Optofluidic Force Induction Meets Raman Spectroscopy and Inductively Coupled Plasma-Mass Spectrometry: A New Hyphenated Technique for Comprehensive and Complementary Characterizations of Single Particles.

Nanoparticles are produced at accelerating rates, are increasingly integrated into scientific and industrial applications, and are widely discharged into the environment. Analytical techniques are required to characterize parameters such as particle number concentrations, mass and size distributions, molecular and elemental compositions, and particle stability. This is not only relevant to investigate their utility for various industrial or medical applications and for controlling the manufacturing processes but also to assess toxicity and environmental fate. Different analytical strategies aim to characterize certain facets of particles but are difficult to combine to retrieve relevant parameters coherently and to provide a more comprehensive picture. In this work, we demonstrate the first online hyphenation of optofluidic force induction (OF2i) with Raman spectroscopy and inductively coupled plasma-time-of-flight-mass spectrometry (ICP-TOFMS) to harness their complementary technology-specific advantages and to promote comprehensive particle characterizations. We optically trapped individual particles on a weakly focused vortex laser beam by aligning a microfluidic flow antiparallelly to the laser propagation direction. The position of particles in this optical trap depended on the hydrodynamic diameter and therefore enabled size calibration as well as matrix elimination. Additionally, laser light scattered on particles was analyzed in a single particle (SP) Raman spectroscopy setup for the identification of particulate species and phases. Finally, particles were characterized regarding elemental composition and their distributions in mass and size using SP ICP-TOFMS. In a proof of concept, we analyzed polystyrene-based microplastic and TiO2 nanoparticles and demonstrated the opportunities provided through the coupling of OF2i with SP Raman and SP ICP-TOFMS.

Coordination chemistry suggests that independently observed benefits of metformin and Zn2+ against COVID-19 are not independent.

Independent trials indicate that either oral Zn2+ or metformin can separately improve COVID-19 outcomes by approximately 40%. Coordination chemistry predicts a mechanistic relationship and therapeutic synergy. Zn2+ deficit is a known risk factor for both COVID-19 and non-infectious inflammation. Most dietary Zn2+ is not absorbed. Metformin is a naked ligand that presumably increases intestinal Zn2+ bioavailability and active absorption by cation transporters known to transport metformin. Intracellular Zn2+ provides a natural buffer of many protease reactions; the variable "set point" is determined by Zn2+ regulation or availability. A Zn2+-interactive protease network is suggested here. The two viral cysteine proteases are therapeutic targets against COVID-19. Viral and many host proteases are submaximally inhibited by exchangeable cell Zn2+. Inhibition of cysteine proteases can improve COVID-19 outcomes and non-infectious inflammation. Metformin reportedly enhances the natural moderating effect of Zn2+ on bioassayed proteome degradation. Firstly, the dissociable metformin-Zn2+ complex could be actively transported by intestinal cation transporters; thereby creating artificial pathways of absorption and increased body Zn2+ content. Secondly, metformin Zn2+ coordination can create a non-natural protease inhibitor independent of cell Zn2+ content. Moderation of peptidolytic reactions by either or both mechanisms could slow (a) viral multiplication (b) viral invasion and (c) the pathogenic host inflammatory response. These combined actions could allow development of acquired immunity to clear the infection before life-threatening inflammation. Nirmatrelvir (Paxlovid®) opposes COVID-19 by selective inhibition the viral main protease by a Zn2+-independent mechanism. Pending safety evaluation, predictable synergistic benefits of metformin and Zn2+, and perhaps metformin/Zn2+/Paxlovid® co-administration should be investigated.

DGet! An open source deuteration calculator for mass spectrometry data.

