Fungal pathogens of cereal crops: Proteomic insights into fungal pathogenesis, host defense, and resistance.

Fungal infections of cereal crops pose a significant risk to global food security through reduced grain production and quality, as well as contamination of animal feed and human products for consumption. To combat fungal disease, we need to understand how the pathogen adapts and survives within the hostile environment of the host and how the host's defense response can be modulated for protection from disease. Such investigations offer insight into fungal pathogenesis, host immunity, the development of resistance, and mechanisms of action for currently-used control strategies. Mass spectrometry-based proteomics provides a technologically-advanced platform to define differences among fungal pathogens and their hosts at the protein level, supporting the discovery of proteins critical for disease, and uncovering novel host responses driving susceptibly or resistance of the host. In this Review, we explore the role of mass spectrometry-based proteomics in defining the intricate relationship between a pathogen and host during fungal disease of cereal crops with a focus on recent discoveries derived from the globally-devastating diseases of Fusarium head blight, Rice blast, and Powdery mildew. We highlight advances made for each of these diseases and discuss opportunities to extrapolate findings to further our fight against fungal pathogens on a global scale.

Proteome and secretome profiling of zinc availability in Cryptococcus neoformans identifies Wos2 as a subtle influencer of fungal virulence determinants.

BACKGROUND: Fungal infections impact over 25% of the global population. For the opportunistic fungal pathogen, Cryptococcus neoformans, infection leads to cryptococcosis. In the presence of the host, disease is enabled by elaboration of sophisticated virulence determinants, including polysaccharide capsule, melanin, thermotolerance, and extracellular enzymes. Conversely, the host protects itself from fungal invasion by regulating and sequestering transition metals (e.g., iron, zinc, copper) important for microbial growth and survival. RESULTS: Here, we explore the intricate relationship between zinc availability and fungal virulence via mass spectrometry-based quantitative proteomics. We observe a core proteome along with a distinct zinc-regulated protein-level signature demonstrating a shift away from transport and ion binding under zinc-replete conditions towards transcription and metal acquisition under zinc-limited conditions. In addition, we revealed a novel connection among zinc availability, thermotolerance, as well as capsule and melanin production through the detection of a Wos2 ortholog in the secretome under replete conditions. CONCLUSIONS: Overall, we provide new biological insight into cellular remodeling at the protein level of C. neoformans under regulated zinc conditions and uncover a novel connection between zinc homeostasis and fungal virulence determinants.

Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production.

BACKGROUND: Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. RESULTS: Analysis of intra- and extracellular changes identified 2380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. CONCLUSIONS: This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

Quantitative proteomic profiling of shake flask versus bioreactor growth reveals distinct responses of Agrobacterium tumefaciens for preparation in molecular pharming.

The preparation of Agrobacterium tumefaciens cultures with strains encoding proteins intended for therapeutic or industrial purposes is an important activity prior to treatment of plants for transient expression of valuable protein products. The rising demand for biologic products such as these underscores the expansion of molecular pharming and warrants the need to produce transformed plants at an industrial scale. This requires large quantities of A. tumefaciens culture, which is challenging using traditional growth methods (e.g., shake flask). To overcome this limitation, we investigate the use of bioreactors as an alternative to shake flasks to meet production demands. Here, we observe differences in bacterial growth among the tested parameters and define conditions for consistent bacterial culturing between shake flask and bioreactor. Quantitative proteomic profiling of cultures from each growth condition defines unique growth-specific responses in bacterial protein abundance and highlights the functional roles of these proteins, which may influence bacterial processes important for effective agroinfiltration and transformation. Overall, our study establishes and optimizes comparable growth conditions for shake flask versus bioreactors and provides novel insights into fundamental biological processes of A. tumefaciens influenced by such growth conditions.

Exposure of Agrobacterium tumefaciens to agroinfiltration medium demonstrates cellular remodelling and may promote enhanced adaptability for molecular pharming.

