Biochemical and molecular responses of the ascorbate-glutathione cycle in wheat seedlings exposed to different forms of selenium.

Biofortification aims to increase selenium (Se) concentration and bioavailability in edible parts of crops such as wheat (Triticum aestivum L.), resulting in increased concentration of Se in plants and/or soil. Higher Se concentrations can disturb protein structure and consequently influence glutathione (GSH) metabolism in plants which can affect antioxidative and other detoxification pathways. The aim of this study was to elucidate the impact of five different concentrations of selenate and selenite (0.4, 4, 20, 40 and 400 mg kg-1) on the ascorbate-glutathione cycle in wheat shoots and roots and to determine biochemical and molecular tissue-specific responses. Content of investigated metabolites, activities of detoxification enzymes and expression of their genes depended both on the chemical form and concentration of the applied Se, as well as on the type of plant tissue. The most pronounced changes in the expression level of genes involved in GSH metabolism were visible in wheat shoots at the highest concentrations of both forms of Se. Obtained results can serve as a basis for further research on Se toxicity and detoxification mechanisms in wheat. New insights into the Se impact on GSH metabolism could contribute to the further development of biofortification strategies.

Commensal protist Tritrichomonas musculus exhibits a dynamic life cycle that induces extensive remodeling of the gut microbiota.

Commensal protists and gut bacterial communities exhibit complex relationships, mediated at least in part through host immunity. To improve our understanding of this tripartite interplay, we investigated community and functional dynamics between the murine protist Tritrichomonas musculus and intestinal bacteria in healthy and B-cell-deficient mice. We identified dramatic, protist-driven remodeling of resident microbiome growth and activities, in parallel with Tritrichomonas musculus functional changes, which were accelerated in the absence of B cells. Metatranscriptomic data revealed nutrient-based competition between bacteria and the protist. Single-cell transcriptomics identified distinct Tritrichomonas musculus life stages, providing new evidence for trichomonad sexual replication and the formation of pseudocysts. Unique cell states were validated in situ through microscopy and flow cytometry. Our results reveal complex microbial dynamics during the establishment of a commensal protist in the gut, and provide valuable data sets to drive future mechanistic studies.

Crosstalk of platelets with macrophages and fibroblasts aggravates inflammation, aortic wall stiffening, and osteopontin release in abdominal aortic aneurysm.

AIMS: Abdominal aortic aneurysm (AAA) is a highly lethal disease with progressive dilatation of the abdominal aorta accompanied by degradation and remodelling of the vessel wall due to chronic inflammation. Platelets play an important role in cardiovascular diseases, but their role in AAA is poorly understood. METHODS AND RESULTS: The present study revealed that platelets play a crucial role in promoting AAA through modulation of inflammation and degradation of the extracellular matrix (ECM). They are responsible for the up-regulation of SPP1 (osteopontin, OPN) gene expression in macrophages and aortic tissue, which triggers inflammation and remodelling and also platelet adhesion and migration into the abdominal aortic wall and the intraluminal thrombus (ILT). Further, enhanced platelet activation and pro-coagulant activity result in elevated gene expression of various cytokines, Mmp9 and Col1a1 in macrophages and Il-6 and Mmp9 in fibroblasts. Enhanced platelet activation and pro-coagulant activity were also detected in AAA patients. Further, we detected platelets and OPN in the vessel wall and in the ILT of patients who underwent open repair of AAA. Platelet depletion in experimental murine AAA reduced inflammation and ECM remodelling, with reduced elastin fragmentation and aortic diameter expansion. Of note, OPN co-localized with platelets, suggesting a potential role of OPN for the recruitment of platelets into the ILT and the aortic wall. CONCLUSION: In conclusion, our data strongly support the potential relevance of anti-platelet therapy to reduce AAA progression and rupture in AAA patients.

FTO Gene Polymorphisms at the Crossroads of Metabolic Pathways of Obesity and Epigenetic Influences.