DGet! is an open-source analysis package written in Python for calculating the degree of deuterium enrichment in isotopically labelled molecules using mass spectrometric data. The nuclear properties of deuterium make it a valuable tracer in metabolic studies and an excellent contrast agent in nuclear spectroscopies. Determination of molecular deuteration levels is typically performed using mass spectrometry, however software options to perform these calculations are scarce. The in-house scripts and spreadsheets currently used rarely account for isotopic interferences from 13C or multi-isotopic elements that impact deuteration calculations. DGet! removes isotopic interferences using de-convolution and both the isotopological makeup and overall deuteration level can be accurately recovered. The software is available as command line and web applications that take a molecular formula and mass spectrometry data and output a graphical representation of the degree of deuteration as well as the distribution of partially deuterated analogues. These applications are designed to be easy to use and enable superior characterisation of deuterated molecules for users of all levels of expertise, without the limitations of techniques currently used by the majority of deuteration laboratories and researchers.

Multi-Chemical Omics Analysis of the Symbiodiniaceae Durusdinium trenchii under Heat Stress.

The urgency of responding to climate change for corals necessitates the exploration of innovative methods to swiftly enhance our understanding of crucial processes. In this study, we employ an integrated chemical omics approach, combining elementomics, metabolomics, and volatilomics methodologies to unravel the biochemical pathways associated with the thermal response of the coral symbiont, Symbiodiniaceae Durusdinium trenchii. We outline the complimentary sampling approaches and discuss the standardised data corrections used to allow data integration and comparability. Our findings highlight the efficacy of individual methods in discerning differences in the biochemical response of D. trenchii under both control and stress-inducing temperatures. However, a deeper insight emerges when these methods are integrated, offering a more comprehensive understanding, particularly regarding oxidative stress pathways. Employing correlation network analysis enhanced the interpretation of volatile data, shedding light on the potential metabolic origins of volatiles with undescribed functions and presenting promising candidates for further exploration. Elementomics proves to be less straightforward to integrate, likely due to no net change in elements but rather elements being repurposed across compounds. The independent and integrated data from this study informs future omic profiling studies and recommends candidates for targeted research beyond Symbiodiniaceae biology. This study highlights the pivotal role of omic integration in advancing our knowledge, addressing critical gaps, and guiding future research directions in the context of climate change and coral reef preservation.

Fine particle pollution during megafires contains potentially toxic elements.

Wildfires that raged across Australia during the 2019-2020 'Black Summer' produced an enormous quantity of particulate matter (PM) pollution, with plumes that cloaked many urban centres and ecosystems along the eastern seaboard. This has motivated a need to understand the magnitude and nature of PM exposure, so that its impact on both built and natural environments can be more accurately assessed. Here we present the potentially toxic fingerprint of PM captured by building heating, ventilation, and air conditioning filters in Sydney, Australia during the peak of the Wildfires, and from ambient urban emissions one year later (Reference period). Atmospheric PM and meteorological monitoring data were also assessed to determine the magnitude and source of high PM exposure. The wildfires were a major source of PM pollution in Sydney, exceeding the national standards on 19 % of days between November-February. Wildfire particles were finer and more spherical compared to Reference PM, with count median diameters of 892.1 ± 23.1 versus 1484.8 ± 96.7 nm (mean ± standard error). On an equal-mass basis, differences in potentially toxic elements were predominantly due to higher SO42--S (median 20.4 vs 4.7 mg g-1) and NO3--N (2.4 vs 1.2 mg g-1) in Wildfire PM, and higher PO43--P (10.4 vs 1.4 mg g-1) in Reference PM. Concentrations of remaining elements were similar or lower than Reference PM, except for enrichments to F-, Cl-, dissolved Mn, and particulate Mn, Co and Sb. Fractional solubilities of trace elements were similar or lower than Reference PM, except for enhanced Hg (12.1 vs 1.0 %) and greater variability in Cd, Hg and Mn solubility, which displayed upper quartiles exceeding that of Reference PM. These findings contribute to our understanding of human and ecosystem exposures to the toxic components of mixed smoke plumes, especially in regions downwind of the source.

Immunolabelling perturbs the endogenous and antibody-conjugated elemental concentrations during immuno-mass spectrometry imaging.