Agroinfiltration is used to treat plants with modified strains of Agrobacterium tumefaciens for the purpose of transient in planta expression of genes transferred from the bacterium. These genes encode valuable recombinant proteins for therapeutic or industrial applications. Treatment of large quantities of plants for industrial-scale protein production exposes bacteria (harboring genes of interest) to agroinfiltration medium that is devoid of nutrients and carbon sources for prolonged periods of time (possibly upwards of 24 h). Such conditions may negatively influence bacterial viability, infectivity of plant cells, and target protein production. Here, we explored the role of timing in bacterial culture preparation for agroinfiltration using mass spectrometry-based proteomics to define changes in cellular processes. We observed distinct profiles associated with bacterial treatment conditions and exposure timing, including significant changes in proteins involved in pathogenesis, motility, and nutrient acquisition systems as the bacteria adapt to the new environment. These data suggest a progression towards increased cellular remodelling over time. In addition, we described changes in growth- and environment-specific processes over time, underscoring the interconnectivity of pathogenesis and chemotaxis-associated proteins with transport and metabolism. Overall, our results have important implications for the production of transiently expressed target protein products, as prolonged exposure to agroinfiltration medium suggests remodelling of the bacterial proteins towards enhanced infection of plant cells.

Fun(gi)omics: Advanced and Diverse Technologies to Explore Emerging Fungal Pathogens and Define Mechanisms of Antifungal Resistance.

The landscape of infectious fungal agents includes previously unidentified or rare pathogens with the potential to cause unprecedented casualties in biodiversity, food security, and human health. The influences of human activity, including the crisis of climate change, along with globalized transport, are underlying factors shaping fungal adaptation to increased temperature and expanded geographical regions. Furthermore, the emergence of novel antifungal-resistant strains linked to excessive use of antifungals (in the clinic) and fungicides (in the field) offers an additional challenge to protect major crop staples and control dangerous fungal outbreaks. Hence, the alarming frequency of fungal infections in medical and agricultural settings requires effective research to understand the virulent nature of fungal pathogens and improve the outcome of infection in susceptible hosts. Mycology-driven research has benefited from a contemporary and unified approach of omics technology, deepening the biological, biochemical, and biophysical understanding of these emerging fungal pathogens. Here, we review the current state-of-the-art multi-omics technologies, explore the power of data integration strategies, and highlight discovery-based revelations of globally important and taxonomically diverse fungal pathogens. This information provides new insight for emerging pathogens through an in-depth understanding of well-characterized fungi and provides alternative therapeutic strategies defined through novel findings of virulence, adaptation, and resistance.

Bioavailability of pollutants in bacterial communities of Rodrigo de Freitas Lagoon, Rio de Janeiro, Brazil.

Processes involving heavy metals and other contaminants continue to present unsolved environmental questions. To advance the understanding of geochemical processes that involve the bioavailability of contaminants, cores where collected in the Rodrigo de Freitas lagoon, and analyzed for bacterial activity and metal concentrations. Results would suggest an extremely reducing environment where organic substances seem to be the predominant agents responsible for this geochemical process. Analytical data showed sulphate reduction to be the main agent driving this process, since this kind of bacteria was found to be active in all of the samples analyzed. Esterase enzyme production did not signal the influence of heavy metals and hydrocarbon concentrations and heavy metals were found to be unavailable for biota. However, correlation between results for bacterial biomass and the potentially mobile percentage of the total Ni concentrations would suggest a negative impact.

Bioavailability of pollutants in bacterial communities of Rodrigo de Freitas Lagoon, Rio de Janeiro, Brazil

Processes involving heavy metals and other contaminants continue to present unsolved environmental questions. To advance the understanding of geochemical processes that involve the bioavailability of contaminants, cores where collected in the Rodrigo de Freitas lagoon, and analyzed for bacterial activity and metal concentrations. Results would suggest an extremely reducing environment where organic substances seem to be the predominant agents responsible for this geochemical process. Analytical data showed sulphate reduction to be the main agent driving this process, since this kind of bacteria was found to be active in all of the samples analyzed. Esterase enzyme production did not signal the influence of heavy metals and hydrocarbon concentrations and heavy metals were found to be unavailable for biota. However, correlation between results for bacterial biomass and the potentially mobile percentage of the total Ni concentrations would suggest a negative impact.

