Methionine sulfoxide reductase 2 regulates Cvt autophagic pathway by altering the stability of Atg19 and Ape1 in Saccharomyces cerevisiae.

The reversible oxidation of methionine plays a crucial role in redox regulation of proteins. Methionine oxidation in proteins causes major structural modifications that can destabilize and abrogate their function. The highly conserved methionine sulfoxide reductases protect proteins from oxidative damage by reducing their oxidized methionines, thus restoring their stability and function. Deletion or mutation in conserved methionine sulfoxide reductases leads to aging and several human neurological disorders and also reduces yeast growth on nonfermentable carbon sources. Despite their importance in human health, limited information about their physiological substrates in humans and yeast is available. For the first time, we show that Mxr2 interacts in vivo with two core proteins of the cytoplasm to vacuole targeting (Cvt) autophagy pathway, Atg19, and Ape1 in Saccharomyces cerevisiae. Deletion of MXR2 induces instability and early turnover of immature Ape1 and Atg19 proteins and reduces the leucine aminopeptidase activity of Ape1 without affecting the maturation process of Ape1. Additonally, Mxr2 interacts with the immature Ape1, dependent on Met17 present within the propeptide of Ape1 as a single substitution mutation of Met17 to Leu abolishes this interaction. Importantly, Ape1 M17L mutant protein resists oxidative stress-induced degradation in WT and mxr2Δ cells. By identifying Atg19 and Ape1 as cytosolic substrates of Mxr2, our study maps the hitherto unexplored connection between Mxr2 and the Cvt autophagy pathway and sheds light on Mxr2-dependent oxidative regulation of the Cvt pathway.

Characterization of the transactivation and nuclear localization functions of Pichia pastoris zinc finger transcription factor Mxr1p.

The zinc finger transcription factor Mxr1p regulates the transcription of genes involved in methanol, acetate, and amino acid metabolism of the industrial yeast Pichia pastoris (a.k.a. Komagataella phaffii) by binding to Mxr1p response elements in their promoters. Here, we demonstrate that Mxr1p is a key regulator of ethanol metabolism as well. Using transcriptomic analysis, we identified target genes of Mxr1p that mediate ethanol metabolism, including ALD6-1 encoding an aldehyde dehydrogenase. ALD6-1 is essential for ethanol metabolism, and the ALD6-1 promoter harbors three Mxr1p response elements to which Mxr1p binds in vitro and activates transcription in vivo. We show that a nine-amino acid transactivation domain located between amino acids 365 and 373 of Mxr1p is essential for the transactivation of ALD6-1 to facilitate ethanol metabolism. Mxr1N250, containing the N-terminal 250 amino acids of Mxr1p, localized to the nucleus of cells metabolizing ethanol dependent on basic amino acid residues present between amino acids 75 and 85. While the N-terminal 400 amino acids of Mxr1p are sufficient for the activation of target genes essential for ethanol metabolism, the region between amino acids 401 and 1155 was also required for the regulation of genes essential for methanol metabolism. Finally, we identified several novel genes whose expression is differentially regulated by Mxr1p during methanol metabolism by DNA microarray. This study demonstrates that Mxr1p is a key regulator of ethanol metabolism and provides new insights into the mechanism by which Mxr1p functions as a global regulator of multiple metabolic pathways of P. pastoris.

Cellular expression and function of naturally occurring variants of the human ABCG2 multidrug transporter.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics; thus the relatively frequent polymorphic and mutant ABCG2 variants in the population may significantly alter disease conditions and pharmacological effects. Low-level or non-functional ABCG2 expression may increase individual drug toxicity, reduce cancer drug resistance, and result in hyperuricemia and gout. In the present work we have studied the cellular expression, trafficking, and function of nine naturally occurring polymorphic and mutant variants of ABCG2. A comprehensive analysis of the membrane localization, transport, and ATPase activity, as well as retention and degradation in intracellular compartments was performed. Among the examined variants, R147W and R383C showed expression and/or protein folding defects, indicating that they could indeed contribute to ABCG2 functional deficiency. These studies and the applied methods should significantly promote the exploration of the medical effects of these personal variants, promote potential therapies, and help to elucidate the specific role of the affected regions in the folding and function of the ABCG2 protein.