Immuno-mass spectrometry imaging uses lanthanide-conjugated antibodies to spatially quantify biomolecules via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The multi-element capabilities allow for highly multiplexed analyses that may include both conjugated antibodies and endogenous metals to reveal relationships between disease and chemical composition. Sample handling is known to perturb the composition of the endogenous elements, but there has been little investigation into the effects of immunolabelling and coverslipping. Here, we used cryofixed muscle sections to examine the impact of immunolabelling steps on the concentrations of a Gd-conjugated anti-dystrophin primary antibody, and the endogenous metals Cu and Zn. Primary antibody incubation resulted in a decrease in Zn, and an increase in Cu. Zn was removed from the cytoplasm where it was hypothesised to be more labile, whereas concentrated locations of Zn remained in the cell membrane in all samples that underwent the immunostaining process. Cu increased in concentration and was found mostly in the cell membrane. The concentration of the Gd-conjugated antibody when compared to the standard air-dried sample was not significantly different when coverslipped using an organic mounting medium, whereas use of an aqueous mounting medium significantly reduced the concentration of Gd. These results build on the knowledge of how certain sample handling techniques change elemental concentrations and distributions in tissue sections. Immunolabelling steps impact the concentration of endogenous elements, and separate histological sections are required for the quantitative analysis of endogenous elements and biomolecules. Additionally, coverslipping tissue sections for complementary immunohistochemical/immunofluorescent imaging may compromise the integrity of the elemental label, and organic mounting media are recommended over aqueous mounting media.

Fuelling phytoremediation: gasoline degradation by green wall systems-a case study.

The capacity for indoor plants including green wall systems to remove specific volatile organic compounds (VOCs) is well documented in the literature; however under realistic settings, indoor occupants are exposed to a complex mixture of harmful compounds sourced from various emission sources. Gasoline vapour is one of the key sources of these emissions, with several studies demonstrating that indoor occupants in areas surrounding gasoline stations or with residentially attached garages are exposed to far higher concentrations of harmful VOCs. Here we assess the potential of a commercial small passive green wall system, commercially named the 'LivePicture Go' from Ambius P/L, Australia, to drawdown VOCs that comprise gasoline vapour, including total VOC (TVOC) removal and specific removal of individual speciated VOCs over time. An 8-h TVOC removal efficiency of 42.45% was achieved, along with the complete removal of eicosane, 1,2,3-trimethyl-benzene, and hexadecane. Further, the green wall also effectively reduced concentrations of a range of harmful benzene derivatives and other VOCs. These results demonstrate the potential of botanical systems to simultaneously remove a wide variety of VOCs, although future research is needed to improve upon and ensure efficiency of these systems over time and within practical applications.

Concentration and Distribution of Toxic and Essential Elements in Traditional Rice Varieties of Sri Lanka Grown on an Anuradhapura District Farm.

Toxic heavy metals have been the focus of many investigations into chronic kidney disease of unknown aetiology (CKDu) within Sri Lanka. It has been hypothesised that exposure to nephrotoxic arsenic, cadmium and lead could play a role in the development of CKDu, and these metals have previously been found in unsafe concentrations in Sri Lankan rice. Traditional varieties of Sri Lankan rice remain popular due to their perceived health benefits, but their uptake of trace and toxic heavy metals remained unexplored. Here, we report a one-time, cross-sectional dataset on the concentrations of essential and toxic elements present in eleven samples of polished and unpolished traditional rice varieties, all regularly grown and sold in the Anuradhapura district, a CKDu hotspot. All rice was sourced from the same farm, with the exception of one store bought sample grown on another, unidentified farm. Cadmium concentrations varied significantly between varieties, and potentially unsafe concentrations of cadmium were detected in the store-bought sample (Suwadel, 113±13 µg kg-1). Elemental imaging of the grains revealed lead to be stored mainly in the rice bran, which is removed during polishing, while cadmium was distributed in the edible portion of the grain. Essential elements were generally higher in the traditional rice varieties than those reported for non-traditional varieties and are a potential source of trace elements for nutrient-deficient communities. The concentration of selenium, an element that plays a protective role in the kidneys, was too low to provide the minimum recommended intake. The methods developed in this study could be applied to a more comprehensive study of elemental uptake of rice under controlled growing conditions.

Analysis of Ti- and Pb-based particles in the aqueous environment of Melbourne (Australia) via single particle ICP-MS.