Eggs as energy: revisiting the scaling of egg size and energetic content among echinoderms.

Marine organisms exhibit substantial life-history diversity, of which egg size is one fundamental parameter. The size of an egg is generally assumed to reflect the amount of energy it contains and the amount of per-offspring maternal investment. Egg size and energy are thought to scale isometrically. We investigated this relationship by updating published datasets for echinoderms, increasing the number of species over those in previous studies by 62%. When we plotted egg energy versus egg size in the updated dataset we found that planktotrophs have a scaling factor significantly lower than 1, demonstrating an overall trend toward lower energy density in larger planktotrophic eggs. By looking within three genera, Echinometra, Strongylocentrotus, and Arbacia, we also found that the scaling exponent differed among taxa, and that in Echinometra, energy density was significantly lower in species with larger eggs. Theoretical models generally assume a strong tradeoff between egg size and fecundity that limits energetic investment and constrains life-history evolution. These data suggest that the evolution of egg size and egg energy content can be decoupled, possibly facilitating response to selective factors such as sperm limitation which could act on volume alone.

Metal and oxalate contamination in a suburban watershed in the greater Toronto area: the benefits of combining acid leach and selective extraction procedures.

Environmentally available concentrations of Fe, Mn, Zn, Cu, Pb, Cr and Ni in soils and sediments from a small suburban catchment, obtained using an acid leach procedure, are compared to the Ontario Ministry of the Environment lowest effect level (LEL) and severe effect levels (SEL) and to Provincial sediment quality guidelines (PSQG's). These data are then compared to the bioavailability, potential bioavailability and non-bioavailability of the same metals, plus oxalate concentration, identified using a selective extraction procedure. This combination of techniques enhanced analytical interpretation with respect to metal mobility and potential metal contamination. Selective extraction highlighted the presence of oxalate as a potential contaminant, especially in poorly drained valley floor deposits (33,633 mg kg(-1) and 26,284 mg kg(-1)) and lakeshore sediments (27,095 mg kg(-1) and 13,729 mg kg(-1)). These levels are considerably in excess of those previously documented in a similar study from Rio de Janeiro, where contamination of urban sediment by sewage is a recognised environmental problem, and could possibly be used both as an indicator of similar contamination and the identification of those areas that warrant further investigation.

Lamellar corneal dissection for visualization of the anterior chamber before triple procedure.

PURPOSE: Open-sky cataract extraction during triple procedure can be associated with higher risk of complications owing to the missing counterbalance by the cornea. Herein, we present a fast and easy technique for visualization of the anterior chamber and the lens in eyes with opaque corneas planed for triple procedure. MATERIALS AND METHODS: Four patients with corneal oedema due to Fuchs' endothelial dystrophy and cataract underwent triple procedure. As the anterior chamber view was limited, the central 7.0 mm of the cornea was marked. Then, 60-80% of the corneal thickness was removed by lamellar dissection and filled with hydroxypropylmethylcellulose. Continuous curvilinear capsulorhexis (CCC) and phacoemulsification with intraocular lens implantation through a corneoscleral tunnel were then performed and at the end the remaining corneal tissue was excised and the donor tissue fixed with a single running continuous suture. RESULTS: Lamellar corneal dissection enhances the anterior chamber view and CCC can be performed under stable anterior chamber condition. Phacoemulsification via sclerocorneal tunnel could be easily performed under good anterior chamber view in all cases. The operation time was 60-75 min in all cases. CONCLUSION: Lamellar corneal dissection in opaque corneas before cataract extraction is a useful technique for enhancing anterior chamber view in cases of triple procedure.

Results 28 months following transpupillary thermotherapy for classic and occult choroidal neovascularization in patients with age-related macular degeneration.