The PI3K subunits, P110α and P110ß are potential targets for overcoming P-gp and BCRP-mediated MDR in cancer.

BACKGROUND: PI3K/AKT is a vital signaling pathway in humans. Recently, several PI3K/AKT inhibitors were reported to have the ability to reverse cancer multidrug resistance (MDR); however, specific targets in the PI3K/AKT pathways and the mechanisms associated with MDR have not been found because many of the inhibitors have multiple targets within a large candidate protein pool. AKT activation is one presumed mechanism by which MDR develops during cancer treatment. METHODS: The effects of inhibiting PI3K 110α and 110ß by BAY-1082439 treatment and CRISPR/Cas9 knockout were examined to determine the possible functions of BAY-1082439 and the roles of PI3K 110α and 110ß in the reversal of MDR that is mediated by the downregulation of P-gp and BCRP. Inhibition of AKT with GSK-2110183 showed that the downregulation of P-gp and BCRP is independent of generalized AKT inactivation. Immunofluorescence, immunoprecipitation, MTT, flow cytometry and JC-1 staining analyses were conducted to study the reversal of MDR that is mediated by P-gp and BCRP in cancer cells. An ATPase assay and a structural analysis were also used to analyze the potential mechanisms by which BAY-1082439 specifically targets PI3K 110α and 110ß and nonspecifically influences P-gp and BCRP. RESULTS: By inhibiting the activation of the PI3K 110α and 110ß catalytic subunits through both the administration of BAY-1082439 and the CRISPR/Cas9 deletion of Pik3ca and Pik3cb, the ATP-binding cassette transporters P-gp/ABCB1 and BCRP/ABCG2 were downregulated, thereby reestablishing the drug sensitivity of human epidermoid carcinoma and non-small cell lung cancer (NSCLC) MDR cells. Inhibition of AKT did not reverse the MDR mediated by P-gp or BCRP. The ABC family proteins and AKT may play MDR-enhancing roles independently. CONCLUSIONS: The reversal of the dual functions of ABC-transporter-mediated and AKT-activation-enhanced MDR through the inhibition or knockout of PI3K 110α or 110ß promises to improve current strategies based on combined drug treatments to overcome MDR challenges.

Targeted editing of transcriptional activator MXR1 on the Pichia pastoris genome using CRISPR/Cas9 technology.

A highly efficient and targeted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system was constructed for Pichia pastoris (syn Komagataella phaffii). Plasmids containing single guide RNA and the methanol expression regulator 1 (MXR1) homology arms were used to precisely edit the transcriptional activator Mxr1 on the P. pastoris genome. At the S215 amino acid position of Mxr1, one, two, and three nucleotides were precisely deleted or inserted, and S215 was also mutated to S215A via a single-base substitution. Sequencing of polymerase chain reaction (PCR) amplicons in the region spanning MXR1 showed that CRISPR/Cas9 technology enabled efficient and precise gene editing of P. pastoris. The expression levels of several of the Mxr1-targeted genes, AOX1, AOX2, DAS1, and DAS2, in strains containing the various mutated variants of MXR1, were then detected through reverse transcription PCR following induction in methanol-containing culture medium. The frameshift mutations of Mxr1 led to almost zero transcription of AOX1, DAS1, and DAS2, while that of AOX2 was reduced to 60%. For the Mxr1 S215A mutant, the transcription of AOX1, AOX2, DAS1, and DAS2 was also reduced by nearly 60%. Based on these results, it is apparent that the transcription of AOX1, DAS1, and DAS2 is exclusively regulated by Mxr1 and serine phosphorylation at Mxr1 residue 215 is not critical for this function. In contrast, the transcription of AOX2 is mainly dependent on the phosphorylation of this residue. CRISPR/Cas9 technology was, therefore, successfully applied to the targeted editing of MXR1 on the P. pastoris genome, and it provided an effective method for the study of this transcription factor and its targets.

Physiological Genomics of Multistress Resistance in the Yeast Cell Model and Factory: Focus on MDR/MXR Transporters.

