Carrier Protein Research Articles

Massively integrated coexpression analysis reveals transcriptional regulation, evolution and cellular implications of the yeast noncanonical translatome

BackgroundRecent studies uncovered pervasive transcription and translation of thousands of noncanonical open reading frames (nORFs) outside of annotated genes. The contribution of nORFs to cellular phenotypes is difficult to infer using conventional approaches because nORFs tend to be short, of recent de novo origins, and lowly expressed. Here we develop a dedicated coexpression analysis framework that accounts for low expression to investigate the transcriptional regulation, evolution, and potential cellular roles of nORFs in Saccharomyces cerevisiae.ResultsOur results reveal that nORFs tend to be preferentially coexpressed with genes involved in cellular transport or homeostasis but rarely with genes involved in RNA processing. Mechanistically, we discover that young de novo nORFs located downstream of conserved genes tend to leverage their neighbors’ promoters through transcription readthrough, resulting in high coexpression and high expression levels. Transcriptional piggybacking also influences the coexpression profiles of young de novo nORFs located upstream of genes, but to a lesser extent and without detectable impact on expression levels. Transcriptional piggybacking influences, but does not determine, the transcription profiles of de novo nORFs emerging nearby genes. About 40% of nORFs are not strongly coexpressed with any gene but are transcriptionally regulated nonetheless and tend to form entirely new transcription modules. We offer a web browser interface (https://carvunislab.csb.pitt.edu/shiny/coexpression/) to efficiently query, visualize, and download our coexpression inferences.ConclusionsOur results suggest that nORF transcription is highly regulated. Our coexpression dataset serves as an unprecedented resource for unraveling how nORFs integrate into cellular networks, contribute to cellular phenotypes, and evolve.

Asymmetric Synthesis and Biological Evaluation of Platensilin, Platensimycin, Platencin, and Their Analogs via a Bioinspired Skeletal Reconstruction Approach.

Platensilin, platensimycin, and platencin are potent inhibitors of β-ketoacyl-acyl carrier protein synthase (FabF) in the bacterial and mammalian fatty acid synthesis system, presenting promising drug leads for both antibacterial and antidiabetic therapies. Herein, a bioinspired skeleton reconstruction approach is reported, which enables the unified synthesis of these three natural FabF inhibitors and their skeletally diverse analogs, all stemming from a common ent-pimarane core. The synthesis features a diastereoselective biocatalytic reduction and an intermolecular Diels-Alder reaction to prepare the common ent-pimarane core. From this intermediate, stereoselective Mn-catalyzed hydrogen atom-transfer hydrogenation and subsequent Cu-catalyzed carbenoid C-H insertion afford platensilin. Furthermore, the intramolecular Diels-Alder reaction succeeded by regioselective ring opening of the newly formed cyclopropane enables the construction of the bicyclo[3.2.1]-octane and bicyclo[2.2.2]-octane ring systems of platensimycin and platencin, respectively. This skeletal reconstruction approach of the ent-pimarane core facilitates the preparation of analogs bearing different polycyclic scaffolds. Among these analogs, the previously unexplored cyclopropyl analog 47 exhibits improved antibacterial activity (MIC80 = 0.0625 μg/mL) against S. aureus compared to platensimycin.

Coenzyme Q10 supplementation affects cellular ionic balance: relevance to aging.

Aging results into disruptive physiological functioning and cellular processes that affect the composition and structure of the plasma membrane. The plasma membrane is the major regulator of ionic homeostasis thatregulates the functioning of membrane transporters and exchangers. Coenzyme Q10 is a lipid-soluble antioxidant molecule that declines during aging and age-associated diseases. The presentstudy aims to explore the role of Coenzyme Q10 supplementation to rats during aging on membrane transporters and redox biomarkers. The studywas conducted on young and old male Wistar rats supplemented with 20 mg/kg b.w. of Coenzyme Q10 per day. After a period of 28days, rats were sacrificed and erythrocyte membrane was isolated. The result exhibits significant decline in biomarkers of oxidative stress in old control rats when compared with young control. The effect of Coenzyme Q10 supplementation was more pronounced in old rats. Thefunctioning of membrane transporters and Na+/H+ exchanger showed potential return to normal levels in the Coenzyme Q10 treated rats. Overall, the results demonstrate that Coenzyme Q10 plays an important role in maintaining redox balance in cells which interconnects with membrane integrity. Thus, Coenzyme Q10 supplementation may play animportant role in protecting age related alterations inerythrocyte membrane physiology.