BACKGROUND AND OBJECTIVE: To report the outcome of patients 28 months following treatment with transpupillary thermotherapy (TTT) for classic and occult choroidal neovascularization (CNV) secondary to age-related macular degeneration. PATIENTS AND METHODS: A nonrandomized pilot trial of 36 eyes of 33 patients was performed. Eyes with angiographically defined CNV, 11 predominantly classical and 25 predominantly occult, were treated with large spot diode laser (810 nm) TTT for 1 minute, the end point being no or minimal visible change. Outcome was assessed with best-corrected LogMAR visual acuity, clinical examination, and fluorescein angiography. RESULTS: Patients were observed for a mean of 28.7 months (range, 18 to 40 months). The mean change in LogMAR visual acuity for predominantly classic membranes was -1.91 (standard deviation [SD] = 4.3) and 5 of 11 (45.5%) eyes had a loss of 3 or more LogMAR lines. Predominantly classic membranes were closed in 9 of 11 eyes and stabilized in 2 of 11 eyes. The mean change in LogMAR visual acuity for predominantly occult membranes was -1.48 (SD = 6.3) and 10 of 25 (40%) patients had a loss of 3 lines or more. Predominantly occult CNV was stabilized in 25 of 25 cases, and recurrence developed in 2 of 25 cases; one of the latter developed classic CNV. CONCLUSIONS: The medium-term results for patients treated with TTT for both occult and classic CNV show good stability, with little visual loss and few recurrences. These data confirm the original findings of this study.

Single intraoperative application of 5-Fluorouracil versus placebo in low-risk trabeculectomy surgery: a randomized trial.

PURPOSE: Trabeculectomy is the procedure of choice for the surgical control of glaucoma, and is often augmented by intraoperative 5-fluorouracil application to reduce scarring. This study aims to assess the validity of this technique with a randomized placebo-controlled trial. METHODS: Patients without risk factors for bleb failure other than previous drop use who were undergoing trabeculectomy surgery as their first ocular procedure were randomized to receive fluorouracil or placebo. The main outcome measures were intraocular pressure and bleb morphology. RESULTS: Results from 40 eyes of 36 patients are presented; 23 eyes were treated with fluorouracil, and 17 eyes were treated with placebo. Mean intraocular pressure at 1 year and at the final follow-up examination was significantly lower than preoperatively, but was not different between fluorouracil and placebo groups. Kaplan-Meier survival analysis also failed to show any difference, though there was a trend toward better survival of an intraocular pressure of 16 mm Hg or lower. Bleb morphology was examined postoperatively in 24 patients, with no differences found between fluorouracil and placebo groups. CONCLUSIONS: No significant difference was found in trabeculectomy outcome between fluorouracil-treated and placebo-treated eyes. The study was too small to state definitively that no such difference exists, but suggests that any treatment effect is likely to be small.

Transpupillary thermotherapy (TTT) for the treatment of choroidal neovascularisation.

AIM: To assess the effectiveness of transpupillary thermotherapy (TTT) for the treatment of classic and occult choroidal neovascularisation (CNV). METHOD: In a retrospective, case selected, open label trial 44 eyes of 42 patients with CNV secondary to age related macular degeneration (ARMD) were studied. 44 eyes with angiographically defined CNV were treated with diode laser (810 nm) TTT. Laser beam sizes ranged between 0.8 and 3.0 mm and power settings between 250-750 mW. Treatment was given in one area for 1 minute, the end point being no visible change, or a slight greying of the retina. Outcome was assessed with Snellen visual acuity and clinical examination; in 24/44 patients angiographic follow up was available. RESULTS: 12 predominantly classic CNV and 32 predominantly occult membranes were followed up for a mean of 6.1 months (range 2-19). Mean change in vision for classic membranes was -0.75 (SD 1.75) Snellen lines and occult membranes was -0.66 Snellen lines (2.1) (p>0.05). Predominantly classic membranes were closed in 75% (95% CI: 62.5-87.5) of eyes, remained persistent in 25% (95% CI: 12.5-37.5); no recurrences occurred. Predominantly occult membranes were closed in 78% (95% CI: 70.1-85.3) of eyes, remained persistent in 12.5% (95% CI: 6.6-18.5), and were recurrent in 5.1% (95% CI: 4.2-14.3). CONCLUSIONS: Transpupillary thermotherapy is a potential treatment for CNV. It is able to close choroidal neovascularisation while maintaining visual function in patients with classic and occult disease. Further trials of TTT are needed to compare this intervention with the natural history and other treatment modalities.