In this review, we summarize the current state of knowledge on the fat mass and obesity-associated (FTO) gene and its role in obesity. The FTO-encoded protein is involved in multiple molecular pathways contributing to obesity as well as other metabolic complexities. This review emphasizes the epigenetic influence on the FTO gene as a new approach in the treatment and management of obesity. Several known substances have a positive effect on reducing FTO expression. Depending on which variant of the single nucleotide polymorphism (SNP) is present, the profile and level of gene expression changes. Implementation of environmental change measures could lead to reduced phenotypic manifestation of FTO expression. Treating obesity through FTO gene regulation will have to include various complex signal pathways in which FTO takes part. Identification of FTO gene polymorphisms may be useful for the development of individual obesity management strategies, including the recommendation of taking certain foods and supplements.

The commensal protist Tritrichomonas musculus exhibits a dynamic life cycle that induces extensive remodeling of the gut microbiota.

Commensal protists and gut bacterial communities exhibit complex relationships, mediated at least in part through host immunity. To improve our understanding of this tripartite interplay, we investigated community and functional dynamics between the murine protist Tritrichomonas musculus ( T. mu ) and intestinal bacteria in healthy and B cell-deficient mice. We identified dramatic, protist-driven remodeling of resident microbiome growth and activities, in parallel with T. mu functional changes, accelerated in the absence of B cells. Metatranscriptomic data revealed nutrient-based competition between bacteria and the protist. Single cell transcriptomics identified distinct T. mu life stages, providing new evidence for trichomonad sexual replication and the formation of pseudocysts. Unique cell states were validated in situ through microscopy and flow cytometry. Our results reveal complex microbial dynamics during the establishment of a commensal protist in the gut, and provide valuable datasets to drive future mechanistic studies.

Myosin-X recruits lamellipodin to filopodia tips.

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

Role of extracellular matrix architecture and signaling in melanoma therapeutic resistance.

The extracellular matrix (ECM) is critical for maintaining tissue homeostasis therefore its production, assembly and mechanical stiffness are highly regulated in normal tissues. However, in solid tumors, increased stiffness resulting from abnormal ECM structural changes is associated with disease progression, an increased risk of metastasis and poor survival. As a dynamic and key component of the tumor microenvironment, the ECM is becoming increasingly recognized as an important feature of tumors, as it has been shown to promote several hallmarks of cancer via biochemical and biomechanical signaling. In this regard, melanoma cells are highly sensitive to ECM composition, stiffness and fiber alignment because they interact directly with the ECM in the tumor microenvironment via cell surface receptors, secreted factors or enzymes. Importantly, seeing as the ECM is predominantly deposited and remodeled by myofibroblastic stromal fibroblasts, it is a key avenue facilitating their paracrine interactions with melanoma cells. This review gives an overview of melanoma and further describes the critical roles that ECM properties such as ECM remodeling, ECM-related proteins and stiffness play in cutaneous melanoma progression, tumor cell plasticity and therapeutic resistance. Finally, given the emerging importance of ECM dynamics in melanoma, future perspectives on therapeutic strategies to normalize the ECM in tumors are discussed.

Toxoplasma infection in male mice alters dopamine-sensitive behaviors and host gene expression patterns associated with neuropsychiatric disease.

During chronic infection, the single celled parasite, Toxoplasma gondii, can migrate to the brain where it has been associated with altered dopamine function and the capacity to modulate host behavior, increasing risk of neurocognitive disorders. Here we explore alterations in dopamine-related behavior in a new mouse model based on stimulant (cocaine)-induced hyperactivity. In combination with cocaine, infection resulted in heightened sensorimotor deficits and impairment in prepulse inhibition response, which are commonly disrupted in neuropsychiatric conditions. To identify molecular pathways in the brain affected by chronic T. gondii infection, we investigated patterns of gene expression. As expected, infection was associated with an enrichment of genes associated with general immune response pathways, that otherwise limits statistical power to identify more informative pathways. To overcome this limitation and focus on pathways of neurological relevance, we developed a novel context enrichment approach that relies on a customized ontology. Applying this approach, we identified genes that exhibited unexpected patterns of expression arising from the combination of cocaine exposure and infection. These include sets of genes which exhibited dampened response to cocaine in infected mice, suggesting a possible mechanism for some observed behaviors and a neuroprotective effect that may be advantageous to parasite persistence. This model offers a powerful new approach to dissect the molecular pathways by which T. gondii infection contributes to neurocognitive disorders.