Structural and molecular dynamics simulation studies of CBL-interacting protein kinase CIPK and its complexes related to plant salinity stress.

Calcium-dependent signaling in plants is responsible for several major cellular events, including the activation of the salinity-responsive pathways. Calcium binds to calcineurin B-like protein (CBL), and the resulting CBL-Ca2+ complex binds to CBL-interacting protein kinase (CIPK). The CBL-CIPK complex enhances the CIPK interaction with an upstream kinase. The upstream kinase phosphorylates CIPK that, in turn, phosphorylates membrane transporters. Phosphorylation influences transporter activity to kick-start many downstream functions, such as balancing the cytosolic Na+-to-K+ ratio. The CBL-CIPK interaction is pivotal for Ca2+-dependent salinity stress signaling. Computational methods are used to model the entire Arabidopsis thaliana CIPK24 protein structure in its autoinhibited and open-activated states. Arabidopsis thaliana CIPK24-CBL4 complex is predicted based on the protein-protein docking methods. The available structural and functional data support the CIPK24 and the CIPK24-CBL4 complex models. Models are energy-minimized and subjected to molecular dynamics (MD) simulations. MD simulations for 500ns and 300ns enabled us to predict the importance of conserved residues of the proteins. Finally, the work is extended to predict the CIPK24-CBL4 complex with the upstream kinase GRIK2. MD simulation for 300ns on the ternary complex structure enabled us to identify the critical CIPK24-GRIK2 interactions. Together, these data could be used to engineer the CBL-CIPK interaction network for developing salt tolerance in crops.

CNS Drug Discovery in Academia: Where Basic Research Meets Innovation.

The involvement of academic research in drug discovery is consistently growing. However, academic projects seldom advance to clinical trials. Here, we assess the landscape of drug discovery within the National Centre of Competence in Research (NCCR) TransCure launched by the Swiss National Science Foundation to foster basic research and early-stage drug discovery on membrane transporters. This included transporters in central nervous system (CNS) disorders, which represent a huge unmet medical need. While idea championship, sustainable funding, collaborations between disciplines at the interface of academia and industry are important for translational research, Popperian falsifiability, strong intellectual property and a motivated startup team are key elements for innovation. This is exemplified by the NCCR TransCure spin-off company Synendos Therapeutics, a clinical stage biotech company developing the first selective endocannabinoid reuptake inhibitors (SERIs) as novel treatment for neuropsychiatric disorders. We provide a perspective on the challenges related to entering an uncharted druggable space and bridging the often mentioned "valley of death". The high attrition rate of drug discovery projects in the CNS field within academia is often due to the lack of meaningful animal models that can provide pharmacological proof-of-concept for potentially disruptive technologies at the earliest stages, and the absence of solid intellectual property.

Nicholas Avrion Mitchison. 5 May 1928 — 28 December 2022

Avrion Mitchison, universally known as Av, made enormous contributions to the field of cellular immunity. Working under Peter Medawar FRS during his PhD in Oxford, Av made the crucial discovery that viable lymphocytes were responsible for rejection of grafts of foreign tissue, initiating the field of cellular immunology, which was to preoccupy him throughout his career. In 1962 he was appointed head of the Division of Experimental Biology at the National Institute for Medical Research and began using proteins decorated with small synthetic molecules termed haptens, enabling him to make his second major discovery. He showed that optimal antibody responses to a hapten required the cooperation of two cell populations: bone-marrow-derived ‘B’ lymphocytes, which synthesized the antibody, and thymus-derived ‘T’ lymphocytes, which recognized moieties in the carrier protein molecule and provided help to the B cells. In 1971 Av became the Jodrell Professor of Zoology at University College London and head of the Imperial Cancer Research Fund Tumour Immunology Unit (TIU), where he continued to investigate how antibody and cytotoxic T cell responses are regulated and the role of major histocompatibility genes in this process. The TIU was a mecca for immunologists from around the world, and Av inspired those who passed through to make major contributions to cellular immunity, neuroimmunology, leukaemogenesis, tumour immunology and autoimmune disease. In 1991 he became the first director of the Rheumatism Research Centre in Berlin, and in a few short years after the fall of the Berlin wall established a flourishing institute. The annual award of the Mitchison Prize for Young Researchers honours his legacy.

