the sources of resistance currently being developed should be effective or partially effective against these races for targeted deployment as soon as they are available.

Characterization and genome sequence of Microbacterium foliorum phage Morrigan, of cluster EA6 with siphovirus morphology.

Bacteriophage Morrigan, which was isolated from soil using Microbacterium foliorum NRRL B-24224, is lytic with siphovirus morphology. Morrigan's 40,509-bp genome has a GC content of 62.8% and 66 putative protein-coding genes, of which 31 could be assigned putative functions. Based on gene content similarity to actinobacteriophages, Morrigan is assigned to subcluster EA6.

First report of Globisporangium uncinulatum (syn. Pythium uncinulatum) causing Pythium wilt of lettuce in Arizona.

In 2021 and 2022, wilt symptoms were observed in lettuce (Lactuca sativa L.) fields in Yuma County, Arizona (AZ). Incidence was < 1% at all locations. Symptoms included stunting, yellowing and wilting of outer leaves. As disease progressed, outer leaves wilted and turned necrotic. In advanced stages, tap roots turned brown-gray, with few feeder roots. The crown remained intact until the plant collapsed. Symptomatic romaine and iceberg plants were collected from two sites. Necrotic roots were washed and then plated on amended corn meal agar (PARP) (Kannwischer et al. 1978). After 2-3 days, slow growing, coenocytic, submerged mycelia grew from these pieces. In culture, profuse oogonia formed with diameters of 30-39 (avg. = 33.7) µm and spiny protuberances (5-8 [avg. = 6.4] µm long) with thickened bases. Oospores were spherical and aplerotic, with diameters of 25-32 (avg. = 27.8) µm. Lettuce with identical symptoms from the Salinas Valley, California (CA) were also tested and similar isolates were recovered. Pathogenicity was tested using six AZ and one CA isolates. Inoculum was grown on barley seeds moistened with sterile distilled water, autoclaved three times (at 24 h intervals), then inoculated with colonized agar plugs and incubated at 20°C. Inoculum was used after two weeks. For each isolate, 12 3-week-old iceberg (cv. Speedway) and romaine (cv. Del Sol) plants were inoculated by placing 3-4 colonized barley seed next to the roots of the potted plants. Plants were maintained in a greenhouse at 24-26°C (daytime high) with ambient light. After eight days, all inoculated plants exhibited chlorotic lower leaves that later wilted. Both feeder roots and taproots showed brown-gray discoloration and were necrotic. Microscopy showed the presence of spiny oogonia in inoculated roots. Symptoms caused by the AZ and CA isolates were indistinguishable from each other. Isolations from necrotic tissue resulted in colonies morphologically identical to the original isolates. Twelve control plants inoculated with uncolonized barley seed developed no symptoms. DNA was extracted from all seven AZ and CA isolates pre-inoculation, and AZ isolate 2 recovered from both lettuce types post-inoculation for molecular characterization. The internal transcribed spacer (ITS) and cytochrome C oxidase subunit 2 (COX II) were amplified for the above isolates using primer sets ITS1/ITS4 (White et al. 1990) and FM66/FM58 (Villa et al. 2006), then sequenced. ITS of pre- and post-inoculated isolates for AZ (OQ054806 and OQ054807) and CA (OQ564388) matched 1078/1078 bases of Globisporangium uncinulatum (syn. Pythium uncinulatum; AY598712.2) with 99.8% identity. There were two SNPs in COX II for AZ isolate 1 (OR069239); all other isolates pre- and post- inoculation for AZ (OR069240 and OR069242) and CA (OR069241) uniformly matched 533/535 bases of G. uncinulatum (KJ595385.1) with 99.4% identity. Based on these molecular and morphological data, the isolates were identified as G. uncinulatum (Blok and Van Der Plaats-Niterink 1978; Van Der Plaats-Niterink 1981). To our knowledge, this is the first report of G. uncinulatum on lettuce in AZ. Designated as Pythium wilt, this disease is reported on lettuce in The Netherlands (Blok and Van Der Plaats-Niterink 1978), Japan (Matsuura, et al. 2010), and CA (Davis, et al. 1995). Arizona is an important lettuce growing region; if this disease becomes more prevalent, lettuce production in this region could be negatively impacted.

