Cation Efflux Research Articles

Investigation on the mechanisms of carbapenem resistance among the non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae.

In Malaysia, an increase in non-carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae (NC-CRKP) has been observed over the years. Previously, four NC-CRKP with increased susceptibility to ciprofloxacin in the presence of phenylalanine-arginine β-naphthylamide (PAβN) were identified. However, no contribution of the PAβN-inhibited efflux pump to carbapenem resistance was observed. All four NC-CRKP harboured non-carbapenemase β-lactamase, with two also exhibiting porin loss. In this study, we further investigated the genomic features and resistance mechanisms of these four isolates. All four NC-CRKP were subjected to whole-genome sequencing, followed by comparative genomic and phylogenetic analyses. Multi-locus sequence typing (MLST) analysis divided the four NC-CRKP into different sequence types: ST392, ST45, ST14, and ST5947. Neither major nor rare carbapenemase genes were detected. Given the presence of non-carbapenemase β-lactamase in all isolates, we further investigated the potential mechanisms of resistance by identifying related chromosomal mutations. Deletion mutation was detected in the cation efflux system protein CusF. Insertion mutation was identified in the nickel/cobalt efflux protein RcnA. Missense mutation of ompK36 porin was detected in two isolates, while the loss of ompK36 porin was observed in another two isolates. This study revealed that NC-CRKP may confer carbapenem resistance through a combination of non-carbapenemase β-lactamase and potential chromosomal mutations including missense mutation or loss of ompK36 porin and/or a frameshift missense mutation in efflux pump systems, such as cation efflux system protein CusF and nickel/cobalt efflux protein RcnA. Our findings highlighted the significance of implementing whole-genome sequencing into clinical practice to promote the surveillance of carbapenem resistance mechanisms among NC-CRKP.

Gene expression profile of Campylobacter jejuni in response to macrolide antibiotics.

Campylobacter jejuni is a foodborne pathogen that causes gastroenteritis in humans and has developed resistance to various antibiotics. The primary objective of this research was to examine the network of antibiotic resistance in C. jejuni. The study involved the wild and antibiotic-resistant strains placed in the presence and absence of antibiotics to review their gene expression profiles in response to ciprofloxacin via microarray. Differentially expressed genes (DEGs) analysis and Protein-Protein Interaction (PPI) Network studies were performed for these genes. The results showed that the resistance network of C. jejuni is modular, with different genes involved in bacterial motility, capsule synthesis, efflux, and amino acid and sugar synthesis. Antibiotic treatment resulted in the down-regulation of cluster genes related to translation, flagellum formation, and chemotaxis. In contrast, cluster genes involved in homeostasis, capsule formation, and cation efflux were up-regulated. The study also found that macrolide antibiotics inhibit the progression of C. jejuni infection by inactivating topoisomerase enzymes and increasing the activity of epimerase enzymes, trying to compensate for the effect of DNA twisting. Then, the bacterium limits the movement to conserve energy. Identifying the antibiotic resistance network in C. jejuni can aid in developing drugs to combat these bacteria. Genes involved in cell division, capsule formation, and substance transport may be potential targets for inhibitory drugs. Future research must be directed toward comprehending the underlying mechanisms contributing to the modularity of antibiotic resistance and developing strategies to disrupt and mitigate the growing threat of antibiotic resistance effectively.

Physiological Responses Revealed Static Magnetic Fields Potentially Improving the Tolerance of Poplar Seedlings to Salt Stress