The analysis of natural and anthropogenic nanomaterials (NMs) in the environment is challenging and requires methods capable to identify and characterise structures on the nanoscale regarding particle number concentrations (PNCs), elemental composition, size, and mass distributions. In this study, we employed single particle inductively coupled plasma-mass spectrometry (SP ICP-MS) to investigate the occurrence of NMs in the Melbourne area (Australia) across 63 locations. Poisson statistics were used to discriminate between signals from nanoparticulate matter and ionic background. TiO2-based NMs were frequently detected and corresponding NM signals were calibated with an automated data processing platform. Additionally, a method utilising a larger mass bandpass was developed to screen for particulate high-mass elements. This procedure identified Pb-based NMs in various samples. The effects of different environmental matrices consisting of fresh, brackish, or seawater were mitigated with an aerosol dilution method reducing the introduction of salt into the plasma and avoiding signal drift. Signals from TiO2- and Pb-based NMs were counted, integrated, and subsequently calibrated to determine PNCs as well as mass and size distributions. PNCs, mean sizes, particulate masses, and ionic background levels were compared across different locations and environments.

Characterisation of microplastics and unicellular algae in seawater by targeting carbon via single particle and single cell ICP-MS.

The discharge of plastic waste and subsequent formation and global distribution of microplastics (MPs) has caused great concern and highlighted the need for dedicated methods to characterise MPs in complex environmental matrices like seawater. Single particle inductively coupled plasma - mass spectrometry (SP ICP-MS) is an elegant method for the rapid analysis of nano- and microparticles and to characterise number concentrations, mass, and size distributions. However, the analysis of carbon (C)-based microstructures such as MPs by SP ICP-MS is at an early stage. This paper investigates various strategies to improve figures of merit to detect and characterise MPs in complex matrices, such as seawater. Ten methods operating distinct acquisition modes with various collision/reaction gases, tandem MS (ICP-MS/MS) and targeting 12C or 13C were developed and compared for the analysis of polystyrene-based MPs standards in ultra-pure water and seawater. The robust analysis of MPs in seawater was accomplished by on-line aerosol dilution enabling repeatable size calibration while minimising drift effects. However, the direct analysis of seawater decreased ion transmission and required matrix-matching for accurate size calibration. Analysis of the 12C isotope instead of 13C improved the size detection limits (sDL) to 0.62 µm in ultra-pure water and to 0.96 µm in seawater. ICP-MS/MS methods decreased ion transmission but also reduced background signal and increased selectivity, particularly in the presence of spectral interferences. In the second part of this study, it was demonstrated that the developed methods were applicable for the analysis of C in unicellular organisms and allowed calibration of physical dimensions. This is relevant for the investigation and understanding of phenotypical traits associated, for example, with climate change resilience as well as oceanic C storage. SP/SC ICP-MS was employed to target five different intact Symbiodiniaceae algae strains with diverse life-histories in seawater and polystyrene-based MPs were used to calibrate cellular C masses, which were between 51 and 83 pg. The C mass distribution across the analysed unicellular cells was used for modelling cell sizes, which were in the range of 7.6 and 10.1 µm. Determined values were in line with values obtained with complementary techniques (Coulter-counting, total organic C analysis and microscopic analysis).

Pew2: Open-Source Imaging Software for Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry.

Open-sourced software is a key component of the mass spectrometry imaging field, where transparency in data processing is vital. Imaging of trace elements and immunohistochemically labeled biomolecules in tissue sections is typically performed using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). However, efficient and facile processing of images is hampered by a lack of verifiable and user-friendly software that supports multiple LA-ICP-MS platforms. In this technical note, we introduce Pew2, a LA-ICP-MS specific and feature-rich open-source image processing software that is compatible with common ICP-MS vendors. Pew2 is designed to be fast and easy to use and adheres to modern visualization philosophies to maximize productivity and to minimize data interpretation errors and image anomalies.

Quantitative immuno-mass spectrometry imaging of skeletal muscle dystrophin.

Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches have poor precision, require large amounts of tissue, and are difficult to standardize. This paper presents an immuno-mass spectrometry imaging method using gadolinium (Gd)-labeled anti-dystrophin antibodies and laser ablation-inductively coupled plasma-mass spectrometry to simultaneously quantify and localize dystrophin in muscle sections. Gd is quantified as a proxy for the relative expression of dystrophin and was validated in murine and human skeletal muscle sections following k-means clustering segmentation, before application to DMD patients with different gene mutations where dystrophin expression was measured up to 100 µg kg-1 Gd. These results demonstrate that immuno-mass spectrometry imaging is a viable approach for pre-clinical to clinical research in DMD. It rapidly quantified relative dystrophin in single tissue sections, efficiently used valuable patient resources, and may provide information on drug efficacy for clinical translation.

RNAi of AGAMOUS genes in sweetgum alters reproductive organ identity and decreases fruit persistence.

Sweetgums (Liquidambar), members of the family Altingiaceae (Altingiales), have inflorescences and floral organs that are distinctive in structure compared with other angiosperms in which the roles of floral homeotic genes have been studied. To begin to understand the role of AGAMOUS (AG)-a floral homeotic gene that has a major role in stamen and carpel development-in development of the monosexual flowers of sweetgum, we used RNAi to reduce the expression of two members of the AG subfamily. Because AG suppression should induce floral sterility, RNAi might also provide a tool to mitigate the risks of invasiveness-and to reduce the production of its nuisance fruits or allergenic pollen-when sweetgum is used as an exotic shade or forest tree. We tested 33 independent transgenic events and non-transgenic controls during 10 years in the field. The RNAi-AG sweetgum trees maintained normal growth, phenology, and vivid fall coloration during the 10 years of study, but 8 insertion events had highly modified inflorescence and floral morphology. The modified flowers had anthers and carpels that were converted to flat leaf-like structures lacking pollen grains and ovules, respectively. The female inflorescences developed into dry papery structures that failed to produce seeds. These infructescences were smaller than control infructescences, and lost a greater percentage of biomass in a controlled decay assay. RNAi against AG genes was highly effective at impairing fertility and modifying reproductive development without significant vegetative effects in sweetgum and gave phenotypes distinct from, but similar to, that of AG loss of function in other angiosperms.

Low background mould-prepared gelatine standards for reproducible quantification in elemental bio-imaging.

Standard preparation for elemental bio-imaging by laser ablation-inductively coupled plasma-mass spectrometry is confounded by the chemical and physical differences between standard and sample matrices. These differences lead to variable ablation, aerosol generation and transportation characteristics and must be considered when designing matrix-matched standards for reliable calibration and quantification. The ability to precisely mimic sample matrices is hampered due to the complexity and heterogeneity of biological tissue and small variabilities in standard matrices and sample composition often negatively impact accuracy, precision and robustness. Furthermore, cumbersome preparation protocols may limit reproducibility and traceability. This work presents novel facile methods for the preparation of gelatine standards using both commercial and laboratory-made moulds. Surface roughness, thickness and robustness of the mould-prepared standards were compared against cryo-sectioned gelatine and homogenised brain tissue standards. The mould-prepared standards had excellent thickness accuracy and signal precision which allowed robust quantification, were easier to prepare and therefore easier to reproduce. We also compared gelatine standards prepared from a variety of animal sources and discuss their suitability to calibrate low level elemental concentrations. Finally, we present a simple method to remove background metals in gelatine using various chelating resins to increase the dynamic calibration range and to improve limits of analysis.

Micro solid-phase extraction for the analysis of per- and polyfluoroalkyl substances in environmental waters.

