Biochemistry

The accelerated recovery of base-poor soils from the legacy effects of acidic deposition may be possible by applying industrial wood ash as a soil amendment. Wood ash may be an effective soil amendment due to its high alkalinity and concentrations of several essential nutrients, such as calcium, magnesium, potassium, and phosphorus, that are retained after the volatilization of the parent material. However, wood ash can also contain trace amounts of metals that could be released into the soil and soil solution. The short-term (<3 years) biogeochemical response of soils, microbial communities, and sugar maple (Acer saccharum Marsh.) trees were assessed following wood ash application at Porridge Lake, Ontario. The study design consisted of five blocks containing three treatment plots each (2.5, 5.0, 7.5 Mg ha-1) and a control. Soil solution pH, base cation, and trace metal concentrations were monitored for three years, using tension lysimeters at depths of 30 and 60 cm and zero-tension lysimeters for forest floor percolate within each plot. In the last year of the trial, soil, foliage, and fine root samples were collected and analyzed for trace elements. Also, soil samples were analyzed for the abundance of 16S and ITS DNA through metabarcoding to ascertain the microbial response to wood ash. Significant changes in soil solution pH were measured within the forest floor horizon in the first year of the trial. Significant increases in calcium (Ca), magnesium (Mg) and calcium/aluminum (Ca/Al) ratios were also observed in the second year of the trial, along with decreases in dissolved organic carbon (DOC), sulphate (SO4) and nitrate (NO3) in the LFH horizon. By the third year of the trial, significant increases in soil solution pH and potassium (K) concentrations and decreases in Al were observed to a depth of 30 cm. Changes in trace metal concentrations in soil water were notably variable, with concentrations of chromium, copper, lead, nickel, and selenium remaining unresponsive, whereas concentrations of cadmium, manganese and zinc decreased by the third year. The metalloid arsenic showed a significant increase in the third year of the trial but remained below regulatory guidelines, similar to all other trace metals. Soil measurements conducted in the third year of the trial showed positive pH responses in the FH horizon and increases in Ca and Mg in the Ah and Bm soil horizons, but foliar base cation and metal concentrations were unchanged. Diversity analysis on the soil prokaryotic and eukaryotic groups indicated increased bacterial alpha diversity in the FH horizon and bacterial dominance in the litter horizon. Analysis of relative abundance at the phylum level for prokaryotes and at the order for eukaryotes did not indicate any compositional shifts due to the wood ash treatments. Changes in the length and diameter of sugar maple and mycorrhizal fine root may point to pH shock being an issue at higher ash doses. The results from this study indicate that wood ash has a strong ameliorative effect on soil properties and does not pose a risk to soil communities. 

Author Keywords: DNA, mycorrhizae, soil acidication, soil amendments, soil solution, sugar maple

Giardia intestinalis is the causative agent of a diarrheal disease in mammals, but the mechanisms of disease pathogenesis are unclear. While proteins secreted by Giardia affect the host cells, the potential of hormone secretion has not been investigated to date. Cytokinins (CKs) are classified as phytohormones, but little is known about their role beyond plants. Mass spectrometry-based intracellular analysis revealed CKs typical of tRNA degradation, and extracellular analysis showed CK-riboside scavenging by Giardia with concurrent secretion of CK-free bases. Metabolomics profiling of culture supernatants showed similar trends where nucleosides were up taken, and nucleobases were secreted. The dynamics of amino acids, nucleosides and nucleobases were altered by CK-supplementation during encystation, along with inhibition of encystation. In summary, this is the first study to report CK synthesis and metabolism by Giardia along with the effects of CKs on the metabolite secretome of Giardia, while establishing a link between CK and nucleoside metabolism.

Author Keywords: Cytokinins, Giardia, mass spectrometry, metabolomics, parasite, secretome

Fungi are a diverse group of organisms that play crucial roles in various ecological processes and have immense economic importance. Understanding the intricate mechanisms underlying fungal growth and development is fundamental to harnessing their potential and exploring their applications in different fields. Signalling molecules, such as hormones, have been identified as key regulators in fungal physiology, orchestrating intricate processes and modulating biological responses to the environment. Phytohormones, commonly associated with plants, have been proposed as potential myco-hormones due to their production in a wide variety of fungi. Metabolomic analyses were performed via LC-MS/MS to investigate the role of phytohormones, specifically cytokinins (CKs) and indole-3-acetic acid (IAA), along with lipids and energetic metabolites such as organic acids during the growth and development of the model fungus Sordaria macrospora. The results revealed a clear switch between CK ribosides and CK free bases during the ascosporegenesis stage, with increased levels of cZ and iP and decreased levels of iPR and cZR. A similar pattern was observed in the mutant strain smgpi1 but demonstrating higher levels of CK free bases and increased fruiting body formation compared to the wild type. These findings provide insights into the regulation of phytohormones especially during fungal fruiting body development. In terms of IAA, the levels increased during the transition to sexual development in all strains, with the per5 mutant, unable to produce lipids via the cytosol, demonstrating a higher concentration than the wild type. The interplay between energetic metabolites and IAA suggests a potential role in the transition to sexual development. Additionally, the dose-dependent effects of exogenous CK application were investigated, showing the potential of low concentrations, from 1 to 10 μM, of CKs in promoting biomass accumulation or sexual development. Furthermore, gene editing in S. macrospora was proposed as a future direction to explore the functions of CKs and other metabolites during fungal development. Overall, this research contributes to our understanding of phytohormone-mediated processes in fungi and opens avenues for future investigations in fungal biology.