Magnetic fields play an important role in regulating plant growth and development, especially in improving plant stress tolerance. However, the physiological mechanism underlying the magnetic effects is still unclear. Here, we examined changes in reactive oxygen species (ROS) levels and ion flux in poplar (Populus × deltoides ‘Lulin-2’) seedling roots under salt stress in a static magnetic field (SMF). SMF treatment significantly increased seedling growth and mitigated the effects of salt stress on root growth. Furthermore, SMF treatment activated ROS and calcium signals in poplar roots. Relative to the SMF treatment group, control plants had significantly higher levels of cytoplasmic free Ca2+ ([Ca2+]cyt) and ROS following exposure to high salt concentrations. Under salt conditions, SMF treatment reduced increases in Na+ concentrations and maintained stable K+ and Ca2+ concentrations and K+/Na+ and Ca2+/Na+ ratios. NMT analysis suggests that SMF treatment may drive cation effluxes in poplar seedling roots. Susceptibility tests of Na+-transport inhibitors indicated that SMF treatment contributed to Na+ repulsion and H+ uptake under salt stress. Moreover, SMF exposure allowed roots to retain the ability to reduce salt-induced K+ and Ca2+ root effluxes, and qRT-PCR results demonstrate that SMF treatment can increase the expression of stress-responsive genes such as PtrRBOHF, PtrNHX1 and PtrHA5 in poplar seedlings. Therefore, we conclude that treating poplar seedlings with SMF can help them establish a stable tolerance to salt stress by regulating ROS, [Ca2+]cyt, and their regulatory networks. This study examined the physiological responses of poplar to SMF exposure under salt stress, providing insights into plant magnetobiological effects.

Development of an immunoassay for the simultaneous detection of GADA and ZnT8A in autoimmune diabetes using a ZnT8/GAD65 chimeric molecule.

The combined presence of autoantibodies to the 65 kDa isoform of glutamic acid decarboxylase (GADA) and to the islet-specific cation efflux transporter ZnT8 (ZnT8A) in serum is the best predictive sign of the loss of immune tolerance and the clinical manifestation of autoimmune diabetes mellitus (DM). The screening of GADA and ZnT8A could help to reach to a correct diagnosis and to start an early and adequate treatment. The aim of the study was to develop an immunoassay for the simultaneous detection of these autoantibodies using a chimera molecule that includes the immunodominant regions of ZnT8 and GAD65, expressed by baculovirus-insect cells system. ZnT8/GAD65 was expressed using the Bac to Bac™ baculovirus expression system. The recombinant chimera was purified by an His6-tag and identified by SDS-PAGE and western blot analysis, and by an indirect ELISA using specific antibodies against ZnT8 and GAD65. A fraction of ZnT8/GAD65 was biotinylated. A bridge ELISA (b-ELISA) was developed using ZnT8/GAD65 immobilized in polystyrene microplates, human sera samples from healthy individuals (n = 51) and diabetic patients (n = 49) were then incubated, and afterwards ZnT8/GAD65-biotin was added. Immune complexes were revealed with Streptavidin-Horseradish Peroxidase. Results were calculated as specific absorbance and expressed as standard deviation scores: SDs. ZnT8/GAD65 was efficiently produced, yielding 30 mg/L culture medium, 80% pure. This recombinant chimera retains the immunoreactive conformation of the epitopes that are recognized by their specific antibodies, so it was used for the development of a high sensitivity (75.51%) and specificity (98.04%) b-ELISA for the detection of ZnT8A and/or GADA, in a one-step screening assay. The ROC curves demonstrated that this method had high accuracy to distinguish between samples from healthy individuals and diabetic patients (AUC = 0.9488); the cut-off value was stablished at 2 SDs. This immunoassay is useful either to confirm autoimmune diabetes or for detection in routine screening of individuals at risk of autoimmune DM. As DM is a slow progress disease, remaining asymptomatic for a long preclinical period, serological testing is of importance to establish a preventive treatment.

Characterization of a cadmium-resistant functional bacteria (Burkholderia sp. SRB-1) and mechanism analysis at physiochemical and genetic level.

In this study, the capacity of cadmium (Cd)-resistant plant growth-promoting bacteria (PGPB) Burkholderia sp. SRB-1 (SRB-1) and its mechanisms were explored through morphological characterizations, biochemical response, plant growth-promoting traits, and functional gene expression patterns. The results showed that SRB-1 was an excellent Cd-resistant bacteria (MIC was 420mg L-1), and its maximum Cd removal rate reached 72.25%. Biosorption was the main removal method of Cd for SRB-1, preventing intracellular Cd accumulation and maintaining cellular metabolism. Various functional groups on the cell wall were involved in Cd binding, which deposited as CdS and CdCO3 on the cell surface according to XPS analysis and might be critical for reducing Cd physiochemical toxicity. Furthermore, metals exporting (zntA, czcA, czcB, czcC), detoxification (dsbA, cysM), and antioxidation (katE, katG, SOD1) related genes were annotated in the SRB-1 genome. The results of Cd distribution and antioxidative enzyme activity in SRB-1 also illustrated that Cd2+ efflux and antioxidative response were the main intracellular Cd-resistant mechanisms. These conclusions were further verified by qRT-PCR analysis. Overall, the strategies of extracellular biosorption, cation efflux, and intracellular detoxification jointly build the Cd-resistant system, which invested Burkholderia sp. SRB-1 with potential for bioremediation in heavily Cd-contaminated environmental sites.