Growing concern over the environmental and health impacts of per- and polyfluoroalkyl substances (PFASs) has led to the development of increasingly stringent regulatory guidelines. To meet these guidelines for the determination of PFASs in surface-water, solid-phase extraction (SPE) is commonly implemented for clean-up and pre-concentration of samples. In this paper a micro-SPE method for the clean-up and pre-concentration of PFASs from surface-water was developed. A micro-SPE packing phase was created to retain 13 long and short chain PFAS after examining combinations of four 3 µm particle size sorbents, with the optimal phase consisting of a 50:50 mixture of C18 and aminopropyl silica. Micro-SPE achieved similar results to conventional SPE methods while reducing sample preparation time to 5 min and using only 2 mL of sample. The method was validated using spiked recoveries (100 ng L-1) from PFAS contaminated surface-water samples with recoveries ranging from 86% to 111% and relative standard deviations below 18%. Concentrations of the PFASs in the samples ranged from below the limit of quantification to 898 ± 15 ng L-1. Automation of sample preparation, including the micro-SPE extraction, was also demonstrated. These results show the potential for automated micro-SPE to replace conventional SPE, with the decreases in sample preparation time, sample and solvent volumes crucial for incorporation into routine analyses in commercial laboratories.

Biguanide is a modifiable pharmacophore for recruitment of endogenous Zn2+ to inhibit cysteinyl cathepsins: review and implications.

Excessive activities of cysteinyl cathepsins (CysCts) contribute to the progress of many diseases; however, therapeutic inhibition has been problematic. Zn2+ is a natural inhibitor of proteases with CysHis dyads or CysHis(Xaa) triads. Biguanide forms bidentate metal complexes through the two imino nitrogens. Here, it is discussed that phenformin (phenylethyl biguanide) is a model for recruitment of endogenous Zn2+ to inhibit CysHis/CysHis(X) peptidolysis. Phenformin is a Zn2+-interactive, anti-proteolytic agent in bioassay of living tissue. Benzoyl-L-arginine amide (BAA) is a classical substrate of papain-like proteases; the amide bond is scissile. In this review, the structures of BAA and the phenformin-Zn2+ complex were compared in silico. Their chemistry and dimensions are discussed in light of the active sites of papain-like proteases. The phenyl moieties of both structures bind to the "S2" substrate-binding site that is typical of many proteases. When the phenyl moiety of BAA binds to S2, then the scissile amide bond is directed to the position of the thiolate-imidazolium ion pair, and is then hydrolyzed. However, when the phenyl moiety of phenformin binds to S2, then the coordinated Zn2+ is directed to the identical position; and catalysis is inhibited. Phenformin stabilizes a "Zn2+ sandwich" between the drug and protease active site. Hundreds of biguanide derivatives have been synthesized at the 1 and 5 nitrogen positions; many more are conceivable. Various substituent moieties can register with various arrays of substrate-binding sites so as to align coordinated Zn2+ with catalytic partners of diverse proteases. Biguanide is identified here as a modifiable pharmacophore for synthesis of therapeutic CysCt inhibitors with a wide range of potencies and specificities. Phenformin-Zn2+ Complex.

Lysosomal metal, redox and proton cycles influencing the CysHis cathepsin reaction.

In the 1930's pioneers discovered that maximal autolysis in tissue homogenates requires metal chelator, sulfhydryl reducing agent and acid pH. However, metals, reducing equivalents and protons (MR&P) have been overlooked as combined catalytic controls. Three categories of lysosomal machinery drive three distinguishable cycles importing and exporting MR&P. Zn(2+) preemptively inhibits CysHis catalysis under otherwise optimal protonation and reduction. Protein-bound cell Zn(2+) concentration is 200-2000 times the non-sequestered inhibitory concentration. Following autophagy, lysosomal proteolysis liberates much inhibitory Zn(2+). The vacuolar proton pump is the driving force for Zn(2+) export, as well as protonation of the peptidolytic mechanism. Other machinery of lysosomal cycles includes proton-driven Zn(2+) exporters (e.g. SLC11A1), Zn(2+) channels (e.g. TRPML-1), lysosomal thiol reductase, etc. The CysHis dyad is a sensor of the vacuolar environment of MR&P, an integrator of these simultaneous variables, and a catalytic responder. Rate-determination can shift between autophagic substrate acquisition (swallowing) and substrate degradation (digesting). Zn(2+) recycling from degraded proteins to new proteins is a fourth cycle that might pace lysosomal function under some conditions. Heritable insufficient or excess functions of CysHis cathepsins are associated with dysfunctional inflammation and immunity/auto-immunity, including diabetic pathogenesis.

The lysosome among targets of metformin: new anti-inflammatory uses for an old drug?

