Extracellular Uptake Research Articles

Discovery and SAR analysis of phenylbenzo[d][1,3]dioxole-based proprotein convertase subtilisin/kexin type 9 inhibitors

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel therapeutic target for the development of cholesterol-lowering drugs. In the discovery of PCSK9/LDLR (low-density lipoprotein receptor) protein-protein interaction (PPI) impairing small molecules, a total of 47 phenylbenzo[d][1,3] dioxole-based compounds were designed and synthesised. The result revealed that the 4-chlorobenzyl substitution in the amino group is important for the PPI disrupting activity. In the hepatocyte-based functional tests, active compounds such as A12, B1, B3, B4 and B14, restored the LDLR levels on the surface of hepatic HepG2 cells and increased extracellular LDL uptake in the presence of PCSK9. It is notable that molecule B14 exhibited good performance in all the evaluations. Collectively, novel structures targeting PCSK9/LDLR PPI have been developed with hypolipidemic potential. Further structural modification of derived active compounds is promising in the discovery of lead compounds with improved activity for the treatment of hyperlipidaemia-related disorders.

The Role of Citrate Homeostasis in Merkel Cell Carcinoma Pathogenesis.

Simple SummaryMerkel cell carcinoma (MCC) is a rare but highly aggressive skin cancer. Despite important progress, overall understanding of the events that drive MCC carcinogenesis remains incomplete. We discovered that the plasma membrane citrate transporter (pmCiC) is upregulated in Merkel cell carcinoma cell lines. Cancer cells import extracellular citrate via pmCiC to support their metabolism, which is critical to support proliferation and metastatic spread. In this study, we show that inhibition of pmCiC can decrease the growth rate of Merkel cell carcinoma cell lines. Targeting pmCiC and thereby the tumor metabolism should be considered further as a potential anti-cancer therapy.Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin with a poor prognosis. The factors driving this cancer must be better understood in order to discover novel targets for more effective therapies. In the search for targets, we followed our interest in citrate as a central and critical metabolite linked to fatty acid synthesis in cancer development. A key to citrate uptake in cancer cells is the high expression of the plasma membrane citrate transporter (pmCiC), which is upregulated in the different adenocarcinoma types tested so far. In this study, we show that the pmCiC is also highly expressed in Merkel cell carcinoma cell lines by western blot and human tissues by immunohistochemistry staining. In the presence of extracellular citrate, MCC cells show an increased proliferation rate in vitro; a specific pmCiC inhibitor (Na+-gluconate) blocks this citrate-induced proliferation. Furthermore, the 3D in vivo Chick Chorioallantoic Membrane (CAM) model showed that the application of Na+-gluconate also decreases Merkel cell carcinoma growth. Based on our results, we conclude that pmCiC and extracellular citrate uptake should be considered further as a potential novel target for the treatment of Merkel cell carcinoma.

Abstract 6167: Pancreatic tumor environmental nutrients alter SREBP-mediated lipid homeostasis and drive reliance on extracellular lipids for growth

Abstract Both the cell-intrinsic metabolic network and the uniquely altered cell-extrinsic tumor microenvironment dictate tumor metabolism. However, while much is known about how cell-intrinsic factors regulate tumor metabolism, the effects of cell-extrinsic factors remain poorly understood. In particular, despite evidence that tumor nutrient availability extrinsically regulates tumor metabolism, further investigation has been hindered by the lack of models to study physiological nutrient availability in vitro.To accurately model the tumor nutrient microenvironment, our group quantified nutrient availability in pancreatic ductal adenocarcinoma (PDAC) tumor perfusate and created a medium to mimic this in vitro, called tumor interstitial fluid medium (TIFM). We compare the effects of TIFM-culture to maintenance in conventional culture medium (RPMI-1640) as a control for standard laboratory conditions. Using this model, I found that TIFM-cultured PDAC cells downregulate lipid synthesis, making TIFM-cultured cells uniquely reliant upon extracellular lipid uptake for growth. Analysis of murine PDAC tumors confirms downregulated lipogenesis in vivo, suggesting cell-extrinsic nutrients render lipogenesis dispensable—and lipid uptake necessary—for PDAC tumor growth. Notably, this contrasts studies in conventional culture media that claim lipogenesis is critical for PDAC growth.I am currently working to: (1) understand how the nutrient microenvironment alters lipid metabolism and (2) determine the extracellular lipids and transporters pancreatic cancer cells require for growth. Regarding the first question, transcriptomic analysis of TIFM-cultured cells revealed significant deactivation of SREBPs, master regulators of intracellular lipid and sterol synthesis, suggesting cell-extrinsic nutrient cues suppress lipogenesis by altering SREBP signaling. I am currently investigating which TIFM nutrients alter this signaling. I am also performing lipidomics analysis and lipid transporter knockdown experiments to identify lipids and transporters that TIFM-cultured cells require for growth. Collectively, these experiments will clarify the role extracellular nutrients play in driving changes to PDAC lipid metabolism and may suggest novel therapeutic treatment options for PDAC patients. Citation Format: Patrick Jonker, Alexander Muir. Pancreatic tumor environmental nutrients alter SREBP-mediated lipid homeostasis and drive reliance on extracellular lipids for growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6167.