Understanding Australian methylamphetamine drug markets through relational, temporal and spatial analyses.

In the area of illicit drugs, forensic case data have proven effective at detecting links between seizures and providing greater insights into illicit drug markets. This research explored the application of mathematical and statistical techniques to several chemical profiles of Australian methylamphetamine seizures. The main aim was to create and deliver a method that would expand the use of illicit drug profiling for strategic intelligence purposes, contributing to the fight against illicit drug trafficking. The use of comparison metrics and clustering analysis to determine links between existing illicit drug specimens and subsequent new specimens was evaluated and automated. Relational, temporal and spatial analyses were subsequently used to gain an insight into illicit drug markets. Relational analysis identified clusters of seizures central to the network. Temporal analysis then provided insights into the behaviour of distribution markets, specifically the emergence and extinction of certain clusters of seizures over time. Spatial analysis aided the understanding of the inter-jurisdictional nature of illicit drug markets. These analyses allowed for the generation of strategic intelligence relating to when and where the Australian methylamphetamine illicit drug market was the most active. Additionally, the strategic level trends identified clusters of seizures that were worth investigating further. These clusters were explored through a case study, which exploited additional chemical profiling data to provide drug market knowledge at an operational level. In turn, the intelligence produced at various levels could allow relevant law enforcement agencies to take necessary measures in disrupting markets.

Chelidonium majus crude extract induces activation of peripheral blood mononuclear cells and enhances their cytotoxic effect toward HeLa cells.

The aim of the study was to examine the immunomodulatory effect of crude Chelidonium majus L ethanolic extract on ex vivo harvested peripheral blood mononuclear cells (PBMNCs). PBMNCs were isolated by density gradient centrifugation. The PBMNC cytotoxicity assay was performed with HeLa tumor cells as target cells. MTT assay was used to estimate the proliferation effect of extract and cytotoxic efficiency of treated PBMNCs. Flow cytometric analysis was used for immunophenotyping. Treatment induced moderate proliferative response, perturbation in PBMNC ratios, and the emergence of some unconventional subpopulations. The percentage ratio of double positive CD4+ and CD8+ T lymphocytes and monocytes, ratio of T and B lymphocytes expressing CD14, and percentage of NK cells expressing CD57 increased after treatment, indicating activation of PBMNC subpopulations. Cytotoxic activity against HeLa cells was enhanced. Activation of PBMNCs and enhancement of their cytotoxic effect toward HeLa cells indicate the immunostimulatory effect of Ch. majus ethanolic extract.

Targeting Discoidin Domain Receptors DDR1 and DDR2 overcomes matrix-mediated tumor cell adaptation and tolerance to BRAF-targeted therapy in melanoma.

Resistance to BRAF/MEK inhibitor therapy in BRAFV600 -mutated advanced melanoma remains a major obstacle that limits patient benefit. Microenvironment components including the extracellular matrix (ECM) can support tumor cell adaptation and tolerance to targeted therapy; however, the underlying mechanisms remain poorly understood. Here, we investigated the process of matrix-mediated drug resistance (MMDR) in response to BRAFV600 pathway inhibition in melanoma. We demonstrate that physical and structural cues from fibroblast-derived ECM abrogate anti-proliferative responses to BRAF/MEK inhibition. MMDR is mediated by drug-induced linear clustering of phosphorylated DDR1 and DDR2, two tyrosine kinase collagen receptors. Depletion and pharmacological targeting of DDR1 and DDR2 overcome ECM-mediated resistance to BRAF-targeted therapy. In xenografts, targeting DDR with imatinib enhances BRAF inhibitor efficacy, counteracts drug-induced collagen remodeling, and delays tumor relapse. Mechanistically, DDR-dependent MMDR fosters a targetable pro-survival NIK/IKKα/NF-κB2 pathway. These findings reveal a novel role for a collagen-rich matrix and DDR in tumor cell adaptation and resistance. They also provide important insights into environment-mediated drug resistance and a preclinical rationale for targeting DDR signaling in combination with targeted therapy in melanoma.