BACKGROUND: Rheumatoid arthritis and type-2 diabetes exhibit progressive co-morbidity. Chloroquine (CQ) reportedly improves both. CQ inhibits lysosomal function in cultured cells at supra-therapeutic concentration; however, this is doubted as target mechanism. Some anti-diabetic biguanides are metal-interactive lysosomal inhibitors; and all bind Zn(2+). OBJECTIVES: i) To bioassay the potency of CQ using (3)H-leucine release from perfused myocardial tissue. ii) To determine whether metformin (MET) is CQ-mimetic, and interactive with Zn(2+). RESULTS: Therapeutic CQ concentration (0.1 - 0.5 microM) clearly does cause lysosomal inhibition although delayed and submaximal. MET alone (10 microM) caused sub-maximal inhibition. Supra-physiological extracellular Zn(2+) (5 - 50 microM) alone increased tissue Zn(2+) content, and inhibited lysosomal proteolysis. Physiological equivalent Zn(2+) (approximately 1 microM) had no effect. MET (

Is dihydrolipoic acid among the reductive activators of parasite CysHis proteases?

Activities of mature CysHis proteases depend upon relative rates of oxidations vs. reductions of catalytic sulfur by multiple enzymatic and non-enzymatic reactions. CysHis peptidolysis is inhibited by Fe3+ but not Fe2+. Others report the paradox that malarial parasites require exogenous free lipoic acid (LA) from human host, although the apicoplast organelle produces it. Extra-cellular LA disulfide can be taken up and reduced to dihydrolipoic acid (DHLA) by reductases of any cell type. Here, the opposing effects of DHLA vs. Fe3+ on the falcipain-2 hemoglobinase were investigated employing Z-Phe-Arg-AMC substrate. Despite limited solubility, non-regenerated DHLA (10 microM, threshold 2 microM) was found to be the most potent activator of the air-inactivated (sulfoxygenated) protease discovered thus far. Activation was preemptively opposed by Fe3+, but not Fe2+. However, cruzain from T. cruzi, and cathepsin B from mammal were indistinguishable in their responsiveness to DHLA and Fe redox. Thus, DHLA activation vs. Fe3+ inhibition is not unique to falcipain-2 or apicomplexans but is rather a primordial feature of CysHis peptidolysis. Free LA and/or unassociated lipoylated enzyme subunits could be among multiple pathways shuttling reducing equivalents to reduction of proteins, including CysHis proteases. It is discussed that opposing DHLA-Fe3+ modification of plasmodial proteolysis might be a specialized adaptation to intra-erythrocytic growth.

Changes in extracellular pH are neither required nor sufficient for activation of mitogen-activated protein kinases (MAPKs) in response to systemin and fusicoccin in tomato.

Leaf wounding and the wound signaling peptide systemin induce expression of wound response genes while the fungal toxin fusicoccin (FC) induces expression of pathogenesis-related genes. Consistent with their functional differences, FC and systemin regulate the extracellular pH in opposite ways, with systemin inducing an alkalinization and FC an acidification response. Here we show that systemin, wounding and FC activate the same mitogen-activated protein kinases (MAPKs; MPKs) MPK1 and 2 in tomato (Lycopersicon esculentum) leaves and L. peruvianum suspension-cultured cells. Wounding and FC activated an additional MAPK, MPK3. Pronounced differences were observed with regard to MAPK activation kinetics. FC induced prolonged, and systemin transient activity of the MAPKs. This shows that functionally different elicitors engage the same signaling components, yet induce signal-specific activation dynamics. A comparative analysis of pH effects and MAPK activity in response to specific treatments revealed that the kinetics of pH changes and MAPK activation did not correlate. Simultaneous application of FC and systemin did not lead to immediate pH changes but resulted in rapid increases in MAPK activity. Furthermore, changes in extracellular pH could be induced without concomitant MAPK activation by exchanging conditioned medium with fresh medium. This shows that changes in the extracellular pH are neither required nor sufficient for MAPK activation, suggesting that signaling pathways involving MAPKs and extracellular pH changes operate in parallel and are not part of the same linear pathway.