Role of Thyrotropin-Releasing Hormone in Regulating Fibroblast Growth Factor 21 in Mouse Pancreatic ß Cells.

Background: Thyrotropin-releasing hormone (TRH) is primarily produced in the hypothalamus and regulates the thyrotropin secretion from the pituitary. TRH is distributed ubiquitously in the extrahypothalamic region, especially in pancreatic islets, while its physiological role remains nebulous. We have previously established a TRH-deficient mouse model, and showed impaired glucose tolerance and downregulated expression of fibroblast growth factor 21 (FGF21) in islets. Recent studies have demonstrated the physiological roles of pancreatic FGF21. Therefore, in this study, we elucidate the direct functions of TRH in pancreatic islets via the regulation of FGF21. Methods: To explore the functions of TRH in pancreatic islets, a microarray analysis using isolated islets from TRH-knockout mice was conducted. The regulatory mechanism of TRH in pancreatic FGF21 was investigated using islet cell lines; reverse transcription-quantitative polymerase chain reaction and Western blotting were used to determine the mRNA and protein expression levels of FGF21 in pancreatic islets and islet cell lines. Induction of FGF21 expression by TRH treatment was examined in vitro. To identify the transcription factors binding to the region responsible for TRH-induced stimulation of the FGF21 promoter, electromobility shift assays were conducted. Results: Among the detected and considerably changed genes in microarray, FGF21 was the most consistently downregulated in TRH-deficient mice islets. FGF21 was strongly co-expressed with insulin in mouse islets, and TRH stimulated endogenous Fgf21 mRNA expression in the islet cell line ßHC9. The E-box site in the FGF21 promoter was responsible for TRH-induced stimulation via the extracellular signal-regulated kinase (ERK)1/2 signaling pathway. The transcription factor upstream stimulatory factor 1 (USF1) could specifically bind to the E-box site. Overexpression of USF1 increased FGF21 promoter activity. Conclusion: FGF21 was transcriptionally upregulated by TRH through the ERK1/2 and USF1 pathways in pancreatic ß cells.

Manipulation of the Plant Host by the Geminivirus AC2/C2 Protein, a Central Player in the Infection Cycle.

Geminiviruses are a significant group of emergent plant DNA viruses causing devastating diseases in food crops worldwide, including the Southern United States, Central America and the Caribbean. Crop failure due to geminivirus-related disease can be as high as 100%. Improved global transportation has enhanced the spread of geminiviruses and their vectors, supporting the emergence of new, more virulent recombinant strains. With limited coding capacity, geminiviruses encode multifunctional proteins, including the AC2/C2 gene that plays a central role in the viral replication-cycle through suppression of host defenses and transcriptional regulation of the late viral genes. The AC2/C2 proteins encoded by mono- and bipartite geminiviruses and the curtovirus C2 can be considered virulence factors, and are known to interact with both basal and inducible systems. This review highlights the role of AC2/C2 in affecting the jasmonic acid and salicylic acid (JA and SA) pathways, the ubiquitin/proteasome system (UPS), and RNA silencing pathways. In addition to suppressing host defenses, AC2/C2 play a critical role in regulating expression of the coat protein during the viral life cycle. It is important that the timing of CP expression is regulated to ensure that ssDNA is converted to dsDNA early during an infection and is sequestered late in the infection. How AC2 interacts with host transcription factors to regulate CP expression is discussed along with how computational approaches can help identify critical host networks targeted by geminivirus AC2 proteins. Thus, the role of AC2/C2 in the viral life-cycle is to prevent the host from mounting an efficient defense response to geminivirus infection and to ensure maximal amplification and encapsidation of the viral genome.

