Sensitivity of Zymoseptoria tritici Isolates from Tunisia to Pyraclostrobin,Fluxapyroxad, Epoxiconazole,Metconazole, Prochloraz and Tebuconazole

Sensitivity of 159 isolates of Zymoseptoria tritici collected from durum wheat fields in Tunisia in 2012 was analysed towards pyraclostrobin, fluxapyroxad, epoxiconazole, metconazole, prochloraz and tebuconazole using microtiter tests. All isolates were found to be highly sensitive to pyraclostrobin with EC₅₀ <0.01 mg/l with the exception of three isolates from the same field with higher EC₅₀ values (>0.5 mg/l). These three isolates carried a mutation in the cytochrome b gene encoding the G143A substitution. This is the first report of quinone outside inhibitors (QoI) resistance in Z. tritici in Tunisia. Sensitivity towards r fluxapyroxad was in a narrow range with EC₅₀ values ranging between 0.013 and 0.125 mg/l, which can serve as baseline sensitivity data for the future. Demethylation inhibitors sensitivity varied across a broad range with the data indicating a slight shift in sensitivity when compared to a previous study on the 2010 population. No highly sensitive strains were isolated from samples from fields, which had received three or four DMI applications.     

Detection and molecular characterization of carbendazim-resistant Colletotrichum truncatum Isolates causing anthracnose of soybean in Thailand

A loss of fungicide efficacy, particularly for carbendazim, was noted in soybean fields in Thailand and was considered to be due to the development of Colletotrichum truncatum resistance. The carbendazim sensitivity of C. truncatum populations isolated from various soybean fields in Thailand was thus evaluated with in vitro sensitivity assays and molecular characterization of mutations in the sequences of the ß₂-tubulin (TUB2) gene that confer carbendazim resistance in the pathogen. Among 52 isolates, 46 isolates were classified as highly resistant (HR) to carbendazim (EC₅₀ > 1,000 µg/ml). All HR isolates grew on PDA amended with carbendazim at 1,000 µg/ml. Six isolates were classified as carbendazim sensitive (S) (EC₅₀ < 1 µg/ml). Mycelial growth on PDA amended with 1 µg/ml carbendazim was inhibited by over 50% compared with growth on PDA alone. When a partial TUB2 gene from the isolates was amplified and analysed using predicted amino acid sequences, an alteration from glutamic acid to alanine at codon 198 (E198A) was found in 45 HR isolates for which the EC₅₀ was higher than 2000 µg/ml. This mutation resulted from a nucleotide substitution from adenine to cytosine (GA G → GC G). The other HR isolate, CtPhS_1, with EC₅₀ of 1,127 µg/ml, had an alteration at codon 200 (F200Y) (TT C → TA C).     

A novel self-cleavable tag Zbasic–∆I-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli

Soluble expression of recombinant therapeutic proteins in Escherichia coli (E. coli) has been a challenging task in biopharmaceutical development. In this study, a novel self-cleavable tag Zbasic–intein has been constructed for the soluble expression and purification of a recombinant cytokine, human interleukin-15 (IL-15). We screened several solubilizing tags fused with the self-cleavable Mycobacterium tuberculosis recA mini-intein ∆I-CM and demonstrated that Zbasic tag can significantly improve the solubility of the product with correspondent to the intein activity. The fusion protein “Zbasic–∆I-CM–IL-15” was expressed with high solubility and easily enriched by the cost-effective cation-exchange chromatography. The self-cleavage of the fusion tag Zbasic–∆I-CM was then induced by a pH shift, with an activation energy of 7.48 kcal/mol. The mature IL-15 with natural N-terminus was released and further purified by hydrophobic interaction and anion-exchange chromatography. High-resolution reverse-phase high-performance liquid chromatography and mass spectrometry analysis confirmed that the product was of high purity and correct mass. With a CTLL-2 cell proliferation-based assay, the EC₅₀ was evaluated to be of about 0.126 ng/mL, similar to the product in clinical trials. By avoiding the time-consuming denaturing-refolding steps in previously reported processes, the current method is efficient and cost-effective. The novel tag Zbasic–∆I-CM can be potentially applied to large-scale manufacturing of recombinant human cytokines as well as other mammalian-sourced proteins in E. coli.