Author Keywords: fungi, LC-MS, metabolomics, myco-hormone, phytohormone, sexual development

Giardia intestinalis is a parasitic protozoan that possesses a flavohemoglobin (gFlHb), an enzyme that plays a role in the detoxification of reactive nitrogen species (RNS) and reactive oxygen species (ROS) via its nitric oxide dioxygenase (NOD) activity as well as its NADH-oxidase activity. This enzyme is a potential target for imidazole-based antigiardial drugs that act as ligands of the iron within its heme cofactor. In this work, two rapid and relatively inexpensive assays, the colorimetric Griess assay and a fluorescence assay, were adapted, optimized, and implemented to screen for flavohemoglobin inhibitors in parallel studies that compared the response of gFlHb to that of Hmp (Escherichia coli flavohemoglobin) when a group of six different imidazole-based compounds was tested. These assays displayed isotype selectivity, showing how the different drugs elicited different responses from the two enzymes. Comparative results for gFlHb and Hmp revealed that bulkier compounds elicited higher inhibition of Hmp, while smaller compounds resulted in better inhibition of gFlHb, which might be explained by the presence of different amino acid residues in the active sites of the enzymes, with two large amino acid sequence inserts being a unique feature of gFlHb, thus blocking the active site from being reached and blocked by larger compounds.

Author Keywords: 2.3-diaminonaphthalene, Flavohemoglobin, Giardia intestinalis, Griess Assay, imidazole-based drugs, nitric oxide detoxification

During the COVID-19 pandemic, nucleic acid and antibody-based testing methods were heavily relied upon, but can be costly, time-consuming and exhibit high false -negative and -positive rates. Thus, alternative strategies are needed. Viral antigens such as the SARS-CoV-2 spike (S) glycoprotein are critical in the function of the virus and useful as diagnostic biomarkers for viral infections. For biosensing applications, aptamers are suitable high-affinity and cost-effective binding partners for their specific targets. Using localized surface plasmon resonance (LSPR), real-time, rapid acquisition of results can be achieved, essential for improving the efficacy of a sensor. Herein, LSPR aptamer sensors were fabricated for the detection of the SARS-CoV-2 protein. Data indicate that the best performing aptasensor was the streptavidin-biotin sensor, while the current gold aptasensor exhibited lower sensitivity and the fabrication of the carboxyl aptasensor was unsuccessful. The S1 aptamer selectively bound the S1 protein with high binding affinity. Excellent shelf-life stability, reusability, and high recovery in complex matrices was also maintained. Additionally, a receptor binding domain (RBD) functionalized sensor was fabricated to examine the interactions with angiotensin converting enzyme 2 (ACE2), for future assessment of inhibitors used in drug therapies. Overall, LSPR has been demonstrated as a viable tool for measuring SARS-CoV-2 related aptamer-protein and protein-protein interactions, and this strategy may be applied to other viral or non-viral antigen targets.

Author Keywords: Antigen-based Detection, Coronavirus, COVID-19, Inhibition, Localized Surface Plasmon Resonance, SARS-CoV-2

Amino acids make up proteins, which are the building blocks of life. A balance of amino acids is needed to maintain a healthy state. Tyrosine (Tyr) is synthesized from the metabolism of phenylalanine, which is an essential amino acid, meaning it can only be obtained from the diet. It is related to many metabolic and neurodegenerative diseases. Tyr can undergo post-translational modifications such as phosphorylation and nitration, which are implicated in cancer and nitrative stress, respectively. Although there are many methods to detect Tyr and its analogues, phosphotyrosine (pTyr) and nitrotyrosine (nTyr), these methods are time-consuming, involve expensive instruments and involve tedious process. This research proposes a new type of nanomaterials, carbon dots (CDs), to detect these amnio acids. Data indicate that CDs can be used to detect nTyr with a limit of detection of 34 μM in the linear range of 20 - 105 μM. The amenability of CD-nTyr assay was also tested in various biological matrices and biological molecules and was shown to be sensitive to nTyr. Nitration of Tyr was carried out in the presence of sodium nitrite and hydrogen peroxide catalyzed by either Cu(II) or Fe(III) to mimic biological reactions and CDs were tested as both inhibitors and indicators of Tyr nitration. Although CDs did not inhibit the nitration reaction of Tyr, they did not serve as indicators of Tyr nitration due to the quenching of CDs by the nitrating agents. This shows the importance of using CDs to detect nTyr and further use it for biological applications to detect diseased states.