The contemporary approach of physiological genomics is vital in providing the indispensable holistic understanding of the complexity of the molecular targets, signalling pathways and molecular mechanisms underlying the responses and tolerance to stress, a topic of paramount importance in biology and biotechnology. This chapter focuses on the toxicity and tolerance to relevant stresses in the cell factory and eukaryotic model yeast Saccharomyces cerevisiae. Emphasis is given to the function and regulation of multidrug/multixenobiotic resistance (MDR/MXR) transporters. Although these transporters have been considered drug/xenobiotic efflux pumps, the exact mechanism of their involvement in multistress resistance is still open to debate, as highlighted in this chapter. Given the conservation of transport mechanisms from S. cerevisiae to less accessible eukaryotes such as plants, this chapter also provides a proof of concept that validates the relevance of the exploitation of the experimental yeast model to uncover the function of novel MDR/MXR transporters in the plant model Arabidopsis thaliana. This knowledge can be explored for guiding the rational design of more robust yeast strains with improved performance for industrial biotechnology, for overcoming and controlling the deleterious activities of spoiling yeasts in the food industry, for developing efficient strategies to improve crop productivity in agricultural biotechnology.

Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system.

The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1-a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter.

The methanol-regulated alcohol oxidase promoter (PAOX1 ) of Pichia pastoris (syn. Komagataella spp. ) is one of the strongest promoters for heterologous gene expression. Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harboring more than two copies of a Rhizopus oryzae lipase gene (ROL) have previously shown a decrease in cell growth, lipase production, and substrate consumption, as well as a significant transcriptional downregulation of methanol metabolism. This pointed to a potential titration effect of key transcriptional factors methanol expression regulator 1 (Mxr1) and methanol-induced transcription factor (Mit1) regulating methanol metabolism caused by the insertion of multiple expression vectors. To prove this hypothesis, a set of strains carrying one and four copies of ROL (1C and 4C, respectively) were engineered to coexpress one or two copies of MXR1*, coding for an Mxr1 variant insensitive to repression by 14-3-3 regulatory proteins, or one copy of MIT1. Small-scale cultures revealed that growth, Rol productivity, and methanol consumption were improved in the 4C-MXR1* and 4C-MIT1, strains growing on methanol as a sole carbon source, whereas only a slight increase in productivity was observed for re-engineered 1C strains. We further verified the improved performance of these strains in glycerol-/methanol-limited chemostat cultures.

Capacity of tissue water regulation is impaired in an osmoconformer living in impacted estuaries?

Estuarine osmoconformes rely on their ability to perform tissue and cell water regulation to cope with daily osmotic challenges that occur in the estuary. In addition, these animals currently must deal with pollutants present in the estuarine environment, which can disturb their capacity of water regulation. We collected the mangrove oyster Crassostrea rhizophorae in two tropical estuaries in the Northeast region of Brazil with different degrees of human interference: the Paraíba Estuary (impacted) and the Mamanguape Estuary (preserved). Tissue water content was analyzed after exposure to salinities 12, 24 and 36 for 24 h. Gill cell volume regulation was analyzed in vitro upon hypo- and hyper-osmotic conditions. We also analyzed gill MXR (multi-xenobiotic resistance) mechanism, as reference of environmental pollution. Gill and muscle of oysters from two sites of Paraíba Estuary, and from one site of Mamanguape Estuary were not able to maintain tissue water content upon hypo- and hyper-osmotic conditions. Gill cells of oyster from the same sites exhibited swelling followed by regulatory volume decrease upon hypo-osmotic condition. Gill MXR activity was increased in oysters from these sites. The best tissue and cell water regulation, and the lowest MXR activity, was found in oyster from downstream of Mamanguape Estuary, our reference site and the one most preserved. Tissue and cell water regulation proved to be a sensitive parameter to environmental pollution and could be considered as biomarker of aquatic contamination.

Transcription factor Mxr1 promotes the expression of Aox1 by repressing glycerol transporter 1 in Pichia pastoris.