Abstract LB126: Functional limitations in respiratory complex I underlie mitochondrial bioenergetics in human colorectal adenocarcinoma

Abstract Typified by oxidative phosphorylation (OXPHOS), mitochondria catalyze a wide variety of cellular processes now understood to be critical for malignant growth. As such, there is considerable interest in targeting mitochondrial OXPHOS in cancer. The present project was based on the premise that the development of efficacious mitochondrial-targeted anti-cancer compounds requires answering two fundamental questions: 1) is mitochondrial bioenergetics in fact different between cancer and non-cancer cells? and 2) If so, what are the underlying mechanisms? To address these questions, we developed a methodological workflow designed to integrate in vitro functional readouts of mitochondrial flux with mass-spectrometry-based proteomics. This allowed us to directly quantitate both total respiratory capacity and OXPHOS kinetics on a per mitochondrion basis. Experiments were performed in isolated mitochondria prepared from freshly resected human colorectal adenocarcinomas (COAD). Relative to adjacent normal colonic mucosa, tumor mitochondria consistently presented with partial disruptions in respiratory complex I (CI). Surprisingly, CI functional limitations did not impair absolute oxidative flux. Instead, the tumor mitochondrial network displayed clear evidence of intrinsic remodeling. Such remodeling maintained respiratory competence at the expense of decreased oxidative ATP synthesis (i.e., OXPHOS inefficiency or increased ‘uncoupling’). Given recent findings demonstrating that mitochondrial OXPHOS is dispensable for tumor growth, our data raise the intriguing possibility that the requirement for mitochondria in COAD may have little to do with oxidative ATP production, but instead reflect a requirement for accelerated mitochondrial flux to support other aspects of anabolic growth (e.g., extracellular nutrient uptake, NAD+ regeneration, nucleotide synthesis). In this respect, rather than functioning as energy producers, COAD mitochondria may primarily function as ‘energy sinks’. Citation Format: Ilya Boykov, McLane M. Montgomery, James T. Hagen, Hannah S. Coalson, Kelsey H. Fisher-Wellman. Functional limitations in respiratory complex I underlie mitochondrial bioenergetics in human colorectal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB126.

Adenosine receptor signaling regulates effector T cell activation through the limitation of aerobic glucose metabolism.

Abstract Background In a mouse model of colitis, we previously reported the protective role of the adenosine axis on effector T (Teff) cell responses and disease progression. Inflammatory reactions are paired with cell metabolic reprogramming from oxidative phosphorylation (OXPHOS) to aerobic glycolysis which is also a hallmark of T cell activation. Thus, we sought to evaluate the role of the adenosine 2A receptor (A2AR) signaling in the regulation of T cell metabolism. Methods Teff cells were reactivated in the presence or absence of A2AR agonists. Gene expression of mTOR, HIF-1α and cMyc (transcriptional factors of glycolysis-related genes) were quantified by qPCR. T cell metabolic phenotype was evaluated by transcriptomic profiling (Nanostring nCounter® Metabolic Pathway Panel), as well as real-time extracellular flux analysis, glucose uptake, and ATP production assays. Results mTOR, HIF-1α and cMyc gene expression increased as soon as 2hr post stimulation. At this time point, transcriptomic analysis revealed an A2AR agonist-induced downregulation of pathways related to T cell activation (i.e. mTOR, MAPK, PI3K, NFκB and cMyc pathways) and energy metabolism (i.e glycolysis, autophagy, fatty acid synthesis) whereas oxidative metabolism pathways (OXPHOS, fatty acid oxidation, glutamine metabolism) were upregulated (Pathway Score q-value <0.05). Functional assays showed a decreased de novo glucose uptake and glycolysis rate after A2AR agonist treatment while OXPHOS capacity and ATP production were not modulated. Conclusion A2AR signaling provokes a rewiring of the activated T cell metabolism towards an oxidative state making adenosine a potential T cell metabolic regulator that can be exploited in the treatment of IBD. Supported by NIH R01AI079145

Extracellular Alpha-Synuclein: Mechanisms for Glial Cell Internalization and Activation.