     

Semisynthesis and Antifungal Activity of Novel Oxime Ester Derivatives of Carabrone Modified at C(4) against Botrytis cinerea

To continuously improve the potential utility of the natural lead compound of carabrone in agrochemistry, carabrone oxime and 36 novel oxime ester derivatives of carabrone modified at C(4) were synthesized, and evaluated for their antifungal activities against Botrytis cinerea in vitro and in vivo. Of these 36 oxime ester derivatives, some compounds exhibited antifungal activities in vitro or in vivo. It was found that compounds with a pyridinyl residue can either efficiently inhibit spore germination or efficiently inhibit hyphal growth of B. cinerea, and compound 9 exhibited the highest activity in vitro and in vivo with IC₅₀ and EC₅₀ values of 1.17 and 12.9 μg/ml, respectively. Further, the structure? activity relationships are also discussed.     

High central venous oxygen saturation is associated with mitochondrial dysfunction in septic shock: A prospective observational study

To test the hypothesis that an impaired mitochondrial function is associated with altered central venous oxygen saturation (ScvO₂), venous-to-arterial carbon dioxide tension difference (delta PCO₂) or serum lactate in sepsis patients. This prospective cohort study was conducted in a single tertiary emergency department between April 2017 and March 2019. Patients with suspected sepsis were included in the study. Serum lactate was obtained in sepsis, ScvO₂ and delta PCO₂ were evaluated in septic shock patients. Mitochondrial function was determined from the peripheral blood mononuclear cells. Forty-six patients with suspected sepsis were included. Of these, twenty patients were septic shock. Mitochondrial oxidative stress levels were increased in the high ScvO₂ group (ScvO₂ > 80%, n = 6), compared with the normal (70%-80%, n = 9) and low ScvO₂ (<70%, n = 5) groups. A strong linear relationship was observed between the mitochondrial oxidative stress and ScvO₂ (r = .75; P = .01). However, mitochondrial respiration was increased in the low ScvO₂ group. In addition, mitochondrial complex II protein levels were significantly decreased in the high ScvO₂ group (P < .05). Additionally, there was no correlation between serum lactate, delta PCO₂, and mitochondria oxidative stress or mitochondria function. ScvO₂ can be potentially useful for developing new therapeutics to reduce mitochondrial dysfunction in septic shock patient.     

Anti‐HSV‐1 and HSV‐2 Flavonoids and a New Kaempferol Triglycoside from the Medicinal Plant Kalanchoe daigremontiana

Kalanchoe daigremontiana (Crassulaceae) is a medicinal plant native to Madagascar. The aim of this study was to investigate the flavonoid content of an aqueous leaf extract from K. daigremontiana (Kd), and assess its antiherpetic potential. The major flavonoid, kaempferol 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside ( 1 ), was isolated from the AcOEt fraction (Kd‐AC). The BuOH‐soluble fraction afforded quercetin 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside ( 2 ) and the new kaempferol 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside‐7‐O‐β‐d ‐glucopyranoside ( 3 ), named daigremontrioside. The crude extract, Kd‐AC fraction, flavonoids 1 and 2 were evaluated using acyclovir‐sensitive strains of HSV‐1 and HSV‐2. Kd‐AC was highly active against HSV‐1 (EC₅₀ = 0.97 μg/ml, SI > 206.1) and HSV‐2 (EC₅₀ = 0.72 μg/ml, SI > 277.7). Flavonoids 1 and 2 showed anti‐HSV‐1 (EC₅₀ = 7.4 μg/ml; SI > 27 and EC₅₀ = 5.8 μg/ml; SI > 8.6, respectively) and anti‐HSV‐2 (EC₅₀ = 9.0 μg/ml; SI > 22.2 and EC₅₀ = 36.2 μg/ml; SI > 5.5, respectively) activities, suggesting the contribution of additional substances to the antiviral activity.     

Haemolytic activity within the species Fibrocapsa japonica (Raphidophyceae)

Strong haemolytic activity was observed for extracts of 15 Fibrocapsa japonica strains collected from different global regions. The EC₅₀ values ranged between 0.4 × 10⁴ and 1.9 × 10⁴ F. japonica cells ml⁻¹. The relationship between the haemolytic activity observed in the cell extracts and the amount and nature of polyunsaturated fatty acids (PUFAs) was investigated. Between 7% and 89% of the haemolytic activity could be attributed to the amount and toxicity of the PUFAs C18:4n3 (OTA), C20:5n3 (EPA), and C20:4n6 (AA). However, the higher the haemolytic activity of a strain the more it appeared to be caused by haemolytic compounds other than the PUFA's. The EC₅₀ of the extracts with the highest haemolytic activity strongly depended on light intensity and was in line with observations made on Chattonella marina.In the cell extracts of the strains studied brevetoxins were always below detection limits. For strains where 20–35% of the haemolytic activity was not caused by PUFAs, the concentration of these compounds was close to LC₅₀ values reported for fish. Overall the haemolytic activity of the F. japonica strains was in the range of known harmful microalgae. Therefore, PUFAs in combination with potent light dependent haemolytic compounds could be a major cause of ichthyotoxicity observed during F. japonica blooms.     