3,3',5-Triiodothyroacetic Acid Transporters.

Introduction: Thyroid hormone transporters are essential for thyroid hormones to enter target cells. Monocarboxylate transporter (MCT) 8 is a key transporter and is expressed at the blood-brain barrier (BBB), in neural cells and many other tissues. Patients with MCT8 deficiency have severe neurodevelopmental delays because of cerebral hypothyroidism and chronic sequelae of peripheral thyrotoxicosis. The T3 analog 3,3',5-triiodothyroacetic acid (TRIAC) rescued neurodevelopmental features in animal models mimicking MCT8 deficiency and improved key metabolic features in patients with MCT8 deficiency. However, the identity of the transporter(s) that facilitate TRIAC transport are unknown. Here, we screened candidate transporters that are expressed at the human BBB and/or brain-cerebrospinal fluid barrier and known thyroid hormone transporters for TRIAC transport. Materials and Methods: Plasma membrane expression was determined by cell surface biotinylation assays. Intracellular accumulation of 1 nM TRIAC was assessed in COS-1 cells expressing candidate transporters in Dulbecco's phosphate-buffered saline (DPBS)/0.1% glucose or Dulbecco's modified Eagle's medium (DMEM) with or without 0.1% bovine serum albumin (BSA). Expression of Slc22a8 was determined by fluorescent in situ hybridization in brain sections from wild-type and Mct8/Oatp1c1 knockout mice at postnatal days 12, 21, and 120. Results: In total, 59 plasma membrane transporters were selected for screening of TRIAC accumulation (n = 40 based on expression at the human BBB and/or brain-cerebrospinal fluid barrier and having small organic molecules as substrates; n = 19 known thyroid hormone transporters). Screening of the selected transporter panel showed that 18 transporters facilitated significant intracellular accumulation of TRIAC in DPBS/0.1% glucose or DMEM in the absence of BSA. In the presence of BSA, substantial transport was noted for SLCO1B1 and SLC22A8 (in DPBS/0.1% glucose and DMEM) and SLC10A1, SLC22A6, and SLC22A24 (in DMEM). The zebrafish and mouse orthologs of these transporters similarly facilitated intracellular accumulation of TRIAC. Highest Slc22a8 mRNA expression was detected in mouse brain capillary endothelial cells and choroid plexus epithelial cells at early postnatal time points, but was reduced at P120. Conclusions: Human SLC10A1, SLCO1B1, SLC22A6, SLC22A8, and SLC22A24 as well as their mouse and zebrafish orthologs are efficient TRIAC transporters. These findings contribute to the understanding of TRIAC treatment in patients with MCT8 deficiency and animal models thereof.

The Competitive Counterflow Assay for Identifying Drugs Transported by Solute Carriers: Principle, Applications, Challenges/Limits, and Perspectives.

The identification of substrates for solute carriers (SLCs) handling drugs is an important challenge, owing to the major implication of these plasma membrane transporters in pharmacokinetics and drug-drug interactions. In this context, the competitive counterflow (CCF) assay has been proposed as a practical and less expensive approach than the reference functional uptake assays for discriminating SLC substrates and non-substrates. The present article was designed to summarize and discuss key-findings about the CCF assay, including its principle, applications, challenges and limits, and perspectives. The CCF assay is based on the decrease of the steady-state accumulation of a tracer substrate in SLC-positive cells, caused by candidate substrates. Reviewed data highlight the fact that the CCF assay has been used to identify substrates and non-substrates for organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs). The performance values of the CCF assay, calculated from available CCF study data compared with reference functional uptake assay data, are, however, rather mitigated, indicating that the predictability of the CCF method for assessing SLC-mediated transportability of drugs is currently not optimal. Further studies, notably aimed at standardizing the CCF assay and developing CCF-based high-throughput approaches, are therefore required in order to fully precise the interest and relevance of the CCF assay for identifying substrates and non-substrates of SLCs.