CTLA4 +49AG (rs231775) and CT60 (rs3087243) gene variants are not associated with alopecia areata in a Mexican population from Monterrey Mexico

Abstract Background: Alopecia areata is an autoimmune disease that produces non-scarring hair loss around the body. Gene variants of the cytotoxic T-lymphocyte antigen 4 (CTLA4) gene, a negative regulator of T-cell response, have been associated with a predisposition to autoimmune diseases in different populations; however, the involvement of these genetic variants in the development of AA is controversial. Objective: The present study evaluated the potential association of two CTLA4 gene variants with alopecia areata in a Mexican population. Methods: We genotyped +49AG (rs231775) and CT60 (rs3087243) variants in 50 AA patients and 100 healthy control participants through PCR-RFLP. Results: No statistical difference was observed for either of the gene variants regarding allele or genotype frequencies between AA patients and the controls when the parameters of family/personal history of autoimmune diseases or gender were considered (p > 0.05). Study limitations: Small sample size of patients and the data were obtained from Northeast Mexico population. Conclusion: The genetic variants rs231775 and rs3087243 of the CTLA4 gene are not a risk factor for the development of alopecia areata in the analyzed Mexican population.

CTLA4 +49AG (rs231775) and CT60 (rs3087243) gene variants are not associated with alopecia areata in a Mexican population from Monterrey Mexico.

BACKGROUND: Alopecia areata is an autoimmune disease that produces non-scarring hair loss around the body. Gene variants of the cytotoxic T-lymphocyte antigen 4 (CTLA4) gene, a negative regulator of T-cell response, have been associated with a predisposition to autoimmune diseases in different populations; however, the involvement of these genetic variants in the development of AA is controversial. OBJECTIVE: The present study evaluated the potential association of two CTLA4 gene variants with alopecia areata in a Mexican population. METHODS: We genotyped +49AG (rs231775) and CT60 (rs3087243) variants in 50 AA patients and 100 healthy control participants through PCR-RFLP. RESULTS: No statistical difference was observed for either of the gene variants regarding allele or genotype frequencies between AA patients and the controls when the parameters of family/personal history of autoimmune diseases or gender were considered (p>0.05). STUDY LIMITATIONS: Small sample size of patients and the data were obtained from Northeast Mexico population. CONCLUSION: The genetic variants rs231775 and rs3087243 of the CTLA4 gene are not a risk factor for the development of alopecia areata in the analyzed Mexican population.

Complementary-sense gene regulation in beet curly top virus-SpCT.

A 278-bp region upstream of the beet curly top virus-SpCT (BCTV-SpCT) C2/C3 genes is necessary for promoter activity and exhibits significant sequence similarity to AL2/3 promoter sequences in tomato golden mosaic virus (TGMV). Maximal expression of the downstream C2/3 genes in BCTV-SpCT requires the presence of the C1 protein, which is supported by observations that mutation of the initiator codon for C1 results in decreased C2/C3 expression. This is similar to TGMV and cabbage leaf curl virus, where AL1 is required for maximal AL2/3 expression. Together, these data suggest a common strategy for complementary-sense gene regulation amongst curtoviruses and begomoviruses.

Improvement of nutrients removal from domestic wastewater by activated-sludge encapsulation with polyvinyl alcohol (PVA).