The Protective Role of <Emphasis Type="SmallCaps">d</Emphasis>-Glucose Against 1-Methyl-4-Phenylpyridinium Ion (MPP<Superscript>+</Superscript>): Induced Mitochondrial Dysfunction in C6 Astroglial Cells

Impaired mitochondrial function in glial and neuronal cells in the substantia nigra is one of the most likely causes of Parkinson’s disease. In this study, we investigated the protective role of glucose on early key events associated with MPP⁺-induced changes in rat C6 astroglial cells. Studies were carried out to examine alterations in mitochondrial respiratory status, membrane potential, glutathione levels, and cell cycle phase inhibition at 48 h in 2 and 10 mM glucose in media. The results obtained suggest that MPP⁺ caused significant cell death in 2 mM glucose with LC₅₀ 0.14 ± 0.005 mM, while 10 mM glucose showed highly significant protection against MPP⁺ toxicity with LC₅₀ 0.835 ± 0.03 mM. This protection was not observed with cocaine, demonstrating its compound specificity. MPP⁺ in 2 mM glucose decreased significantly mitochondrial respiration, membrane potential and glutathione levels in a dose dependent manner, while 10 mM glucose significantly restored them. MPP⁺ in 2 mM glucose arrested the cells at G0/G1 and G2/M phases, demonstrating its dual inhibitory effects. However, in 10 mM glucose, MPP⁺ caused G0/G1 arrest only. In summary, the results suggest that loss of cell viability in 2 mM glucose group with MPP⁺ treatments was due to mitochondrial dysfunction caused by multilevel mechanism, involving significant decrease in mitochondrial respiration, membrane potential, glutathione levels, and dual arrest of cell phases, while 10 mM glucose rescued astroglial cells from MPP⁺ toxicity by significant maintenance of these factors.     

Establishment of an EC50 database of pesticides using a Vibrio fischeri bioluminescence method

An EC₅₀ database was established to assess the acute toxicity of 16 PESTANAL pesticide standards and of seven pesticide commercial formulations using a Vibrio fischeri bioluminescence method. Half maximal effective concentration ( EC₅₀) is defined as the concentration of pollutant (in this case, pesticide) destroying 50% of the bacteria population and causing 50% bioluminescence inhibition, after a specified exposure time. Linear curves of bioluminescence inhibition versus pesticide concentration and EC₅₀ values were obtained for exposure times (t) of 5 or 15 min for these pesticides. The EC₅₀ values ranged from 6.90 × 10^?4 to 0.83 mg/ml (t = 5 min), and from 9.00 × 10^?4 to 0.37 mg/ml (t = 15 min) for pesticide standards, plus from 0.0077 to 0.74 mg/ml (t = 5 min), and from 0.0076 and 0.57 mg/ml (t = 15 min) for pesticide commercial formulations. The EC₅₀ database allowed classification of the pesticides under study into three categories according to their toxicity: very toxic, toxic and moderately toxic. These results demonstrated that the establishment of an EC₅₀ database and of linear curves of bioluminescence inhibition versus the pesticide concentration resulted in very important and irreplaceable tools to estimate the global and individual toxicity of pesticides present in environmental samples.     

Population Structure and Development of Resistance to Hymexazol Among Fusarium solani Populations from Date Palm,Citrus and Cucumber

A study was conducted to investigate genetic diversity and sensitivity to hymexazol among 80 isolates of Fusarium solani complex obtained from date palm (30), citrus (31) and cucumber (19). Characterization based on sequences of the EF1α and ITS rRNA showed that isolates belong to F. solani complex MLST type 3 + 4. AFLP analysis produced 980 polymorphic loci, 80 AFLP genotypes and moderate levels of genetic diversity (H = 0.2494). Clustering of the isolates was not related to the host or the geographical origin of the isolates. Analysis of molecular variance (amova ) indicated the existence of a low level of genetic differentiation among populations obtained from different hosts (F_st = 0.0162) and regions (F_st = 0.0066). This may provide evidence for frequent movement of inoculum among hosts and regions in Oman, which could be attributed to cultural practices employed by farmers. Isolates of F. solani displayed variation in sensitivity to hymexazol, with EC₅₀ values ranging from 2 to 5745 μg/ml (mean = 878 μg/ml); 19% of the isolates have an EC₅₀ value of more than 1000 μg/ml. Findings are discussed in terms of the factors that affect diversity in F. solani isolates. The study reports for the first time the development of resistance to hymexazol among F. solani isolates from date palm, citrus and cucumber.     