Determining expression changes of ANO7 and SLC38A4 membrane transporters in colorectal cancer

Membrane transporters are proteins responsible for facilitating the movement of molecules within biological membranes. They play a vital role in maintaining cellular homeostasis by regulating the transport of nutrients, ions, and other molecules into and out of cells. Our aim is to identify biomarkers in colorectal cancer using membrane transporter proteins. We utilized COAD TCGA data for this purpose. Subsequently, we conducted differential gene analysis and feature selection using membrane transporter proteins. Furthermore, we identified two potential genes, including ANO7 and SLC38A4. To validate the expression profiles of ANO7 and SLC38A4, key genes in this context, RT-qPCR was employed on colorectal cancer samples and adjacent normal tissues. Additionally, utilizing GEPIA2, Kaplan-Meier survival analysis, and cBioPortal, we assessed the status of these genes in various cancers, examining their methylation and mutation patterns. In conclusion, we suggest that ANO7 and SLC38A4 serve as prognostic biomarkers in colorectal cancer.

Reconsidering red blood cells as the diagnostic potential for neurodegenerative disorders.

Red blood cells (RBCs) are usually considered simple cells and transporters of gases to tissues. However, recent research has suggested that RBCs may have diagnostic potential in major neurodegenerative disorders (NDDs). This review summarizes the current knowledge on changes in RBC in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and other NDDs. It discusses the deposition of neuronal proteins like amyloid-β, tau, and α-synuclein, polyamines, changes in the proteins of RBCs like band-3, membrane transporter proteins, heat shock proteins, oxidative stress biomarkers, and altered metabolic pathways in RBCs during neurodegeneration. It also highlights the comparison of RBC diagnostic markers to other in-market diagnoses and discusses the challenges in utilizing RBCs as diagnostic tools, such as the need for standardized protocols and further validation studies. The evidence suggests that RBCs have diagnostic potential in neurodegenerative disorders, and this study can pave the foundation for further research which may lead to the development of novel diagnostic approaches and treatments.

Induction of the intestinal membrane transporters ABCA1 and ABCG8 by 27-hydroxycholesterol through a redox mechanism

Graphical abstract Redox-induction of expression and synthesis of ATP binding cassette transporters in CaCo-2 cells by externally added 27-hydroxycholesterol. 27OHC: 27-hydroxycholesterol; NOX: NADPH oxidase; ROS: reactive oxygen species; ABCA1: ATP binding cassette A1; ABCG8: ATP binding cassette G8; DPI: diphenylene iodonium. Abstract Objective We tested the effect of 27-hydroxycholesterol (27OHC) on the expression and synthesis of two membrane transporters involved in sterols extrusion from the intestinal epithelium into the gut lumen: ATP-binding cassette A1 (ABCA1) and G8 (ABCG8). Special attention was given to ABCG8, a key player in the intestinal cell discharge of plant sterols. Methods Differentiated CaCo-2 intestinal cells were supplemented with 27OHC, and added to the cell incubation medium at a final concentration of 1 or 5 µM. These 27OHC externally added amounts were proven to reach intracellular oxysterol levels within the range of those normally recovered in the human peripheral blood. Results An up-regulation of the ABCA1 and ABCG8 mRNAs was observed in the CaCo-2 cells supplemented with 27OHC. Moreover, both 1 µM and 5 µM 27OHC induced a net, and steady, statistically significant, increase of both ABCA1 and ABCG8 protein levels. Of interest, the cellular pre-treatment with diphenylene iodonium, a selective inhibitor of NADPH oxidase, i.e. a major intracellular source of reactive oxygen species, fully inhibited the 27OHC enhancement of both ABCA1 and ABCG8 protein synthesis. Conclusion This in vitro study shows for the first time that the addition of 27OHC to intestinal epithelial cells up-regulates ABCG8, the transporter discharging plant sterols into the gut lumen, besides confirming to induce ABCA1 as well. Importantly, the 27OHC-dependent up-regulation of the two transporters appears to involve a redox mechanism rather than the canonical liver-X-receptors-dependent pathway. Significance statement The 27OHC introduced with the diet might modulate the plant sterol extrusion in the gut, in parallel with that of cholesterol.

