Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

An extensive review on antifungal approaches in the treatment of mucormycosis

During the period of COVID-19, the occurrences of mucormycosis in immunocompromised patients have increased significantly. Mucormycosis (black fungus) is a rare and rapidly progressing fungal infection associated with high mortality and morbidity in India as well as globally. The causative agents for this infection are collectively called mucoromycetes which are the members of the order Mucorales. The diagnosis of the infection needs to be performed as soon as the occurrence of clinical symptoms which differs with types of Mucorales infection. Imaging techniques magnetic resonance imaging or computed tomography scan, culture testing, and microscopy are the approaches for the diagnosis. After the diagnosis of the infection is confirmed, rapid action is needed for the treatment in the form of antifungal therapy or surgery depending upon the severity of the infection. Delaying in treatment declines the chances of survival. In antifungal therapy, there are two approaches first-line therapy (monotherapy) and combination therapy. Amphotericin B ( 1 ) and isavuconazole ( 2 ) are the drugs of choice for first-line therapy in the treatment of mucormycosis. Salvage therapy with posaconazole ( 3 ) and deferasirox ( 4 ) is another approach for patients who are not responsible for any other therapy. Adjunctive therapy is also used in the treatment of mucormycosis along with first-line therapy, which involves hyperbaric oxygen and cytokine therapy. There are some drugs like VT-1161 ( 5 ) and APX001A ( 6 ), Colistin, SCH 42427, and PC1244 that are under clinical trials. Despite all these approaches, none can be 100% successful in giving results. Therefore, new medications with favorable or little side effects are required for the treatment of mucormycosis.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Capillary reversed-phase high-performance liquid chromatographic determination of acetyl-methylprednisolone in feline spinal cord

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) was used to determine acetylmethylprednisolone (A-MP) that had been administered to feline spinal cord tissue. The method used a 300 mm × 0.32 mm I.D. packed capillary octadecylsilyl (ODS) column and an isocratic mobile phase of 40 mM triethylamine formate (TEAF, pH 3.2)-acetonitrile (50:50, v:v). The chromatographic behavior of A-MP was evaluated with respect to peak-area and peak-height by varying the A-MP concentration (12–190 μM) with a fixed injection volume (1 μl), and by varying the injection volume (1–10 μl) with a fixed concentration (12 μM) of A-MP. The limit of detection (signal-to-noise ratio, 3:1) was 250 pg (600 fmol) of synthetic A-MP. Various amounts of A-MP directly spiked into feline spinal cord segments were solvent extracted, separated, and plotted against peak-area (r² = 1.00). Background tissue without A-MP gives minimal (<1%) interference at 243 nm. The method also detects exogenous A-MP that was administered into feline spinal cord via an intrathecal injection. Furthermore, the presence of A-MP was confirmed via its molecular ion and corresponding product ions that were obtained by fast-atom bombardment tandem mass spectrometry (FAB-MS-MS).     

Mass spectrometric quantification of the mu opioid receptor agonist Tyr-d-Arg-Phe-Lys-NH2 (DALDA) in high-performance liquid chromatography-purified ovine plasma

The mu opioid receptor agonist Tyr-d-Arg-Phe-Lys-Amide (d-Arg²-Lys⁴-Dermorphin_1-4amide=DALDA) was infused continuously for 2 h into sheep. The presence of DALDA in ovine plasma was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) in plasma samples that were obtained at different times during and following that infusion. A stable isotope-incorporated internal standard, deuterated DALDA (d₅-DALDA), was used for the MS quantification of DALDA via the protonated molecule ion, (M+H)⁺, of DALDA and of d₅-DALDA. Time-course data (μg DALDA ml⁻¹ plasma vs. time) were obtained. Tandem MS (MS–MS) provided the product-ion spectrum of the (M+H)⁺ ion of DALDA in one of the samples to confirm the amino acid sequence of DALDA.     