Entire nucleotide sequences of Gossypium raimondii and G. arboreum mitochondrial genomes revealed A‐genome species as cytoplasmic donor of the allotetraploid species

• Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups, designated A–G and K, and an allotetraploid genomic group, AD. Gossypium raimondii (D₅) and G. arboreum (A₂) are the putative contributors to the progenitor of G. hirsutum (AD₁), the economically important fibre‐producing cotton species.
• Mitochondrial DNA from week‐old etiolated seedlings was extracted from isolated organelles using discontinuous sucrose density gradient method. Mitochondrial genomes were sequenced, assembled, annotated and analysed in orderly.
• Gossypium raimondii (D₅) and G. arboreum (A₂) mitochondrial genomes were provided in this study. The mitochondrial genomes of two diploid species harboured circular genome of 643,914 bp (D₅) and 687,482 bp (A₂), respectively. They differ in size and number of repeat sequences, both contain illuminating triplicate sequences with 7317 and 10,246 bp, respectively, demonstrating dynamic difference and rearranged genome organisations. Comparing the D₅ and A₂ mitogenomes with mitogenomes of tetraploid Gossypium species (AD₁, G. hirsutum; AD₂, G. barbadense), a shared 11 kbp fragment loss was detected in allotetraploid species, three regions shared by G. arboreum (A₂), G. hirsutum (AD₁) and G. barbadense (AD₂), while eight regions were specific to G. raimondii (D₅). The presence/absence variations and gene‐based phylogeny supported that A‐genome is a cytoplasmic donor to the progenitor of allotetraploid species G. hirsutum and G. barbadense.
• The results present structure variations and phylogeny of Gossypium mitochondrial genome evolution.

     

A Comparison of Different Estimation Methods for Fungicide EC50 and EC95 Values

EC₅₀ and EC₉₅ (the effective concentrations to cause inhibitions by 50 and 95%, respectively) are commonly used to express fungicide potency. Different methods are currently employed to calculate EC₅₀ and EC₉₅ values. In this study, EC₅₀ and EC₉₅ values for fungicide epoxiconazole against 34 isolates of Sclerotinia sclerotiorum were calculated with seven different methods. Results showed that for both EC₅₀ and EC₉₅ calculations, there was no significant difference among three statistical programs IBM spss ^®, GraphPad Prism^® and dps ^® (P ≥ 0.066). Methods linear log (linear regression of mycelial growth inhibition vs. logarithmic concentration) and interpolation log (linear interpolation from inhibition and logarithmic concentration data) were not significantly different (P ≥ 0.058) from IBM spss in EC₅₀ calculations. These results indicate that among the seven methods, the three statistical programs IBM spss , GraphPad Prism, dps and linear log method are appropriate for EC₅₀ calculations. But for EC₉₅ calculations, only the three statistical programs are recommended, and GraphPad Prism is likely to give a little higher values than spss and dps .     

Synthesis,In Vitro Anti-HIV and Anti-hepatitis B Activities and Pharmacokinetic Properties of Amphiphilic Heterodinucleoside Phosphates Containing ddC and AZT

Abstract

Amphiphilic heterodinucleoside phosphates containing AZT and ddC as antiviral monomer were synthesized according to the hydrogenphosphonate method and evaluated in vitro against HIV. dT-N⁴-pamddC was the most active (IC₅₀ = 40 μM, EC₅₀ = 80 nM) and least toxic (TI = 524) dimer and it exhibited also strong antiviral effects against eight AZT-resistant HIV strains. The ddC-containing heterodimers additionally inhibited HBV replication by 50–80% at 50 μM in Hep G2 2.2.15 cells.     

Mitochondrial respiration and succinate dehydrogenase are suppressed early during entrance into a hibernation bout,but membrane remodeling is only transient

We examined respiration and lipid composition of liver mitochondria purified from a hibernator (Ictidomys tridecemlineatus) in different stages of a torpor bout. Between interbout euthermia (body temperature, T _b, 37°C) and early entrance (T _b 30°C), state 3 and state 4 respirations, fueled by 6 mM succinate, fell by over 50%. Mitochondrial respiration did not decline any further in the late entrance and torpor stages (T _b 15 and 5°C, respectively). Succinate dehydrogenase (SDH) activity declined in a similar pattern as mitochondrial respiration, and there was a significant positive correlation between state 3 respiration and SDH activity. However, unlike during arousal from torpor, oxaloacetate was not a major factor in inhibition of SDH. Analysis of mitochondrial lipids showed little change in neutral lipids or phospholipid classes, except for a transient decrease in phosphatidylethanolamine content in early entrance. In the transition from interbout euthermia to early entrance, we found transient increases in some saturated phospholipid fatty acids (16:0, 18:0) and decreases in some unsaturates (18:2, 20:4). These changes resulted in transient increases in total saturates and the ratio of saturates to unsaturates, and transient decreases in total unsaturates, total polyunsaturates, total n-6, the ratio of monounsaturates to polyunsaturates, and unsaturation index. None of these changes persisted into late entrance or torpor, nor did they correlate with mitochondrial respiration. We conclude that mitochondrial metabolic suppression during entrance into a torpor bout occurs very early and is likely related to acute regulation of electron transport chain enzymes rather than changes in membrane phospholipid composition.     