The membrane transporter SLC25A48 enables transport of choline into human mitochondria

Choline has important physiological functions as a precursor for essential cell components, signaling molecules, phospholipids, and the neurotransmitter acetylcholine. Choline is a water-soluble charged molecule requiring transport proteins to cross biological membranes. Although transporters continue to be identified, membrane transport of choline is incompletely understood and knowledge about choline transport into intracellular organelles such as mitochondria remains limited. Here we show that SLC25A48 imports choline into human mitochondria. Human loss-of-function mutations in SLC25A48 show impaired choline transport into mitochondria and are associated with elevated urine and plasma choline levels. Thus, our studies may have implications for understanding and treating conditions related to choline metabolism.

Interplay between photosynthetic electron flux and organic carbon sinks in sucrose-excreting Synechocystis sp. PCC 6803 revealed by omics approaches

BackgroundAdvancing the engineering of photosynthesis-based prokaryotic cell factories is important for sustainable chemical production and requires a deep understanding of the interplay between bioenergetic and metabolic pathways. Rearrangements in photosynthetic electron flow to increase the efficient use of the light energy for carbon fixation must be balanced with a strong carbon sink to avoid photoinhibition. In the cyanobacterium Synechocystis sp. PCC 6803, the flavodiiron protein Flv3 functions as an alternative electron acceptor of photosystem I and represents an interesting engineering target for reorganizing electron flow in attempts to enhance photosynthetic CO2 fixation and increase production yield.ResultsWe have shown that inactivation of Flv3 in engineered sucrose-excreting Synechocystis (S02:Δflv3) induces a transition from photoautotrophic sucrose production to mixotrophic growth sustained by sucrose re-uptake and the formation of intracellular carbon sinks such as glycogen and polyhydroxybutyrate. The growth of S02:Δflv3 exceeds that of the sucrose-producing strain (S02) and demonstrates unforeseen proteomic and metabolomic changes over the course of the nine-day cultivation. In the absence of Flv3, a down-regulation of proteins related to photosynthetic light reactions and CO2 assimilation occurred concomitantly with up-regulation of those related to glycolytic pathways, before any differences in sucrose production between S02 and S02:Δflv3 strains were observed. Over time, increased sucrose degradation in S02:Δflv3 led to the upregulation of respiratory pathway components, such as the plastoquinone reductase complexes NDH-11 and NDH-2 and the terminal respiratory oxidases Cyd and Cox, which transfer electrons to O2. While glycolytic metabolism is significantly up-regulated in S02:Δflv3 to provide energy for the cell, the accumulation of intracellular storage compounds and the increase in respiration serve as indirect sinks for photosynthetic electrons.ConclusionsOur results show that the presence of strong carbon sink in the engineered sucrose-producing Synechocystis S02 strain, operating under high light, high CO2 and salt stress, cannot compensate for the lack of Flv3 by directly balancing the light transducing source and carbon fixing sink reactions. Instead, the cells immediately sense the imbalance, leading to extensive reprogramming of cellular bioenergetic, metabolic and ion transport pathways that favor mixotrophic growth rather than enhancing photoautotrophic sucrose production.

Overcoming flumatinib resistance in chronic myeloid leukaemia: Insights into cellular mechanisms and ivermectin's therapeutic potential.

Chronic myeloid leukaemia (CML) is a haematological malignancy characterized by the constitutive tyrosine kinase activity of the BCR-ABL1 fusion protein. Flumatinib, a second-generation tyrosine kinase inhibitor, has exhibited superior clinical efficacy compared to its precursor, imatinib. However, with increased clinical use, resistance to flumatinib has emerged as a significant challenge. To investigate the mechanisms of flumatinib resistance in CML, we induced the human CML cell line K562 using a flumatinib concentration gradient method invitro, successfully establishing a flumatinib-resistant K562/FLM cell line. This cell line exhibited cross-resistance to imatinib and doxorubicin, but remained sensitive to the antiparasitic agent ivermectin, which possesses antitumoural effects. Through cellular experimentation, we explored the resistance mechanisms, which indicated that K562/FLM cells evade flumatinib cytotoxicity by enhancing autophagy, increasing the expression of membrane transport proteins, particularly P-glycoprotein, ABCC1 and ABCC4, as well as enhancing phosphorylation of p-EGFR, p-ERK and p-STAT3 proteins. Moreover, it was found that ivermectin effectively suppressed the expression of autophagy and transport proteins in K562/FLM cells, reduced the activity of the aforementioned phosphoproteins, and promoted apoptotic cell death. Collectively, the increased autophagy, higher expression of drug-efflux proteins and hyperactivation of the EGFR/ERK/STAT3 signalling pathway were identified as pivotal elements promoting resistance to flumatinib. The significant effects of ivermectin might offer a novel therapeutic strategy to overcome flumatinib resistance and optimize the treatment outcomes of CML.