In the methylotrophic yeast Pichia pastoris (P. pastoris), the efficient promoter of alcohol oxidase (PAox1) is induced by methanol and repressed by glycerol, but the molecular mechanism is not clear. In this study, the relationship between alcohol oxidase 1 (aox1), methanol expression regulator 1 (mxr1) and glycerol transporter 1 (gt1) was studied. By RT-PCR, it was found that the overexpression of gt1 could increase the glycerol content in cells and repress the expression of mxr1 and aox1, and the deletion of gt1 reduced the glycerol content in cells and promoted the expression of aox1. The overexpression of mxr1 could repress the expression of gt1, and the deletion of mxr1 could promote the expression of gt1 to some extent. By EMSA, Mxr1 binding sites were found in the promoter of gt1 (PGt1) (-141 to -138, CCCC), and Mxr1 could regulate the expression of gt1 by binding to PGt1. The relationships among aox1, mxr1 and gt1 revealed here provide a reference for the understanding of the mechanism of glycerol repression of PAox1.

ABCB1 and ABCC1-like transporters in immune system cells from sea urchins Echinometra lucunter and Echinus esculentus and oysters Crassostrea gasar and Crassostrea gigas.

ABC transporters activity and expression have been associated with the multixenobiotic resistance phenotype (MXR). The activity of these proteins leads to a reduction in the intracellular concentration of several xenobiotics, thus reducing their toxicity. However, little attention has been given to the expression of ABC transporters in marine invertebrates and few studies have investigated their role in immune system cells of sea urchins and shellfish bivalves. The aim of the present study was to investigate the activity of the ABC transporters ABCB1 and ABCC1 in immune system cells of sea urchins (coelomocytes) and oysters (hemocytes) from different climatic regions (Brazil and France). Sea urchins and oysters were collected at Paraíba coast; Brazil (Echinometra lucunter and Crassostrea gasar) and Rade of Brest; France (Echinus esculentus and Crassostrea gigas). Coelomocytes and hemocytes were stained with the ABC transporter substrate calcein-AM and dye accumulation analyzed under flow cytometry. Reversin 205 (ABCB1 transporter blocker) and MK571 (ABCC1 transporter blocker) were used as pharmacological tools to investigate ABC transporter activity. A different pattern of calcein accumulation was observed in coelomocytes: phagocytes > colorless spherulocytes > vibrate cells > red spherulocytes. The treatment with MK571 increased calcein fluorescence levels in coelomocytes from both species. However, reversin 205 treatment was not able to increase calcein fluorescence in E. esculentus coelomocytes. These data suggest that ABCC1-like transporter activity is present in both sea urchin species, but ABCB1-like transporter activity might only be present in E. lucunter coelomocytes. The activity of ABCC1-like transporter was observed in all cell types from both bivalve species. However, reversin 205 only increased calcein accumulation in hyalinocytes of the oyster C. gasar, suggesting the absence of ABCB1-like transporter activity in all other cell types, including hyalinocytes from the oyster C. gigas. Additionally, our results showed that C. gigas exhibited higher activity of ABCC1-like transporter in all hemocyte types than C. gasar. The present work is the first to characterize ABCB1 and ABCC1-like transporter activity in the immune system cells of sea urchins E. lucunter and E. esculentus and oysters. Our findings encourage the performing studies regarding ABC transporters activity/expression in immune system cells form marine invertebrates under stress conditions and the possible use of ABC transporters as biomarkers.

Mesodesma mactroides Gill Cells Exposed to Copper: Does Hyposmotic Saline Increase Cytotoxicity or Cellular Defenses?

Gill cells of filter feeding mollusks have cellular defense mechanisms, such as multixenobiotic resistance (MXR), that allow them to extrude possible contaminants. To analyze the cytotoxicity and cellular defenses of gills in the clam Mesodesma mactroides, gill cells were exposed to copper in both iso- and hyposmotic solutions. Analysis of MXR activity by fluorescence microscopy showed that hyposmotic saline activated defenses, whereas the presence of copper in isosmotic solution inhibited the activation of defenses. Cell viability was decreased in cells exposed to copper in isosmotic saline, but not in cells exposed to hyposmotic saline. We conclude that when cells cannot defend themselves due to decreased MXR, cell death occurs. In addition, gill cells under hyposmotic conditions have a greater capacity for defense and a lower rate of cellular mortality than when they are maintained under isosmotic conditions.