Alpha-synuclein (α-syn) is a small protein composed of 140 amino acids and belongs to the group of intrinsically disordered proteins. It is a soluble protein that is highly expressed in neurons and expressed at low levels in glial cells. The monomeric protein aggregation process induces the formation of oligomeric intermediates and proceeds towards fibrillar species. These α-syn conformational species have been detected in the extracellular space and mediate consequences on surrounding neurons and glial cells. In particular, higher-ordered α-syn aggregates are involved in microglial and oligodendrocyte activation, as well as in the induction of astrogliosis. These phenomena lead to mitochondrial dysfunction, reactive oxygen and nitrogen species formation, and the induction of an inflammatory response, associated with neuronal cell death. Several receptors participate in cell activation and/or in the uptake of α-syn, which can vary depending on the α-syn aggregated state and cell types. The receptors involved in this process are of outstanding relevance because they may constitute potential therapeutic targets for the treatment of PD and related synucleinopathies. This review article focuses on the mechanism associated with extracellular α-syn uptake in glial cells and the consequent glial cell activation that contributes to the neuronal death associated with synucleinopathies.

Nickel ion extracellular uptake by the phototrophic bacterium Rhodobacter sphaeroides: new insights from Langmuir modelling and X-ray photoelectron spectroscopic analysis

The presence of anionic functional groups on the surface of microbial cells makes the bacterial biomass very attractive as metal cation removal tool for environmental bioremediation. To go insight to the heavy metal sorption abilities of Gram-negative bacteria, we characterized the extracellular uptake of Ni2+ ions by the phototrophic bacterium Rhodobacter sphaeroides, whose ability to sequester toxic metal ions has been widely documented. The sorption process proved to be well described by the Langmuir model, suggesting that the cell envelope behaves as an ideal adsorbent surface, showing a layer of chemically equivalent binding sites with high affinity for Ni2+. X-ray photoelectron spectroscopic data reveal that, at binding site saturation, Ni2+ ions fully displace alkali metal ions from the bacterial surface, while they are unable to displace Ca2+ and Mg2+ from their tight binding sites. The analysis of carbon, nitrogen and oxygen speciation, as arisen from high resolution spectra fitting, highlights that the Ni2+/K+ cation exchange affects the surface distribution of main chemical components, with increased contribution by peptides and phosphatidylcholines. A role of Ni2+ ions in favoring the stacking and/or packing of lipopolysaccharide sugar chains by interaction with carboxylate groups is proposed and discussed.

Circ_0072008, an oncogene in pancreatic ductal adenocarcinoma, contributes to tumour cell malignant progression and glycolysis by regulating miR-545-3p/SLC7A11 axis

Background The hsa_circRNA_103809 (circ_0072088) has been an emerging tumour regulator in human cancers, and is identified as one most aberrantly expressed circRNA in patients with pancreatic ductal adenocarcinoma (PDAC). However, the role of circ_0072088 remains unclear in PDAC cells. Methods Expression of circ_0072088, microRNA (miR)-545-3p and solute carrier family 7 member 11 (SLC7A11) was detected by real-time quantitative PCR and western blotting. Cell progression was measured by cell counting kit (CCK)-8 assay, transwell assays and flow cytometry, as well as xenograft tumour models. Glycolysis was evaluated by commercial assay kits. The interaction among circ_0072088, miR-545-3p and SLC7A11 was confirmed by dual-luciferase reporter assay. Results Circ_0072088 was upregulated in PDAC tumours and cells; besides, high circ_0072088 level was associated with high tumour-node-metastasis (TNM) stage. The circ_0072088 siRNA suppressed cell viability, migration, invasion, extracellular acidification rate (ECAR), lactate production, glucose uptake, and ATP generation, but promoted apoptosis rate and oxygen consumption rate (OCR) in SW1990 and PANC-1 cells. In vivo, circ_0072088 knockdown retarded tumour growth of PANC-1 cells. Overexpressing miR-545-3p mimicked circ_0072088 siRNA-induced actions, and inhibited cell progression and glycolysis of SW1990 and PANC-1 cells. Moreover, SLC7A11 downregulation could be mediated by both circ_0072008 siRNA and miR-545-3p mimic, and participating in suppressive role in cell progression and glycolysis of SW1990 and PANC-1 cells. In mechanism, miR-545-3p was targeted by circ_0072008, and SLC7A11 was target of miR-545-3p. Conclusion Circ_0072088 elicited oncogenic role in malignant cell progression and glycolysis of PDAC cells through circ_0072088/miR-545-3p/SLC7A11 pathway. Highlights Circ_0072088 was upregulated in PDAC tumours and was associated with high tumour burden. Blocking circ_0072088 suppressed cell proliferation, migration, invasion, and glycolysis in PDAC cells. Circ_0072088 could directly regulate miR-545-3p, and SLC7A11 was a target of miR-545-3p. Restoring miR-545-3p mimicked the effects of circ_0072088 knockdown in PDAC cell in vitro.