Deciphering structural variation upon biotinylation of biotin carboxyl carrier protein domain in Streptococcus pneumoniae

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and is responsible for acute invasive and non-invasive infections. Fight against pneumococcus is currently hampered by insufficient vaccine coverage and rising antimicrobial resistance, making the research necessary on novel drug targets. High-throughput mutagenesis has shown that acetyl-CoA carboxylase (ACC) is an essential enzyme in S. pneumoniae which converts acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis. ACC has four subunits; Biotin carboxyl carrier protein (BCCP), Biotin carboxylase (BC), Carboxyl transferase subunit α and β. Biotinylation of S. pneumoniae BCCP (SpBCCP) is required for the activation of ACC complex. In this study, we have biophysically characterized the apo- and holo- biotinylating domain SpBCCP80. We have performed 2D and 3D NMR experiments to analyze the changes in amino acid residues upon biotinylation of SpBCCP80. Further, we used NMR backbone chemical shift assignment data for bioinformatical analyses to determine the secondary and tertiary structure of proteins. We observed major changes in AMKVM motif and thumb region of SpBCCP80 upon biotinylation. Overall, this work provides structural insight into the apo- to holo- conversion of SpBCCP80 which can be further used as a drug target against S. pneumoniae.

Role of functional group orientation in the drug partitioning and micelle-mediated delivery of quinoline family drugs to the carrier protein

The understanding of the characteristics of the carrier vehicle and the specific drug it transports is required for designing an effective drug delivery system. We have done a comprehensive analysis of the distribution of three quinolines − quinine, quinidine, and cinchonidine − within hexadecyltrimethyl-ammonium bromide (HTAB) micelles. Further, we tracked their journey as they are transported to the carrier protein human serum albumin (HSA), using spectroscopy, calorimetry, and molecular docking methodologies. The mechanism of partitioning of the quinolines in HTAB micelles is explained based on thermodynamic parameters obtained from isothermal titration calorimetry (ITC). The partitioning coefficient obtained for the quinolines is in the range of 102-104 M−1 and is strongest for quinidine [(1.17 ± 0.78) × 104 M−1]. The stereochemistry and hydroxyl group orientation modulates the partitioning of the quinolines and also the delivery of the quinolines via micellar HTAB. The polar/electrostatic/H-bonding interactions of the hydrophilic polar head of HTAB hinders in efficient quinine delivery, while hydrophobic interactions through the micellar palisade layer facilitate the delivery of quinidine and cinchonidine. Such thorough exploration into the delivery of drugs via micelles is crucial in identifying functionalities necessary to optimize drug delivery systems for the successful delivery of drugs.

Protective Effect of Ergothioneine against Oxidative Stress-Induced Chondrocyte Death.

Reactive oxygen species (ROS) induce oxidative stress in cells and are associated with various diseases, including autoimmune diseases. Ergothioneine (EGT) is a natural amino acid derivative derived from the ergot fungus and has been reported to exhibit an effective antioxidant function in many models of oxidative stress-related diseases. Recently, mutations in OCTN1, a membrane transporter of EGT, have been reported to be associated with rheumatoid arthritis. Therefore, we investigated the chondrocyte-protective function of EGT using a model of oxidative stress-induced injury of chondrocytes by hydrogen peroxide (H2O2). Human chondrocytes were subjected to oxidative stress induced by H2O2 treatment, and cell viability, the activity of lactate dehydrogenase (LDH) released into the medium, dead cell ratio, intracellular ROS production, and mitochondrial morphology were assessed. EGT improved chondrocyte viability and LDH activity in the medium and strongly suppressed the dead cell ratio. EGT also exerted protective effects on intracellular ROS production and mitochondrial morphology. These results provide evidence to support the protective effects of EGT on chondrocytes induced by oxidative stress.

Environmental control and metabolic strategies of organic-matter-responsive bacterioplankton in the Weddell Sea (Antarctica).