Assessment of domestic landfill leachate toxicity to the Asian clam Corbicula fluminea via biomarkers.

In order to evaluate the effects of domestic landfill leachate to bivalves Corbicula fluminea, clams were exposed to different leachate concentrations (v/v): 2, 3, 6 and 10 percent, corresponding to dilutions observed along a stream that receives this effluent, or only to clean water for comparisons. After 5 and 15 days of exposure the activity of the biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST), the multixenobiotic resistance mechanism (MXR) and lipid peroxidation (LPO) in gills and digestive gland and metallothionein (MT) content in gills were evaluated. Differences in biomarkers responses were observed between gills and digestive gland, except for MXR that decreased in both tissues of clams exposed to 6 percent for 5 days. EROD activity in gills was reduced in all leachate concentrations after 5 days and only in 2 percent after 15 days exposure, while an EROD increase was observed in digestive gland after 15 days exposure to 6 percent. GST activity increased only in the gills of clams exposed to 10 percent for 5 days. LPO varied between tissues and different conditions. A significant increase in LPO was observed in the gills, after 5 days exposure to 2 and 6 percent, and in digestive gland after 5 and 15 days exposure to 2 and 3 percent. MT content in the gills increased after 15 days exposure to 2 percent. In conclusion, different leachate concentrations tested here caused biochemical changes in C. fluminea, but due to the observed variability in biomarkers responses among leachate concentrations, it was difficult to determine patterns or thresholds concentrations.

Genotoxic and biochemical effects of atrazine and Roundup(®), alone and in combination, on the Asian clam Corbicula fluminea.

The present study aimed to evaluate biochemical and genotoxic effects of the herbicides atrazine (ATZ) and Roundup(®) (RD) separately, as well as their mixture, on the freshwater clam Corbicula fluminea after 96 h exposure. Animals were exposed to 2 and 10 ppb of ATZ (ATZ2 and ATZ10), 2 and 10 ppm of RD (RD2 and RD10) and the following mixtures: 2 ppb ATZ+2ppm RD (AR2) and 10 ppb ATZ+10 ppm RD (AR10). Activities of ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the multixenobiotic resistance mechanism (MXR), reduced glutathione concentrations (GSH) and lipid peroxidation (LPO) were measured in gills and digestive gland. DNA damage was determined in clams hemocytes through the comet assay. The gills were more susceptible to the action of the herbicides and the results showed that ATZ2 and ATZ10 caused a significant reduction in EROD and the mixture leads to a significant decrease in EROD and MXR. No significant change in the biotransformation parameters was observed in the digestive gland. Regarding the primary antioxidant defenses, SOD activity increased in the gills of clams exposed to ATZ10 and RD10 and in the digestive gland of animals exposed to RD2 and RD10, CAT activity was significantly reduced only in digestive gland of clams exposed RD10 while GPX increased in the gills after exposure to ATZ2 and RD10. The exposure to RD10 caused a significant increase in LPO in both gills and digestive gland. While the exposure to ATZ and RD separately did not increase DNA damage, the exposure to AR2 and AR10 caused a significant increase in the occurrence of DNA damage. In conclusion, this study showed that both herbicides applied alone caused effects on C. fluminea; ATZ interfered mostly in biotransformation while RD interfered mainly in antioxidant defenses leading to lipid peroxidation. The herbicides mixture showed antagonistic effects on the gills EROD and on lipid peroxidation in gills and digestive gland and synergistic effects on the gills MXR and on DNA damage in the hemocytes.

What Is Medical Extended Reality? A Taxonomy Defining the Current Breadth and Depth of an Evolving Field.