Pb(II)-phycoremediation mechanism using Scenedesmus obliquus: cells physicochemical properties and metabolomic profiling

This study highlights the mechanisms of Pb(II)-phycoremediation using the Pb(II) tolerant strain of Scenedesmus obliquus. First, monitoring of cell growth kinetics in control and Pb(II)-doped medium revealed significant growth inhibition, while the analyses through flow cytometry and Zetasizer revealed no difference in cell viability and size. Residual weights of control and Pb(II)-loaded cells assessed by thermogravimetric analysis were 31.34% and 57.8%, respectively, indicating the uptake of Pb(II) into S. obliquus cells. Next, the use of chemical extraction to distinguish between the intracellular and extracellular uptake indicated the involvement of both biosorption (85.5%) and bioaccumulation (14.5%) mechanisms. Biosorption interaction of Pb(II) ions and the cell wall was confirmed using SEM-EDX, FTIR, zeta potential, zero-charge pH, and contact angle analyses. Besides, the biochemical characterization of control and Pb(II)-loaded cells revealed that the bioaccumulation of Pb(II) induces significant increases in the carotenoids and lipids content, while it decreases in the chlorophyll, carbohydrates, and proteins content. Finally, the metabolomic analysis indicated an increase in the relative abundance of fatty acid methyl esters, alkanes, aromatic compounds, and sterols. However, the alkenes and monounsaturated fatty acids decreased. Such metabolic adjustment may represent an adaptive strategy that prevents high Pb(II)-bioaccumulation in cellular compartments.

A Simple and Rapid Bioluminescence-Based Functional Assay of Organic Anion Transporter 1 as a D-Luciferin Transporter.

Organic anion transporter 1 (SLC22A6/OAT1) plays a key role in renal tubular excretion of endo- and exogenous anionic substances including drugs. Since the inhibition of OAT1 function by a concomitant drug may cause pharmacokinetic drug-drug interactions (DDIs) in clinical practice, an in vitro uptake study to evaluate the inhibition potency of OAT1 is useful for the prediction and avoidance of DDIs and recommended for drug candidates in drug development. In this chapter, we describe a rapid and highly sensitive functional assay of OAT1 based on bioluminescence (BL) detection using D-luciferin as a substrate in living cells. The principle of measurement simply relies on the biochemical feature of D-luciferin to be recognized as a substrate of OAT1, and the BL intensity depending on intracellular D-luciferin level and luciferase activity, thereby allowing the quantitative analysis of OAT1-mediated D-luciferin transport. The BL measurement can be completed within 1min without experimental procedures for removing extracellular uptake solution and washing cells, both of which involve in the conventional uptake studies using isotope-labeled or fluorescent compounds. The present method is applicable to high-throughput screening to identify and avoid potential OAT1 inhibitors in drug development.

Knockdown of OLR1 weakens glycolytic metabolism to repress colon cancer cell proliferation and chemoresistance by downregulating SULT2B1 via c-MYC