Heterotrophic microbial communities play a significant role in driving carbon fluxes in marine ecosystems. Despite their importance, these communities remain understudied in remote polar oceans, which are known for their substantial contribution to the biological drawdown of atmospheric carbon dioxide. Our research focused on understanding the environmental factors and genetic makeup of key bacterial players involved in carbon remineralization in the Weddell Sea, including its coastal polynyas. Our experiments demonstrated that the combination of labile organic matter supply and temperature increase synergistically boosted bacterial growth. This suggests that, besides low seawater temperature, carbon limitation also hinders heterotrophic bacterial activity. Through the analysis of metagenome-assembled genomes, we discovered distinct genomic adaptation strategies in Bacteroidia and Gammaproteobacteria, both of which respond to organic matter. Both natural phytoplankton blooms and experimental addition of organic matter favoured Bacteroidia, which possess a large number of gene copies and a wide range of functional membrane transporters, glycoside hydrolases, and aminopeptidases. In contrast, the genomes of organic-matter-responsive Gammaproteobacteria were characterized by high densities of transcriptional regulators and transporters. Our findings suggest that bacterioplankton in the Weddell Sea, which respond to organic matter, employ metabolic strategies similar to those of their counterparts in temperate oceans. These strategies enable efficient growth at extremely low seawater temperatures, provided that organic carbon limitation is alleviated.

Solute Transporter OCTN1/Slc22a4 Affects Disease Severity and Response to Infliximab in Experimental Colitis: Role of Gut Microbiota and Immune Modulation

Abstract Background Inflammatory bowel diseases are chronic disabling conditions with a complex and multifactorial etiology, still incompletely understood. OCTN1, an organic cation transporter, could have a role in modulating the inflammatory response, and some genetic polymorphisms of this molecule have been associated with increased risk of inflammatory bowel diseases. Until now, limited information exists on its potential in predicting/modulating patient’s response to therapies. The aim of this study was to evaluate the role of OCTN1 in modifying gut microbiota and mucosal immunity in response to infliximab therapy in murine colitis. Methods A dextran sodium sulphate model of colitis was used to assess the clinical efficacy of infliximab administered intravenously in ocnt1 gene knockout mice and their C57BL/6 controls. Stool, colon, and mesenteric lymph node samples were collected to evaluate differences in gut microbiota composition, histology, and T cell populations, respectively. Results Octn1 -/- influences the microbiota profile and is associated with a worse dysbiosis in mice with colitis. Infliximab treatment attenuates colitis-associated dysbiosis, with an increase of bacterial richness and evenness in both strains. In comparison with wild type, octn1-/- mice have milder disease and a higher baseline percentage of Treg, Tmemory, Th2 and Th17 cells. Conclusions Our data support the murine model to study OCTN1 genetic contribution to inflammatory bowel diseases. This could be the first step towards the recognition of this membrane transporter as a biomarker in inflammatory conditions and a predictor of response to therapies.

Alterations to the catalytic properties of methylketone synthase 2 from eggplant (Solanum melongena) by mutating the conserved aspartate into glutamate

Methylketone synthase 2 (MKS2) has been widely found in the plant kingdom and identified as a single-hotdog-fold acyl-lipid thioesterase (ALT) which mainly hydrolyzes the thioester bond in 3-ketoacyl-acyl carrier protein (3-ketoacyl-ACP) intermediates of the fatty acid biosynthetic pathway into free 3-keto fatty acids. Our previous study identified SmMKS2–2 as one of two functional ALTs in eggplant Solanum melongena. To gain mechanistic insights into catalysis by this enzyme, we herein combined biochemical and in silico structural analyses on SmMKS2–2. While SmMKS2–2 is capable of producing a wide range of 3-ketoacids from corresponding 3-ketoacyl-ACP substrates, SmMKS2–2-D77E mutant variant drops its thioesterase activity to the undetectable level. Consistently, the structural modelling of the D77E mutant displays that the orientation of the side chain carboxylate group of the replacing amino acid has been shifted compared to that of the native residue, resulting in smaller surface area of binding pocket that would dismiss nucleophilic catalysis of the mutant protein. Together, these data suggested that D77 is critical and specific for SmMKS2–2 to hydrolyze the thioester bond of acyl-ACP.