Use of complementary therapies by patients attending musculoskeletal clinics.

Patients with musculoskeletal disorders commonly seek treatment outside orthodox medicine (complementary therapy). In patients attending hospital clinics we investigated the prevalence of such behaviour and the reasons for it. Patients attending rheumatology and orthopaedic clinics who agreed to participate were interviewed on the same day by means of a structured questionnaire in three sections: the first section about demographic characteristics; the second about the nature and duration of the complaint, the length of any treatment and whether the patient was satisfied with conventional treatment; and the third about the use of complementary medicine, the types of therapy that had been considered and the reasoning behind these decisions. The data were examined by univariate and bivariate analysis as well as logistic regression multivariate analysis. 166 patients were interviewed (99% response rate) and the predominant diagnosis was rheumatoid arthritis (22.3%). 109 patients (63%) were satisfied with conventional medical treatment; 63 (38%) had considered the use of complementary therapies, and 47 (28%) had tried such a therapy. 26 of the 47 who had used complementary therapy said they had gained some benefit. Acupuncture, homoeopathy, osteopathy and herbal medicine were the most popular types of treatment to be considered. Patients of female gender (P = 0.009) and patients who had expressed dissatisfaction with current therapies (P = 0.01) were most likely to have considered complementary medicine. These results indicate substantial use of complementary therapy in patients attending musculoskeletal disease clinics. The reasons for dissatisfaction with orthodox treatment deserve further investigation, as does the effectiveness of complementary treatments, which must be demonstrated before they are integrated with orthodox medical practice.

Nutrient enrichment of white spruce seedlings during nursery culture and initial plantation establishment.

Growth and nutrient dynamics of bare-root white spruce (Picea glauca (Moench) Voss) seedlings were monitored for three years in a nursery and for one season after planting to assess effects of three nursery fertilization regimes. Nitrogen (totaling 0, 650, and 1300 kg N ha(-1) for 3 years) was applied conventionally in equal amounts during the growing season. The high-N treatment, representing a nutrient loading regime, was also applied seasonally at exponentially increasing rates as a fourth treatment. By the end of the 3-year nursery rotation, the N treatments had stimulated growth by 104-180% and nutrient uptake by as much as 381, 224 and 145% for N, P and K, respectively, inducing large accumulations of N and P in both conventionally and exponentially loaded seedlings. Compared with exponentially loaded seedlings, the concentrations of nutrients were less in conventionally loaded seedlings although their biomass was larger (31%). High nutrient reserves in the seedlings at the end of the nursery rotation resulted in increased biomass production (40-190%) after planting, which was related to the ability of the seedlings to retranslocate internal reserves for new growth, despite little or no net uptake of nutrients during the first season after planting. Compared with conventionally loaded seedlings, retranslocation was greater in exponentially loaded seedlings, which had accumulated larger and more readily available nutrient reserves during the nursery phase. The shortness of this study limits its usefulness for predicting the persistence of the loading response after planting, but we postulate that the high nutrient status of loaded seedlings at the end of the nursery rotation will contribute to future growth through increased nutrient storage and retranslocation, thus prolonging the loading response.

S(N)2 Mechanism for Alcoholysis, Aminolysis, and Hydrolysis of Acetyl Chloride.