Chemoresistance is one of the major problems of colon cancer treatment. In tumors, glycolytic metabolism has been identified to promote cell proliferation and chemoresistance. However, the molecular mechanisms underlying glycolytic metabolism and chemoresistance in colon cancer remains enigmatic. Hence, this research was designed to explore the mechanism underlying the OLR1/c-MYC/SULT2B1 axis in the regulation of glycolytic metabolism, to affect colon cancer cell proliferation and chemoresistance. Colon cancer tissues and LoVo cells were attained, where OLR1, c-MYC, and SULT2B1 expression was detected by immunohistochemistry, RT-qPCR, and western blot analysis. Next, ectopic expression and knockdown assays were implemented in LoVo cells. Cell proliferation was detected by MTS assay and clone formation. Extracellular acidification, glucose uptake, lactate production, ATP/ADP ratio, and GLUT1 and LDHA expression were measured to evaluate glycolytic metabolism. Then, the transfected cells were treated with chemotherapeutic agents to assess drug resistance by MTS experiments and P-gp and SMAD4 expression by RT-qPCR. A nude mouse model of colon cancer transplantation was constructed for in vivo verification. The levels of OLR1, c-MYC, and SULT2B1 were upregulated in colon cancer tissues and cells. Mechanistically, OLR1 increased c-MYC expression to upregulate SULT2B1 in colon cancer cells. Moreover, knockdown of OLR1, c-MYC, or SULT2B1 weakened glycolytic metabolism, proliferation, and chemoresistance of colon cancer cells. In vivo experiments authenticated that OLR1 knockdown repressed the tumorigenesis and chemoresistance in nude mice by downregulating c-MYC and SULT2B1. Conclusively, knockdown of OLR1 might diminish SULT2B1 expression by downregulating c-MYC, thereby restraining glycolytic metabolism to inhibit colon cancer cell proliferation and chemoresistance.

Co-option of an extracellular protease for transcriptional control of nutrient degradation in the fungus Aspergillus nidulans

Nutrient acquisition is essential for all organisms. Fungi regulate their metabolism according to environmental nutrient availability through elaborate transcription regulatory programs. In filamentous fungi, a highly conserved GATA transcription factor AreA and its co-repressor NmrA govern expression of genes involved in extracellular breakdown, uptake, and metabolism of nitrogen nutrients. Here, we show that the Aspergillus nidulans PnmB protease is a moonlighting protein with extracellular and intracellular functions for nitrogen acquisition and metabolism. PnmB serves not only as a secreted protease to degrade extracellular nutrients, but also as an intracellular protease to control the turnover of the co-repressor NmrA, accelerating AreA transcriptional activation upon nitrogen starvation. PnmB expression is controlled by AreA, which activates a positive feedback regulatory loop. Hence, we uncover a regulatory mechanism in the well-established controls determining the response to nitrogen starvation, revealing functional evolution of a protease gene for transcriptional regulation and extracellular nutrient breakdown.

In Vivo crispr Screening Identifies GLUT1 As a Key Metabolic Regulator of MLL-AF9-Driven Acute Myeloid Leukemia