Medical extended reality (MXR) has emerged as a dynamic field at the intersection of health care and immersive technology, encompassing virtual, augmented, and mixed reality applications across a wide range of medical disciplines. Despite its rapid growth and recognition by regulatory bodies, the field lacks a standardized taxonomy to categorize its diverse research and applications. This American Medical Extended Reality Association guideline, authored by the editorial board of the Journal of Medical Extended Reality, introduces a comprehensive taxonomy for MXR, developed through a multidisciplinary and international collaboration of experts. The guideline seeks to standardize terminology, categorize existing work, and provide a structured framework for future research and development in MXR. An international and multidisciplinary panel of experts was convened, selected based on publication track record, contributions to MXR, and other objective measures. Through an iterative process, the panel identified primary and secondary topics in MXR. These topics were refined over several rounds of review, leading to the final taxonomy. The taxonomy comprises 13 primary topics that jointly expand into 180 secondary topics, demonstrating the field's breadth and depth. At the core of the taxonomy are five overarching domains: (1) technological integration and innovation; (2) design, development, and deployment; (3) clinical and therapeutic applications; (4) education, training, and communication; and (5) ethical, regulatory, and socioeconomic considerations. The developed taxonomy offers a framework for categorizing the diverse research and applications within MXR. It may serve as a foundational tool for researchers, clinicians, funders, academic publishers, and regulators, facilitating clearer communication and categorization in this rapidly evolving field. As MXR continues to grow, this taxonomy will be instrumental in guiding its development and ensuring a cohesive understanding of its multifaceted nature.

Bioaccumulation of algal toxins and changes in physiological parameters in Mediterranean mussels from the North Adriatic Sea (Italy).

The Northwestern Adriatic Sea is a commercially important area in aquaculture, accounting for about 90% of the Italian mussel production, and it was subjected to recurring cases of mussel farm closures due to toxic algae poisoning. A spatial and temporal survey of four sites along the North Adriatic Sea coasts of Emilia Romagna (Italy) was undertaken to study the possible impairments of physiological parameters in Mytilus galloprovincialis naturally exposed to algal toxins. The sites were selected as part of the monitoring network for the assessment of algal toxins bioaccumulation by the competent Authority. Samples positive to paralytic shellfish toxins and to lipophilic toxins were detected through the mouse bioassay. Lipophilic toxins were assessed by HPLC. Decreasing yessotoxins (YTX) levels were observed in mussels from June to December, while homo-YTX contents increased concomitantly. Lysosome membrane stability (LMS), glutathione S-transferase and catalase activities, and multixenobiotic resistance (MXR)-related gene expressions were assessed as parameters related to the mussel health status and widely utilized in environmental biomonitoring. Levels of cAMP were also measured, as possibly involved in the algal toxin mechanisms of action. Low LMS values were observed in hemocytes from mussels positive to the mouse bioassay. MXR-related gene expressions were greatly inhibited in mussels positive to the mouse bioassay. Clear correlations were established between increasing homo-YTX contents (and decreasing YTX) and increasing cAMP levels in the tissues. Similarly, significant correlations were established between the increase of homo-YTX and cAMP levels, and the expressions of three MXR-related genes at submaximal toxin concentrations. In conclusion, YTXs may affect mussel physiological parameters, including hemocyte functionality, gene expression and cell signaling.

Multixenobiotic defence mechanism in native and exotic freshwater snails as a biomarker for land uses-changes.

Human activities such as agriculture and urbanization generate a large number of substances like personal care products, pharmaceutical compounds, and pesticides, which often reach aquatic environments and represent a threat to biodiversity. Many organisms have developed different evolutionary strategies to remove pervasive substances from their bodies, allowing them to persist even in polluted environments, and one of these is the multixenobiotic resistance (MXR) mechanism associated with the expression of membrane proteins like P-glycoprotein (P-gp). Numerous chemical compounds with diverse functions and structures can modulate this mechanism, which can be employed as a pollution biomarker. We examined the MXR activity in two species of snails that inhabit Patagonian freshwaters. Functional assay measurements of MXR were conducted on the native Chilina dombeiana and the exotic Physella acuta in stream reaches affected by anthropogenic impacts. Results indicated that at agricultural sites, C. dombeiana snails had a more active MXR system than organisms sampled at reference and moderately disturbed urban sites, whereas P. acuta snails from agricultural and highly disturbed urban sites showed better detoxifying activity than organisms from reference sites. Only in exotic snails, part of this activity was due to the action of P-gp. The most important environmental variables explaining MXR activity were ammonium, nitrate and nitrite, phosphates, and electrical conductivity. These results show the promise of measuring MXR activity in native and exotic snails, as a biomarker in the environmental monitoring of Patagonian freshwaters.