Micronutrient supplements can promote disruptive protozoan and fungal communities in the developing infant gut.

Supplementation with micronutrients, including vitamins, iron and zinc, is a key strategy to alleviate child malnutrition. However, association of gastrointestinal disorders with iron has led to ongoing debate over their administration. To better understand their impact on gut microbiota, we analyse the bacterial, protozoal, fungal and helminth communities of stool samples collected from a subset of 80 children at 12 and 24 months of age, previously enrolled into a large cluster randomized controlled trial of micronutrient supplementation in Pakistan (ClinicalTrials.gov identifier NCT00705445). We show that while bacterial diversity is reduced in supplemented children, vitamins and iron (as well as residence in a rural setting) may promote colonization with distinct protozoa and mucormycetes, whereas the addition of zinc appears to ameliorate this effect. We suggest that the risks and benefits of micronutrient interventions may depend on eukaryotic communities, potentially exacerbated by exposure to a rural setting. Larger studies are needed to evaluate the clinical significance of these findings and their impact on health outcomes.

Myosin-X and talin modulate integrin activity at filopodia tips.

Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin regulation in filopodia are poorly defined. Here, we report that active integrins accumulate at the tip of myosin-X (MYO10)-positive filopodia, while inactive integrins are uniformly distributed. We identify talin and MYO10 as the principal integrin activators in filopodia. In addition, deletion of MYO10's FERM domain, or mutation of its ß1-integrin-binding residues, reveals MYO10 as facilitating integrin activation, but not transport, in filopodia. However, MYO10's isolated FERM domain alone cannot activate integrins, potentially because of binding to both integrin tails. Finally, because a chimera construct generated by swapping MYO10-FERM by talin-FERM enables integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a two-step integrin activation model in filopodia: receptor tethering by MYO10 followed by talin-mediated integrin activation.

TBX3 Promotes Melanoma Migration by Transcriptional Activation of ID1, which Prevents Activation of E-Cadherin by MITF.

In melanoma, a phenotype switch from proliferation to invasion underpins metastasis, the major cause of melanoma-associated death. The transition from radial to vertical growth phase (invasive) melanoma is characterized by downregulation of both E-cadherin (CDH1) and MITF and upregulation of the key cancer-associated gene TBX3 and the phosphatidylinositol 3 kinase signaling pathway. Yet, whether and how these diverse events are linked remains poorly understood. Here, we show that TBX3 directly promotes expression of ID1, a dominant-negative regulator of basic helix-loop-helix transcription factors, and that ID1 decreases MITF binding and upregulation of CDH1. Significantly, we show that TBX3 activation of ID1 is necessary for TBX3 to enhance melanoma cell migration, and the mechanistic links between TBX3, ID1, MITF, and invasion revealed here are reflected in their expression in human melanomas. Our results reveal that melanoma migration is promoted through a TBX3-ID1-MITF-E-cadherin axis and that ID1-mediated repression of MITF activity may reinforce maintenance of an MITFLow phenotype associated with disease progression and therapy resistance.

Interpreting the link value of similarity scores between illicit drug specimens through a dual approach, featuring deterministic and Bayesian frameworks.

Illicit drug trafficking and in particular amphetamine-type stimulants continue to be a major problem in Australia. With the constant evolution of illicit drugs markets, it is necessary to gain as much knowledge about them to disrupt or reduce their impact. Illicit drug specimens can be analysed to generate forensic intelligence and understand criminal activities. Part of this analysis involves the evaluation of similarity scores between illicit drug profiles to interpret the link value. Most studies utilise one of two prominent score evaluation approaches, i.e. deterministic or Bayesian. In previous work, the notion of a dual approach was suggested, which emphasised the complementary nature of the two mentioned approaches. The aim of this study was to assess the operational capability of a dual approach in evaluating similarity scores between illicit drug profiles. Utilising a practical example, link values were generated individually from both approaches, then compared in parallel. As a result, it was possible to generate more informed hypotheses, relating to specimen linkage, due to the greater wealth of information available from the two approaches working concurrently. Additionally, it was shown that applying only one approach led to less information being generated during analysis as well as potentially important links between illicit drug specimens being missed.