Disease relapse in patients with acute myeloid leukemia (AML) is associated with a failure of current treatments to eradicate leukemia stem cells (LSCs), a self-renewing population of cells responsible for disease progression and maintenance. Thus, novel therapeutic strategies designed to specifically target LSCs while sparing normal hematopoietic stem cells are needed. To identify dependencies in LSCs that may reveal new treatment opportunities, we performed an in vivo CRISPR/Cas9 dropout screen in the widely used MLL-AF9-driven AML murine model. The pooled lentiviral CRISPR library was designed to target 960 genes encoding cell surface proteins expressed on MLL-AF9 AML cells as these are accessible for therapeutic targeting.The facilitated glucose transporter member 1(GLUT1), a major mediator of cellular glucose uptake, emerged as the highest ranked dependency in the screen, with all 6 sgRNAs depleted more than 10-fold in vivo. Consistent with the results from the screen, validation experiments confirmed that sgRNA-mediated GLUT1 disruption in c-Kit +Cas9 +dsRed +MLL-AF9 cells led to a 5-fold reduction in the establishment of leukemia in both the bone marrow and spleen of recipient mice. In line with these in vivo observations, leukemia cells expressing GLUT1 sgRNAs were rapidly depleted over time in an ex vivo competition assay (p<0.0001). GLUT1 disruption also led to a marked increase in mean survival from 28 to 73 days in mice transplanted with sorted GLUT1 sgRNA-expressing leukemia cells relative to controls. Notably, while GLUT1 loss did not affect apoptosis or cell-cycle state, it led to a more than two-fold increase in the surface expression of the myeloid differentiation marker Gr-1 (p=0.0002). Interestingly, knockdown of GLUT1 lead to reduced mRNA expression levels of key downstream genes of MLL-driven leukemia Meis1 (p<0.0001) and Hoxa9 (p=0.0013) , both of which are commonly downregulated upon differentiation. These findings suggest that GLUT1 ablation arrests AML cell growth at least in part via accelerated differentiation and attenuated cell proliferation.Given GLUT1-mediated glucose transfer constitutes the first rate-limiting step for glucose metabolism, we assessed the metabolic profile of MLL-AF9 AML cells following loss of GLUT1. Bioenergetic profiling revealed that the rate of glycolysis was significantly decreased upon GLUT1 knockdown, as measured by a decrease in extracellular acidification rate (ECAR), glucose uptake, hexokinase activity and extracellular lactate production.To further assess the feasibility of GLUT1 inhibition as a therapy for AML patients, we treated murine cKit +MLL-AF9 leukemia cells with BAY-876, a potent and highly selective GLUT1 inhibitor. BAY-876 impaired tumor growth following 24hr (IC 50 60.3 nM) and 48hr (IC 50 68.8 nM) treatment ex vivo in a dose-dependent manner. Interestingly, the inhibitory effect on the counterpart healthy bone marrow c-Kit + cells was significantly weaker (24hr IC 50 347.7 nM; 48hr IC 50 258.4nM), indicating selective targeting of LSCs. To test the efficacy of BAY-876 as an anti-leukemic agent in vivo, sublethally irradiated mice were transplanted with c-Kit +MLL-AF9 AML cells and 3 days post-injection, were randomised into two groups (Veh n=4; BAY-876 n=6) and orally treated with either vehicle or 4mg/kg of BAY-876 daily. Following 10 days of treatment, mice were sacrificed and leukemia burden was assessed. Notably, substantially lower levels of leukemia cells in the bone marrow (p=0.0095), spleen (p=0.0095), and peripheral blood (p=0.036) were observed in the BAY-876 treatment group with no significant loss of body weight. Consistent with these findings, the average spleen weight was reduced by 66% upon BAY-876 treatment (p=0.0136).Collectively, we demonstrate that MLL-AF9-driven AML cells are dependent on GLUT1 for continued growth and survival. Targeting of GLUT1 downregulates glycolysis and induces cellular differentiation. We report that genetic or pharmacological inhibition of GLUT1 is sufficient to impair leukemic growth in vitro and in vivo, highlighting a potential therapeutic opportunity for disarming intrinsic metabolic dependencies of LSCs. Ongoing studies are aimed at translating these findings to the human disease and exploring combinatorial therapies that may act synergistically to overcome mechanisms of therapy resistance and metabolic plasticity. DisclosuresNo relevant conflicts of interest to declare.

Transcriptional Profile of the Industrial Hybrid Saccharomyces pastorianus Reveals Temperature-Dependent Allele Expression Bias and Preferential Orthologous Protein Assemblies.

Saccharomyces pastorianus is a natural yeast evolved from different hybridization events between the mesophilic S. cerevisiae and the cold-tolerant S. eubayanus. This complex aneuploid hybrid carries multiple copies of the parental alleles alongside specific hybrid genes and encodes for multiple protein isoforms which impart novel phenotypes, such as the strong ability to ferment at low temperature. These characteristics lead to agonistic competition for substrates and a plethora of biochemical activities, resulting in a unique cellular metabolism. Here, we investigated the transcriptional signature of the different orthologous alleles in S. pastorianus during temperature shifts. We identified temperature-dependent media-independent genes and showed that 35% has their regulation dependent on extracellular leucine uptake, suggesting an interplay between leucine metabolism and temperature response. The analysis of the expression of ortholog parental alleles unveiled that the majority of the genes expresses preferentially one parental allele over the other and that S. eubayanus-like alleles are significantly over-represented among the genes involved in the cold acclimatization. The presence of functionally redundant parental alleles may impact on the nature of protein complexes established in the hybrid, where both parental alleles are competing. Our expression data indicate that the majority of the protein complexes investigated in the hybrid are likely to be either exclusively chimeric or unispecific and that the redundancy is discouraged, a scenario that fits well with the gene balance hypothesis. This study offers the first overview of the transcriptional pattern of S. pastorianus and provides a rationalization for its unique industrial traits at the expression level.