Expression, Function and Trafficking of the Human ABCG2 Multidrug Transporter Containing Mutations in an Unstructured Cytoplasmic Loop.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354-367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357-360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter.

Implications of immersive technologies in healthcare sector and its built environment.

Objectives: This research focuses on how built environment experts can contribute to the MXR-enabled digital innovation as part of the multidisciplinary team effort to ensure post-pandemic resilience in healthcare built environment. The goal of this research is to help healthcare providers, built environment experts, and policy makers respectively: (1) Advocate the benefits of MXR for innovating health and social care; (2) Spark debate across networks of expertise to create health-promoting environment; and (3) Understand the overriding priorities in making effective pathways to the implementation of MXR. Methods: To highlight the novelty of this research, the study relies on two qualitative methodologies: exploratory literature review and semi-structured interviews. Based on the evaluation of prior works and cross-national case studies, hypotheses are formulated from three arenas: (1) Cross-sectional Initiatives for Post-pandemic Resilience; (2) Interoperability and Usability of Next-gen Medicines; and (3) Metaverse and New Forms of Value in Future Healthcare Ecosystems. To verify those hypotheses, empirical findings are derived from in-depth interviews with nine key informants. Results: The main findings are summarized under the following three themes: (1) Synergism between Architecture and Technology; (2) Patient Empowerment and Staff Support; and (3) Scalable Health and Wellbeing in Non-hospital and Therapeutic Settings. Firstly, both built environment and healthcare sectors can benefit from the various capabilities of MXR through cross-sectional initiatives, evidence-based practices, and participatory approaches. Secondly, a confluence of knowledge and methods of HCI and HBI can increase the interoperability and usability of MXR for the patient-centered and value-based healthcare models. Thirdly, the MXR-enabled technological regime will largely affect the new forms of value in healthcare premises by fostering more decentralized, preventive, and therapeutic characteristics in the future healthcare ecosystems. Conclusion: Whether it's virtual or physical, our healthcare systems have placed great emphasis on the rigor of evidence-based approach linking health outcome to a clinical environment. Henceforth, built environment experts should seek closer ties with the MXR ecosystems for the co-production of scalable health and wellbeing in non-hospital and therapeutic settings. Ultimately, this is to improve resource efficiency in the healthcare sector while considering the transition of health resources towards in silico status by increasing the implementation of MXR.

Deletion analysis of Pichia pastoris alcohol dehydrogenase 2 (ADH2) promoter and development of synthetic promoters.

Pichia pastoris (Komagataella phaffii) is a non-conventional Crabtree-negative yeast with the capability of reaching very high cell densities in a fed-batch fermentation process. The alcohol dehydrogenase (ADH) genes of P. pastoris involved in ethanol metabolism were identified and were previously characterized. This work aimed to extend current knowledge of the regulation of the ADH2 promoter. To this end, we first determined the upstream activator (UAS) and repressor (URS) sequences of the promoter by deletion assays. Two upstream activator sites have been identified, positioned between -900 and -801 bp, and -284 and -108 bp upstream of the ADH2 transcription start site. The sequences positioned between -361 and -262 bp had a negative effect on the promoter activity and designated a repressor sequence (URS). We then demonstrated that Mxr1 (methanol expression regulator 1) transcription factor activates the ADH2 promoter through the direct interaction with UAS regions in response to ethanol. Furthermore, five different synthetic promoters were constructed by adding or deleting the regulatory sites. These synthetic promoters were tested for extracellular xylanase production at shake flask level by inducing with ethanol. These promoter variants improved the xylanase production ranging between 165% and 200% of the native promoter. The synthetic promoter 5 (SNT5) that displayed the highest activity was further evaluated at the fermenter scale. The modification in the promoter features might have several implications for industrial processes where decoupling the cell growth and product formation is advantageous.