Effect of ß-cyclodextrin encapsulation on cytotoxic activity of acetylshikonin against HCT-116 and MDA-MB-231 cancer cell lines.

Acetylshikonin (AcSh), as a red colored pigment found in roots of the plants from family Boraginaceae, showed excellent cytotoxic activity. Due to its hydrophobic nature, and thus poor bioavailability, the aim of this study was to prepare acetylshikonin/ß-cyclodextrin (AcSh/ß-CD) inclusion complex by using coprecipitation method, characterize obtained system by using UV/VIS, IR and 1H NMR spectroscopy, and determine cytotoxic activity. Phase solubility test indicated formation of AL-type binary system (substrate/ligand ratio was 1:1 M/M), with stability constant Ks of 306.01 M-1. Formation of noncovalent bonds between inner layer of the hole of ß-CD and AcSh was observed using spectroscopic methods. Notable changes in chemical shifts of two protons (-0.020 ppm) from naphthoquinone moiety (C6-H and C7-H), as well as protons from hydroxyl groups (-0.013 and -0.009, respectively) attached to C5 and C8 carbons from naphthoquinone part indicate that the molecule of AcSh enters the ß-CD cavity from the aromatic side. Cytotoxic activity against HCT-116 and MDA-MB-231 cell lines was measured by MTT test and clonogenic assay. Mechanisms of action of free AcSh and inclusion complex were assessed by flow cytometry. In comparison to free AcSh, AcSh/ß-CD showed stronger short-term effect on HCT-116 cells and superior long-term effect on both cell lines. Inclusion complex induced more pronounced cell cycle arrest and autophagy inhibition, and induced increase in accumulation of intracellular ROS more effectively than free AcSh. In conclusion, AcSh/ß-CD binary system showed better performances regarding cytotoxic activity against tested tumor cell lines.

Novel amino-functionalized lignin microspheres: High performance biosorbent with enhanced capacity for heavy metal ion removal.

Novel highly effective amino-functionalized lignin-based biosorbent in the microsphere geometry (A-LMS) for removal of heavy metal ions, was synthesized via inverse suspension copolymerization of kraft lignin with poly(ethylene imine) grafting-agent and epoxy chloropropane cross-linker. Optimization of A-LMS synthesis, performed with respect to the quantity of sodium alginate emulsifier (1, 5 and 10 wt%), provides highly porous microspheres A-LMS_5, using 5 wt% emulsifier, with 800 ± 80 µm diameter, 7.68 m2 g-1 surface area and 7.7 mmol g-1 of terminal amino groups. Structural and surface characteristics were obtained from Brunauer-Emmett-Teller method, Fourier Transform-Infrared spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy and porosity determination. In a batch test, the influence of pH, A-LMS_5 dose, temperature, contact time on adsorption efficiency of Ni2+, Cd2+, As(V) and Cr(VI) ions were studied. The adsorption is spontaneous and feasible with maximum adsorption capacity of 74.84, 54.20, 53.12 and 49.42 mg g-1 for Cd2+, Cr(VI), As(V) and Ni2+ ions, respectively, obtained by using Langmuir model. Modeling of kinetic data indicated fast adsorbate removal rate with pore diffusional transport as rate limiting step (pseudo-second order model and Weber-Morris equations), thus further confirming high performances of produced bio-adsorbent for heavy metal ions removal.

Review of the most common chemometric techniques in illicit drug profiling.

The information generated through drug profiling can be used to infer a common source between one or several seizures as well as drug trafficking routes to provide insights into drug markets. Although well established, it is time-consuming and ineffective to compare all drug profiles manually. In recent years, there has been a push to automate processes to enable a more efficient comparison of illicit drug specimens. Various chemometric methods have been employed to compare and interpret forensic case data promptly. The intelligence that is produced can be used by decision-makers to disrupt or reduce the impact of illicit drug markets. This review highlights the most common chemometric techniques used in drug profiling and more specifically, the most efficient comparison metrics and pattern recognition techniques outlined in the literature.