Sex-related Differences in Glial Fibrillary Acidic Protein-positive GABA Regulate Neuropathology Following Pilocarpine-induced Status Epilepticus

Status epilepticus (SE) is a life-threatening neurological disorder that causes neuronal death and glial activation. Studies have explained the clinical side effects and lack of effectiveness of neurological disorder treatments based on sex-related differences in brain structure and function. However, the sex-specific outcomes of seizure disorders and the underlying mechanisms remain unknown. We compared SE-induced behavioral and pathophysiological changes in male and female mice. The time taken to reach stage 6 seizure following pilocarpine injection was shorter in male mice than in female mice, and the prevalence of SE was higher in male mice than in female mice. Fluoro-Jade B staining revealed more extensive SE-induced hippocampal neuronal death in male mice than in female mice. Glial cells were more activated in male mice than in female mice. In contrast, astrocyte-derived γ-aminobutyric acid (GABA)-immunostaining was less expressed in male mice than in female mice. Moreover, the mRNA levels of inflammatory cytokines released from activated glial cells were higher in male mice than in female mice. Notably, the mRNA level of astrocytic γ-aminobutyric acid transporter (GAT-3) involved in extracellular GABA uptake was lower in female mice than in male mice, while the mRNA levels of glutamate/aspartate transporter (GLAST (EAAT1)) and glutamate transporter (GLT-1 (EAAT2)) involved in extracellular glutamate uptake were higher in female mice. Our findings suggest that male mice are more vulnerable to SE than female mice, resulting in more extensive neuronal cell death and glial activation in male mice, partly due to increased GAT-3 expression that subsequently leads to reduced glial fibrillary acidic protein (GFAP)-positive GABA content assessed with anti-GABA antibodies.

A myo-inositol bioassay utilizing an auxotrophic strain of S. cerevisiae

Myo-inositol is a six‑carbon sugar that is essential for the growth of mammalian cells and must be obtained through either extracellular uptake or de novo biosynthesis. The physiological importance of myo-inositol stems from its incorporation into phosphoinositides and inositol phosphates, which serve a variety of signaling, regulatory, and structural roles in cells. To study myo-inositol metabolism and function, it is essential to have a reliable method for assaying myo-inositol levels. However, current approaches to assay myo-inositol levels are time-consuming, expensive, and often unreliable. This article describes a simple new myo-inositol bioassay that utilizes an auxotrophic strain of S. cerevisiae to measure myo-inositol concentration in solutions. The accuracy of this method was confirmed by comparing assay values to those obtained by tandem mass spectrometry (LC-MS/MS). It is easy to perform, inexpensive, does not require sophisticated equipment, and is specific for myo-inositol.

Innovative mycoremediation technique for treating unsterilized PCDD/F-contaminated field soil and the exploration of chlorinated metabolites

Mycoremediation of unsterilized PCDD/F-contaminated field soil was successfully demonstrated by solid-state fermentation coupled with Pleurotus pulmonarius utilizing a patented incubation approach. The experiments were carried out in four setups with two as controls. The contaminated soil was homogenously mixed with solid inocula, 1:0.5 dry w/w, resulting in an initial concentration of 4432 ± 623 ng WHO-TEQ kg−1. After a 30-day incubation under controlled conditions, the overall removal (approx. 60%) was non-specific. The removal was attributed to degradation by extracellular ligninolytic enzymes and uptake into the fruiting tissue (~110 ng WHO-TEQ kg−1 of mushroom). Furthermore, less recalcitrant chlorinated metabolites were found, implying ether bond cleavage and dechlorination happened during the mycoremediation. These metabolites resulted from the complex interaction between P. pulmonarius and the indigenous microbes from the unsterilized soil. This study provides a new step toward scaling up this mycoremediation technique to treat unsterilized PCDD/F-contaminated field soil.

LncRNA XIST Contributes to Cisplatin Resistance of Lung Cancer Cells by Promoting Cellular Glycolysis through Sponging miR-101-3p