Host Plant Physiology and Mycorrhizal Functioning Shift across a Glacial through Future [CO2] Gradient

Rising atmospheric carbon dioxide concentration ([CO₂]) may modulate the functioning of mycorrhizal associations by altering the relative degree of nutrient and carbohydrate limitations in plants. To test this, we grew Taraxacum ceratophorum and Taraxacum officinale (native and exotic dandelions) with and without mycorrhizal fungi across a broad [CO₂] gradient (180–1,000 µL L⁻¹). Differential plant growth rates and vegetative plasticity were hypothesized to drive species-specific responses to [CO₂] and arbuscular mycorrhizal fungi. To evaluate [CO₂] effects on mycorrhizal functioning, we calculated response ratios based on the relative biomass of mycorrhizal (M_Bio) and nonmycorrhizal (NM_Bio) plants (R_Bio = [M_Bio − NM_Bio]/NM_Bio). We then assessed linkages between R_Bio and host physiology, fungal growth, and biomass allocation using structural equation modeling. For T. officinale, R_Bio increased with rising [CO₂], shifting from negative to positive values at 700 µL L⁻¹. [CO₂] and mycorrhizal effects on photosynthesis and leaf growth rates drove shifts in R_Bio in this species. For T. ceratophorum, R_Bio increased from 180 to 390 µL L⁻¹ and further increases in [CO₂] caused R_Bio to shift from positive to negative values. [CO₂] and fungal effects on plant growth and carbon sink strength were correlated with shifts in R_Bio in this species. Overall, we show that rising [CO₂] significantly altered the functioning of mycorrhizal associations. These symbioses became more beneficial with rising [CO₂], but nonlinear effects may limit plant responses to mycorrhizal fungi under future [CO₂]. The magnitude and mechanisms driving mycorrhizal-CO₂ responses reflected species-specific differences in growth rate and vegetative plasticity, indicating that these traits may provide a framework for predicting mycorrhizal responses to global change.Atmospheric carbon dioxide concentration ([CO₂]) has more than doubled over the past 20,000 years, rising from a minimum value of approximately 180 µL L⁻¹ during the Last Glacial Maximum (LGM; Augustin et al., 2004) to a current value of 401 µL L⁻¹. Due to ongoing fossil fuel emissions, [CO₂] is expected to reach 700 to 1,000 µL L⁻¹ by the end of this century (IPCC, 2013). Rising [CO₂] has greatly impacted plant physiology since the LGM (Sage and Coleman, 2001; Ainsworth and Rogers, 2007; Gerhart and Ward, 2010), likely altering interactions between plants and their microbial symbionts over geologic and contemporary time scales.Mycorrhizal associations are ancient plant-fungal symbioses (Remy et al., 1994) where host plants and their fungal partners exchange photosynthetically derived carbohydrates for soil nutrients (Smith and Read, 2008). These associations play a critical role in modern ecosystems via their effects on plant physiology, species coexistence, carbon and nutrient cycling, and net primary productivity (Hodge and Fitter, 2010; Clemmensen et al., 2015; Lin et al., 2015). Temporal changes in [CO₂] since the LGM have likely influenced the functioning of mycorrhizal associations along a continuum from mutualism to parasitism, hereafter referred to as the M-P continuum (Johnson et al., 1997), by altering plant carbohydrate production and nutrient demand. Previous mycorrhizal-CO₂ studies have focused mainly on the effects of modern versus future conditions (Alberton et al., 2005; Mohan et al., 2014), and little is known about mycorrhizal responses to low [CO₂] of the past (Treseder et al., 2003; Procter et al., 2014). Assessing mycorrhizal responses to a broad, temporal [CO₂] gradient is critical to establish a baseline for how these symbioses functioned prior to anthropogenic forcing, which will provide insight into potential constraints on mycorrhizal responses to future conditions (Ogle et al., 2015). In addition, rising [CO₂] is known to cause nonlinear shifts in plant physiology and growth (Gerhart and Ward, 2010); therefore, nonlinear shifts in mycorrhizal functioning also are likely to occur. Characterizing such responses is critical to accurately predict how these symbioses will impact plant physiology and growth in the future and