Author Keywords: amino acids, carbon dots, nanomaterials, sensor, spectroscopy, tyrosine

The onset of neurodegenerative diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) are typically characterised by the aggregation of protein biomarkers into cytotoxic fibrils. Novel means of analysing these biomarkers are needed to expand the literature toward earlier diagnosis of these conditions. Electrochemical sensors could offer the sensitivity and selectivity needed for specialised analysis, including potential point-of-care applications. The AD biomarker Tau, and ALS biomarker TDP-43 proteins are explored here by using a label-free electrochemical sensors. Tau protein was covalently bound to gold electrode surface to study the in vitro mechanisms of aggregation for this protein. An immunosensor to TDP-43 was developed by covalently binding primary TDP-43 antibodies (Abs) on gold electrode surface. A novel direct ELISA sensor for TDP-43 with visual detection and electrochemical quantification was also developed. The results validated the experimental designs toward specialised and selective analysis of these biomarkers and their aggregation mechanisms.

Author Keywords: ALS, Alzheimer's, Biosensors, Electrochemistry, Tau, TDP-43

Transactive response (TAR) DNA-binding protein of 43 kDa (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS) and is characterized by hyperphosphorylation and aggregation patterns, a mechanism largely understudied. In addition, ALS remains without a cure. Herein, in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Additionally, in vitro phosphorylation of TDP-43 by protein kinases was conducted to identify which protein kinases catalyze phosphorylation. The aggregation of phosphorylated and unphosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) fluorescence spectroscopy. The protein aggregates were largely insoluble, ThT-positive and characterized with heterogeneous morphologies. Antibodies specific to epitopes within the RNA-recognition motifs and the C-terminal domains reduced the formation of β-sheets and insoluble aggregates, with outcomes highly dependent on the type of antibodies, indicating dual functionality. The only protein kinase able to phosphorylate TDP-43 at S410 was MARK4, indicating its role in the onset of PTMs in the protein. Thus, targeting TDP-43 epitopes for inhibition of aggregation and in vitro phosphorylation represent viable biochemical assays for screening protein kinase inhibitors as potential drugs against ALS.

Author Keywords: aggregation, ALS, antibody-based inhibition, phosphorylation, protein kinase, TDP-43

Giardia intestinalis possesses four isotypes of cytochrome b5 (gCYTB-I-IV) that differ from their mammalian counterparts, suggesting different functions in this protozoan parasite. Although the recently discovered Giardia flavoenzyme, GiOR-1, reduces these cytochromes, its properties have not been thoroughly studied, owing to the difficulty in its expression. Here I describe successful conditions for expression of GiOR-1 using autoinduction. GiOR-1 is obtained with flavins bound as indicated by its UV-visible spectrum. Its ability to catalyze electron transfer from donors (NADH, NADPH) to acceptors (oxygen, ferricyanide, cytochrome c, gCYTB5-III) were studied in spectrophotometric rate assays. NADPH is the preferred electron donor, while cytochromes are the preferred electron acceptors. Interestingly, the His-tag used to purify gCYTB5-III decreases its reaction rate with GiOR-1, as an untagged version has slightly faster rates. These findings establish the appropriate conditions for further studies on GiOR-1, including the identification of endogenous electron acceptors.

Author Keywords: Autoinduction, Cytochrome b5, Cytochrome P450 oxidoreductase, Giardia intestinalis, GiOR-1, Polyhistidine tag

Postclassic (AD 900-1500) Maya diet at Ka'kabish, Belize was examined using stable carbon and nitrogen isotope analysis of human bone collagen, and stable carbon isotope analysis of bone structural carbonate. Isotope data were compared to skeletal and dental indicators of diet and disease, and dietary differences among burials excavated from chultuns (B-2, C-1, C-2, and C-3) at Ka'kabish. Varying in dimensions, chultuns are characterized as multiple subterranean chambers carved into limestone bedrock, where re-entry was permitted through the removal of a capstone placed over a circular entrance. Due to poor preservation and commingling of human remains, diet and its relation to age, sex, and social status could not be explored. The general diet at Ka'kabish is consistent with the consumption of a diverse range of terrestrial plants and animals, in addition to marine resources. Relative to the other chultun burials, Chultun C-2 is an outlier, with a noticeably different diet, evidence for skeletal pathology, and absence of dental modifications. This study demonstrates a lack of significant dietary differences among Postclassic Maya sites in northern Belize, along with an apparent reliance on marine resources, further supporting the notion of a close association, and equal participation in a regional trading system with coastal sites that allowed for populations in this region to thrive during the Postclassic period.

Author Keywords: Ancient Maya, Bioarchaeology, Ka'kabish, Osteology, Postclassic, Stable Isotopes