Background: Non-small-cell lung carcinoma is one of the most frequently diagnosed cancers. Cisplatin (CDDP) is a currently applied standard anticancer agent for advanced lung cancers. Although effectively clinical response was achieved initially, a large fraction of lung cancer patients developed cisplatin resistance. Therefore, understanding the molecular mechanisms of chemoresistance is crucial for anti-lung cancer therapy. Long non-coding RNA (lncRNA)-X-inactive-specific transcript (XIST) has been reported to be positively associated with multiple cancers. Currently, the precise role and mechanism of XIST in cisplatin resistance of lung cancer have not been elucidated. Methods: The expression levels of miR-101-3p and lncRNA XIST were detected by qRT-PCR. Cisplatin-resistant lung cancer cell line was established by selecting the survival cells under gradually increased cisplatin treatments. The cell proliferation was detected by MTT assay, and the cellular glucose metabolism rate was evaluated by Seahorse metabolic flux analysis and glucose uptake and lactate product assays. Glycolysis-related protein expression levels were detected by Western blot. Dual luciferase reporter was constructed to determine the lncRNA-miRNA interaction. Results: Here, we report XIST is significantly upregulated in lung cancer tissues compared with normal lung tissues. In addition, cisplatin-resistant lung cancer cells displayed remarkably elevated XIST expression. We demonstrated that miR-101-3p functioned as a tumor suppressor in lung cancer and sensitized lung cancer cells to cisplatin. Bioinformatics analysis predicted miR-101-3p could be a potential target of XIST through direct binding with it as a competing endogenous RNA, which was further validated from lung tumor tissues and cell lines by luciferase assay. Intriguingly, XIST significantly promoted cellular glycolysis rate of lung cancer cells. The extracellular acidification rate, glucose uptake, and lactate product were elevated by XIST overexpression. On the contrary, miR-101-3p effectively suppressed glycolysis rate. Finally, we demonstrated silencing XIST significantly recovered miR-101-3p expression and downregulated expression of glycolysis key enzymes, a phenotype could be further overridden by miR-101-3p inhibition. Conclusions: This study reveals a new molecular mechanism for the lncRNA-XIST-promoted cisplatin resistance via sponging miR-101-3p, leading to de-repression of cellular glycolysis. Moreover, these findings warrant further in vivo investigations to study XIST as a potential target to overcome cisplatin resistance.

Transcription-Based Multidimensional Regulation of Fatty Acid Metabolism by HIF1α in Renal Tubules.

Lipid metabolism plays a basic role in renal physiology, especially in tubules. Hypoxia and hypoxia-induced factor (HIF) activation are common in renal diseases; however, the relationship between HIF and tubular lipid metabolism is poorly understood. Using prolyl hydroxylase inhibitor roxadustat (FG-4592), we verified and further explored the relationship between sustained HIF1α activation and lipid accumulation in cultured tubular cells. A transcriptome and chromatin immunoprecipitation sequencing analysis revealed that HIF1α directly regulates the expression of a number of genes possibly affecting lipid metabolism, including those associated with mitochondrial function. HIF1α activation suppressed fatty acid (FA) mobilization from lipid droplets (LDs) and extracellular FA uptake. Moreover, HIF1α decreased FA oxidation and ATP production. A lipidomics analysis showed that FG-4592 caused strong triglyceride (TG) accumulation and increased some types of phospholipids with polyunsaturated fatty acyl (PUFA) chains, as well as several proinflammatory lipids. Nevertheless, the overall FA level was maintained. Thus, our study indicated that HIF1α reduced the FA supply and utilization and reconstructed the composition of lipids in tubules, which is likely a part of hypoxic adaptation but could also be involved in pathological processes in the kidney.

Extracellular silicate uptake and deposition induced by oxidative burst in Saccharina japonica sporophytes (Phaeophyceae)

The process of biogenic silicon (Si) deposition in sporophytes of kelp (Saccharina japonica) was investigated by culturing sporophyte disks in the medium with and without hydrogen peroxide (H2O2). Culture with H2O2 remarkably promoted silicate-Si uptake by the disks. Silicate-Si uptake was completely inhibited by the treatment with potassium bromide (KBr), a competitive inhibitor of iodoperoxidase (IPO), even in the medium with H2O2. In addition, disk culture with phenol red in the medium with H2O2 resulted in the formation of bromophenol blue, indicating the presence of extracellular IPO. However, KBr treatment inhibited the formation of bromophenol blue in the medium with H2O2. These results showed that silicate-Si uptake is activated by extracellular IPO in the presence of radical oxygen species (ROS), such as H2O2. Interestingly, 1 mM salicylic acid also promoted silicate-Si uptake, even in the medium without H2O2. Furthermore, the allocation of silicate-Si to the soluble and cell wall-bound phlorotannin fractions using rhodamine 123 (R123), a tracer of biogenic Si, was estimated. Tracer fluorescence was detected in not only the soluble but also the cell wall-bound phlorotannin fraction. R123 fluorescence was histologically detected in the apoplasts of the epidermal and outer cortical layers of the disk, overlapping the histochemical distribution of IPO activity. These results suggested that silicate-Si uptake is activated by an oxidative burst induced by various stresses, and Si deposition is catalyzed by extracellular IPO in the presence of both ROS and phlorotannin in the extracellular matrix of sporophytes. Based on these findings, the hypothetical mechanisms of the extracellular silicate-Si uptake for defense responses in kelp were discussed.