The complete mitochondrial genome of the oriental fruit moth <Emphasis Type="Italic">Grapholita molesta</Emphasis> (Busck) (Lepidoptera: Tortricidae)

The oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae) currently is one of the economically most destructive pest species of stone and pome fruits worldwide. Here we sequenced the complete mitochondrial genome of this pest. This genome is 15,776 bp long, with an A + T content of 81.24%, containing 37 typical animal mitochondrial genes and an A + T-rich region. All gene are arranged as hypothesized ancestral gene order of insects except for trnM, which was shuffled from 3′ downstream of trnQ to 5′ upstream of trnI. cox1 gene uses unusual CGA start codon, as that in all other sequenced lepidopteran mitochondrial genome. The secondary structures for the two rRNA genes were predicted. All helices typically present in insect mitochondrial rRNA genes are generated. A microsatellite sequence was inserted into the region of H2347 in rrnL in G. molesta and two other sequenced tortricid mitochondrial genomes, indicating that the insertion event in this helix might occurred anciently in family Tortricidae. All of the 22 typical animal tRNA genes have a typical cloverleaf structure except for trnS2, in which the D-stem pairings in the DHU arm are absent. An intergenic sequence is present between trnQ and nad2 as well as in other sequenced lepidopteran mitochondrial genomes, which was presumed to be a remnant of trnM gene and its boundary sequences after the duplication of trnM to the upstream of trnI in Lepidoptera. The A + T-rich region is 836 bp, containing six repeat sequences of “TTATTATTATTATTAAATA(G)TTT.”     

Tea waste as a supplement for the cultivation of <Emphasis Type="Italic">Ganoderma</Emphasis> <Emphasis Type="Italic">lucidum</Emphasis>

Tea waste (TW) was investigated as a new supplement for substrate mixtures in Ganoderma lucidum cultivation. The effects of sawdust (S) based substrates supplemented with TW at the various levels (75S:25TW, 80S:20TW, 85S:15TW, and 90S:10TW) and Ganoderma lucidum strains on yield, biological efficiency (BE) and the chemical composition of fruiting bodies were determined in solid-state fermentation. Significant differences were found among substrates regarding yield and BE, while yield and BE of the strains were not different. The substrate formulations producing highest yield and BE were 80S:20TW (87.98 g/kg substrate and 34.90%) and 75S:25TW (82.30 g/kg substrate and 31%). Yield and BE of substrates containing TW were generally higher than that of the control (80sawdust:18wheat bran:1sucrose:1 CaCO₃). Nitrogen, potassium, iron, and manganese contents and C:N ratios of substrates were strongly correlated with yield. BE showed positive and significant correlations with potassium, iron and manganese. Moisture content, potassium, magnesium, calcium, iron, and zinc contents of the fruiting bodies were affected by both strain and substrate. It was concluded that TW can be used as a supplement for substrate preparation in G. lucidum cultivation.     

Annual sediment respiration in estuarine sandy intertidal flats in the Seto Inland Sea, Japan

To quantify organic matter mineralization at estuarine intertidal flats, we measured in situ sediment respiration rates using an infrared gas analyzer in estuarine sandy intertidal flats located in the northwestern Seto Inland Sea, Japan. In situ sediment respiration rates showed spatial and seasonal variations, and the mean of the rates is 38.8 mg CO₂-C m⁻² h⁻¹ in summer. In situ sediment respiration rates changed significantly with sediment temperature at the study sites (r ² = 0.70, p < 0.05), although we did not detect any significant correlations between the rates and sediment characteristics. We prepared a model for estimating the annual sediment respiration based on the in situ sediment respiration rates and their temperature coefficient (Q ₁₀ = 1.8). The annual sediment respiration was estimated to be 92 g CO₂-C m⁻² year⁻¹. The total amount of organic carbon mineralization for the entire estuarine intertidal flats through sediment respiration (43 t C year⁻¹) is equivalent to approximately 25% of the annual organic carbon load supplied from the river basin of the estuary.