First-order solvolysis rate constants are reported for solvolyses of acetyl chloride in methanol and MeOD, and in binary aqueous mixtures with acetone, acetonitrile, ethanol, methanol, and trifluoroethanol at 0 degrees C. Product selectivities (S = [MeCOOR]/[MeCOOH] x [water]/[alcohol]) are reported for solvolyses in ethanol/ and methanol/water at 0 degrees C. Solvolyses of acetyl chloride show a high sensitivity to changes in solvent ionizing power, consistent with C-Cl bond cleavage. As the solvent is varied from pure ethanol (or methanol) to water, S values and rate-rate profiles show no evidence for the change in reaction channel observed for solvolyses of benzoyl and trimethylacetyl chlorides. However, using rate ratios in 40% ethanol/water and 97% trifluoroethanol/water (solvents of similar ionizing power but different nucleophilicities) to compare sensitivities to nucleophilic attack, solvolyses of acetyl chloride are over 20-fold more sensitive to nucleophilic attack than benzoyl chloride. The solvent isotope effect of 1.29 (MeOH/MeOD) for acetyl chloride is similar to that for p-methoxybenzoyl chloride (1.22) and is lower than for benzoyl chloride (1.55). Second-order rate constants for aminolyses of acetyl chloride with m-nitroaniline in methanol at 0 degrees C show that acetyl chloride behaves similarly to p-methoxybenzoyl chloride, whereas benzoyl chloride is 40-fold more sensitive to the added amine. The results indicate mechanistic differences between solvolyses of acetyl and benzoyl chlorides, and an S(N)2 mechanism is proposed for solvolyses and aminolyses by m-nitroaniline of acetyl chloride (i.e. these reactions are probably not carbonyl additions, but a strong sensitivity to nucleophilic attack accounts for their high rates).

Effect of flurbiprofen on acetylcholine-induced pupillary contraction.

Preoperative treatment with topical nonsteroidal anti-inflammatory agents such as flurbiprofen (Ocufen) is used to maintain pupil dilation during cataract surgery. Flurbiprofen maintains pupil dilatation by inhibiting release of prostaglandins and other modulators of surgical miosis. Some reports suggest that these agents may reduce the miotic effect of intra-operative acetylcholine (Miochol). It is hard to explain this effect unless there is a pharmacological interaction between the two drugs. This study aimed to investigate the possibility of a direct interface by flurbiprofen with the action of acetylcholine on the pupil sphincter. Iris tissue of seven cadaver eyes were isolated and maintained in an organ bath containing Kreb's solution. Pupil contraction force induced by acetylcholine was measured in the presence and absence of flurbiprofen. Acetylcholine produced a mean pupillary contraction force of 52.4 x 10(-3) N. When it was added to the organ bath in the presence of flurbiprofen the mean contraction force was 50.5 x 10(-3) N (paired t-test P = 0.68). This study does not find any evidence to suggest that flurbiprofen reduces the pupil sphincter contraction that is induced by acetylcholine. It is likely that the apparent reduction in miosis is due to factors other than the mechanism of action of acetylcholine on the sphincter pupillae.

Frozen histologic section as a guide to sepsis in revision joint arthroplasty.

The accurate prediction of occult sepsis is very important in evaluating the patient who has a painful prosthetic joint. Unfortunately, there is no single test that is able to consistently differentiate mechanical from septic loosening. Authors have suggested that the presence of acute inflammation on frozen histologic sections correlated well with positive intraoperative cultures. The authors' experience with frozen sections was examined as a guide to the intraoperative determination of occult sepsis in revision joint surgery. One hundred seven consecutive total joint revisions were performed from 1986 to 1989. All patients had a complete histologic analysis of tissue obtained from multiple surgical sites. The relationship between the frozen section, final culture, and complete histologic analysis was evaluated in a comparative fashion. Using a positive culture as the criterion for the presence of occult infection, a statistical analysis comparing frozen sections and the complete histologic analyses were performed. The sensitivity of a frozen section as a diagnostic test to detect occult periprosthetic infection when present was 18.2%. The specificity of a frozen section to correctly identify the absence of infection was 89.5%. The frozen section is a fairly specific, but not a sensitive, screening test for the detection of occult sepsis. Over reliance on this technique in a revision setting should be avoided.