requires experimentation that manipulates mycorrhizal associations at both low and elevated [CO₂]. Furthermore, to fully understand the physiological mechanisms driving plant responses to mycorrhizal fungi, linkages between host plant physiology and mycorrhizal functioning across the full M-P continuum need to be assessed. A broad [CO₂] gradient will likely generate both mutualistic and parasitic symbioses and provide insight into physiological traits that vary with mycorrhizal functioning.In this study, we examined arbuscular mycorrhizal (AM) associations in two closely related C₃ plant species, Taraxacum ceratophorum and Taraxacum officinale (native and exotic common dandelions; Asteraceae), across a glacial through future [CO₂] gradient. AM fungi are the predominant, ancestral type of mycorrhizal fungi (Smith and Read, 2008), and approximately 80% of modern plant species form AM associations (Brundrett, 2009). These fungi are primarily associated with increased phosphorus (P) uptake (Johnson, 2010), and past studies show that as much as 90% of plant P is acquired via fungal symbionts (Pearson and Jakobsen, 1993; Smith et al., 2009). AM fungi also can increase nitrogen (N) uptake, especially NH₄⁺, although the fungal contribution to plant N uptake is challenging to measure and highly variable among studies (e.g. 0%–74% of total plant N; Hodge and Storer, 2015). In exchange for these nutrients, plants allocate an estimated 5% to 10% of their carbohydrates to AM fungi (Bryla and Eissenstat, 2005).The net effect of AM fungi on plants is often measured in terms of plant growth, with some associations promoting faster growth rates and larger, more competitive plants (mutualism), while other associations restrict plant growth, resulting in smaller, less competitive plants (parasitism; Johnson et al., 1997). Where an interaction falls along the M-P continuum is highly dependent on physiological tradeoffs and resource limitations in the host. In general, nutrient availability is thought to be the primary driver of mycorrhizal functioning, with AM fungi increasing plant growth when nutrients, especially P, are more limiting than carbohydrates (Johnson et al., 2015). Changes in [CO₂] will likely modulate nutrient effects on AM associations by altering the relative degree of nutrient and carbohydrate limitations in plants (Fig. 1; Johnson, 2010). More specifically, there is strong evidence that low [CO₂] during the LGM produced major carbon (C) limitations within C₃ plants, and modern plants grown at glacial [CO₂] generally exhibit greater than 50% reductions in growth and photosynthetic rates relative to modern [CO₂] (Polley et al., 1993; Tissue et al., 1995; Sage and Coleman, 2001; Beerling, 2005; Gerhart and Ward, 2010; Gerhart et al., 2012). Furthermore, the majority of C₃ plants show some level of increased photosynthetic rates, leaf carbohydrate levels, and biomass when grown at future [CO₂] (Ainsworth and Rogers, 2007; Prior et al., 2011). However, plants may require more nutrients in order to maintain high rates of photosynthesis and growth at elevated [CO₂] (Campbell and Sage, 2006; Lewis et al., 2010). Thus, rising [CO₂] may cause mycorrhizal associations to shift along the M-P continuum by reducing plant carbohydrate limitation while simultaneously increasing plant nutrient limitation.Open in a separate windowFigure 1.[CO₂] is predicted to mediate nutrient effects on mycorrhizal associations by altering relative resource limitation in host plants. Specifically, the mycorrhizal response ratio (R_Bio) is predicted to increase with rising [CO₂] due to simultaneous increases in plant carbohydrate production and plant nutrient limitation. R_Bio is calculated from the biomass of mycorrhizal (M_Bio) and nonmycorrhizal (NM_Bio) plants (R_Bio = [M_Bio − NM_Bio]/NM_Bio). The dashed line represents a neutral association (no difference in plant size). R_Bio may be negative (solid line) or marginally positive (dotted line) at low [CO₂], depending on mycorrhizal effects on plant physiological responses to a CO₂-limiting environment.Most mycorrhizal-CO₂ studies compared the effects of current (340–400 µL L⁻¹) and future (540–750 µL L⁻¹) [CO₂] (Alberton et al., 2005; Mohan et al., 2014), but