Conventional activated-sludge (AS) technologies are deficient for nutrient removal because they require specific floc characteristics. Therefore, the encapsulated AS with polyvinyl alcohol (PVA) will favor floc's formation that removes nutrients. The applied method was based on monitoring the removal of organic matter and nutrients (NH4+, NO3-, NO2-, PO43-) from synthetic domestic wastewater using laboratory-scale AS. The experimental reactors were operated at 8 h as optimized Hydraulic Retention Time (HRT). The sludge characteristics evaluation was carried out through the Sludge Volumetric Index (SVI), Food/Microorganism ratio (F/M), and Mixed Liquor Volatile Suspended Solids (MLVSS). Other specific floc characteristics, such as zeta potential and effective diameter were also evaluated. The results showed that the encapsulated AS with PVA favors nitrogen and phosphorous removal up to 35% but it did not improve organic matter removal. In addition, encapsulated AS with PVA has the characteristics of filamentous sludge (F/M: 0.7 g COD g-1 MLVSS d-1) with good settleability conditions (SVI: 43 mL g-1 MLSVS h-1) and low zeta potential (ZP: -0.9 mV), which favors its separation from the liquid phase. In conclusion, the encapsulation of AS with PVA improves nutrient removal by improving floc characteristics.

Emerging technologies for protease engineering: New tools to clear out disease.

Proteases regulate many biological processes through their ability to activate or inactive their target substrates. Because proteases catalytically turnover proteins and peptides, they present unique opportunities for use in biotechnological and therapeutic applications. However, many proteases are capable of cleaving multiple physiological substrates. Therefore their activity, expression, and localization are tightly controlled to prevent unwanted proteolysis. Currently, the use of protease therapeutics has been limited to a handful of proteases with narrow substrate specificities, which naturally limits their toxicity. Wider application of proteases is contingent upon the development of methods for engineering protease selectivity, activity, and stability. Recent advances in the development of high-throughput, bacterial and yeast-based methods for protease redesign have yielded protease variants with novel specificities, reduced toxicity, and increased resistance to inhibitors. Here, we highlight new tools for protease engineering, including methods suitable for the redesign of human secreted proteases, and future opportunities to exploit the catalytic activity of proteases for therapeutic benefit. Biotechnol. Bioeng. 2017;114: 33-38. © 2016 Wiley Periodicals, Inc.

Intracellular FRET-based Screen for Redesigning the Specificity of Secreted Proteases.

Proteases are attractive as therapeutics given their ability to catalytically activate or inactivate their targets. However, therapeutic use of proteases is limited by insufficient substrate specificity, since off-target activity can induce undesired side-effects. In addition, few methods exist to enhance the activity and specificity of human proteases, analogous to methods for antibody engineering. Given this need, a general methodology termed protease evolution via cleavage of an intracellular substrate (PrECISE) was developed to enable engineering of human protease activity and specificity toward an arbitrary peptide target. PrECISE relies on coexpression of a protease and a peptide substrate exhibiting Förster resonance energy transfer (FRET) within the endoplasmic reticulum of yeast. Use of the FRET reporter substrate enabled screening large protease libraries using fluorescence activated cell sorting for the activity of interest. To evolve a human protease that selectively cleaves within the central hydrophobic core (KLVF↓F↓AED) of the amyloid beta (Aß) peptide, PrECISE was applied to human kallikrein 7, a protease with Aß cleavage activity but broad selectivity, with a strong preference for tyrosine (Y) at P1. This method yielded a protease variant which displayed up to 30-fold improvements in Aß selectivity mediated by a reduction in activity toward substrates containing tyrosine. Additionally, the increased selectivity of the variant led to reduced toxicity toward PC12 neuronal-like cells and 16-1000-fold improved resistance to wild-type inhibitors. PrECISE thus provides a powerful high-throughput capability to redesign human proteases for therapeutic use.

Cardiac hormones are c-Jun-N-terminal kinase 2-inhibiting peptides.