very few studies included more than two [CO₂] treatments, preventing the assessment of potential nonlinear patterns. These studies indicate that AM fungi generally increase plant growth at elevated [CO₂] and that rising [CO₂] promotes stronger mutualism (Mohan et al., 2014). However, the functioning of mycorrhizal associations at low [CO₂] of the past remains unclear. One possibility is that AM fungi reduce plant growth at low [CO₂] because these symbionts are a major sink for carbohydrates that are expensive to produce when CO₂ is limiting (Gerhart and Ward, 2010; Gerhart et al., 2012). Studies that show reduced AM fungal abundance in soils exposed to preindustrial [CO₂] provide tentative support for this hypothesis (Treseder et al., 2003; Procter et al., 2014). Alternatively, some plants may partially compensate for low [CO₂] by increasing their investment in the photosynthetic machinery, including the enzyme Rubisco (Sage and Coleman, 2001; Becklin et al., 2014). This strategy enhances CO₂ uptake but comes at the cost of greater demand for N (Sage and Coleman, 2001). P limitation also has been shown to restrict the rate of ribulose bisphosphate regeneration, with subsequent effects on photosynthesis under preindustrial [CO₂] (Campbell and Sage, 2006). Thus, by alleviating nutrient limitations on plant physiology, AM fungi may promote plant growth even under low [CO₂].Characterizing CO₂ effects on AM associations is critical for understanding the unique properties of these symbioses as well as for predicting how these interactions respond to both past and future environments. Here, we examined mycorrhizal responses across a glacial through future [CO₂] gradient in a controlled-environment experiment. We predicted that mycorrhizal associations would become more beneficial (i.e. have a larger positive effect on plant growth) with rising [CO₂] (Fig. 1). We further hypothesized two possible outcomes for mycorrhizal functioning under low [CO₂] of the past: (1) AM fungi will exacerbate carbohydrate constraints and restrict plant growth under low [CO₂], resulting in parasitic mycorrhizal associations when CO₂ is limiting (Fig. 1, solid line); and (2) fungal effects on nutrient uptake will alleviate nutrient constraints on plant physiological responses to low [CO₂], resulting in mutualistic mycorrhizal associations under glacial conditions (Fig. 1, dotted line). Additionally, studies indicate that many C₃ plants respond nonlinearly to rising [CO₂], with stronger responses to changes in [CO₂] below the modern value compared with above (Gerhart and Ward, 2010). These nonlinear shifts in plant physiology and growth will likely alter plant resource limitations and mycorrhizal functioning across a broad [CO₂] gradient. Thus, we further predicted that plants will be most responsive to increases from glacial to modern [CO₂], resulting in nonlinear shifts in plant physiology, plant growth, and mycorrhizal functioning across the full [CO₂] gradient. To better understand the mechanisms that alter plant responses to mycorrhizal fungi and overall patterns of plant productivity with rising [CO₂], we conducted detailed studies of plant physiology, intraradical fungal growth, and plant biomass allocation in mycorrhizal plants. We then tested for causal relationships among these traits and overall plant responses to mycorrhizal fungi using structural equation modeling (Fig. 2).Open in a separate windowFigure 2.A, Piecewise structural equation models describing [CO₂] effects on R_Bio via changes in plant and fungal traits. B, Model results for T. ceratophorum indicate that both direct and indirect effects of [CO₂] on plant traits contributed to shifts in R_Bio. C, Model results for T. officinale indicate that primarily direct effects of [CO₂] on plant traits contributed to shifts in R_Bio. In B and C, boxes represent measured traits and solid arrows indicate significant pathways in the model (P < 0.05). Black and red arrows indicate positive and negative correlations, respectively. The thickness of solid arrows was scaled to reflect the magnitude of the standardized regression coefficients. r² values for each component model and standardized pathway coefficients are listed in Supplemental Table S4.