BACKGROUND: Four cardiac peptide hormones, namely vessel dilator, long-acting natriuretic peptide (LANP), kaliuretic peptide, and atrial natriuretic peptide (ANP) have anticancer effects. MATERIALS AND METHODS: The effects of these four cardiac hormones on human c-Jun-N-terminal kinase 2 (JNK2) were examined in human small cell lung cancer and human prostate cancer cells. RESULTS: Vessel dilator, LANP, kaliuretic peptide and ANP maximally reduced expression of JNK2 by 89%, 56%, 45%, and 28%, respectively (each at p<0.0001) in human small cell lung cancer cells. In human prostate adenocarcinoma cells, JNK2 was maximally decreased 76%, 56%, 45%, (each at p<0.0001), and 28% (p<0.01) secondary to vessel dilator, LANP, kaliuretic peptide and ANP, respectively. CONCLUSION: These results indicate that four cardiac hormones are significant inhibitors (by up to 89%) of JNK2 in human small cell lung cancer cells and up to 76% in human prostate adenocarcinoma cells as part of their anticancer mechanism(s) of action.

Rethinking the role of automaticity in cognitive control.

Behaviour that is assumed to be guided by strategy can, in fact, be based on the implicit learning of regularities in the environment. We demonstrate this point in the context of a Stroop experiment. It has been shown previously that performance on this measure of cognitive control varies as a function of the relative proportions of congruent and incongruent trials in a block. Here we provide evidence that this modulation of performance is largely based on implicit, rather than explicit, knowledge of these proportions. This result has important implications for our understanding of cognitive control.

[Methicillin-resistant Staphylococcus aureus at a general hospital: epidemiological overview between 2000-2007]. / Staphylococcus aureus resistente a meticilina en un hospital general: panorama epidemiológico del 2000 al 2007.

INTRODUCTION: The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitalized patients is a significant problem due to its associated morbidity and mortality. It is important to know the recent MRSA epidemiology at a General Hospital. OBJECTIVES: To determine the MRSA epidemiology at a Mexican general hospital from 2000 to 2007, in order to know if there is a significant trend in its proportion. MATERIAL AND METHODS: Prevalence survey. The resistance to oxacillin was identified by the Kirby-Bauer's method. The specimens were classified by type and year of isolation. Trend statistics were used for analysis. RESULTS: S. aureus was identified in 1,008 samples, being 301 resistant to oxacillin (30%, 95% Confidence Interval [CI], 25 to 35%). The proportion of MRSA went from 37% (95% CI, 29 to 44%) to 49% (95% CI, 40 to 58%) in the period of study (chi2 for trends = 6.676, p < 0.01). Specimens with the highest proportion of MRSA were blood and sterile liquids with 32% (95% CI, 26 to 39%), secretions with 29% (95% CI, 24 to 33%), and catheters with 21% (95% CI, 16 to 26%). CONCLUSIONS: The proportion of MRSA has increased significantly. This leads to higher costs and morbi-mortality for the hospitalized patients. We require stricter policies to prevent transmission and to control the use of antibiotics.

Rational improvement of simvastatin synthase solubility in Escherichia coli leads to higher whole-cell biocatalytic activity.

Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility of LovD without decreasing its catalytic activity can therefore elevate the performance of the whole-cell biocatalyst. Using a combination of homology structural prediction and site-directed mutagenesis, we identified two cysteine residues in LovD that are responsible for nonspecific intermolecular crosslinking, which leads to oligomer formation and protein aggregation. Replacement of Cys40 and Cys60 with alanine residues resulted in marked gain in both protein solubility and whole-cell biocatalytic activities. Further mutagenesis experiments converting these two residues to small or polar natural amino acids showed that C40A and C60N are the most beneficial, affording 27% and 26% increase in whole cell activities, respectively. The double mutant C40A/C60N combines the individual improvements and displayed approximately 50% increase in protein solubility and whole-cell activity. Optimized fed-batch high-cell-density fermentation of the double mutant in an E. coli strain engineered for simvastatin production quantitatively (>99%) converted 45 mM MJSS to SS within 18 h, which represents a significant improvement over the performance of wild-type LovD under identical conditions. The high efficiency of the improved whole-cell platform renders the biocatalytic synthesis of SS an attractive substitute over the existing semisynthetic routes.