Identification and Characterization of Dmct: A Cation Transporter in Yarrowia lipolytica Involved in Metal Tolerance

Yarrowia lipolytica is a dimorphic fungus used as a model organism to investigate diverse biotechnological and biological processes, such as cell differentiation, heterologous protein production, and bioremediation strategies. However, little is known about the biological processes responsible for cation concentration homeostasis. Metals play pivotal roles in critical biochemical processes, and some are toxic at unbalanced intracellular concentrations. Membrane transport proteins control intracellular cation concentrations. Analysis of the Y. lipolytica genome revealed a characteristic functional domain of the cation efflux protein family, i.e., YALI0F19734g, which encodes YALI0F19734p (a putative Yl-Dmct protein), which is related to divalent metal cation tolerance. We report the in silico analysis of the putative Yl-Dmct protein’s characteristics and the phenotypic response to divalent cations (Ca2+, Cu2+, Fe2+, and Zn2+) in the presence of mutant strains, Δdmct and Rdmct, constructed by deletion and reinsertion of the DMCT gene, respectively. The absence of the Yl-Dmct protein induces cellular and growth rate changes, as well as dimorphism differences, when calcium, copper, iron, and zinc are added to the cultured medium. Interestingly, the parental and mutant strains were able to internalize the ions. Our results suggest that the protein encoded by the DMCT gene is involved in cell development and cation homeostasis in Y. lipolytica.

Genome-wide identification and expression profile analysis of metal tolerance protein gene family in Eucalyptus grandis under metal stresses

Metal tolerance proteins (MTPs) as Me2+/H+(K+) antiporters participate in the transport of divalent cations, leading to heavy metal stress resistance and mineral utilization in plants. In the present study, to obtain better knowledge of the biological functions of the MTPs family, 20 potential EgMTPs genes were identified in Eucalyptus grandis and classified into seven groups belonging to three cation diffusion facilitator groups (Mn-CDFs, Zn/Fe-CDFs, and Zn-CDFs) and seven groups. EgMTP-encoded amino acids ranged from 315 to 884, and most of them contained 4–6 recognized transmembrane domains and were clearly prognosticated to localize into the cell vacuole. Almost all EgMTP genes experienced gene duplication events, in which some might be uniformly distributed in the genome. The numbers of cation efflux and the zinc transporter dimerization domain were highest in EgMTP proteins. The promoter regions of EgMTP genes have different cis-regulatory elements, indicating that the transcription rate of EgMTP genes can be a controlled response to different stimuli in multiple pathways. Our findings provide accurate perception on the role of the predicted miRNAs and the presence of SSR marker in the Eucalyptus genome and clarify their functions in metal tolerance regulation and marker-assisted selection, respectively. Gene expression profiling based on previous RNA-seq data indicates a probable function for EgMTP genes during development and responses to biotic stress. Additionally, the upregulation of EgMTP6, EgMTP5, and EgMTP11.1 to excess Cd2+ and Cu2+ exposure might be responsible for metal translocation from roots to leaves.

Physical and biogeochemical drivers of solute mobilization and flux through the critical zone after wildfire

A nine-year time series of nutrient cation and anion concentration and efflux from three forested catchments in the Jemez River Basin Critical Zone Observatory (JRB-CZO) in northern New Mexico was used to quantify the pulse of chemical denudation resulting from varying levels stand-replacing wildfire intensity in May-June of 2013. The 3 years of pre-fire and 6 years of postfire data were also probed to shed light on the mechanisms underlying the pulsed release and the subsequent recovery. The initial large solute pulse released to the streams—K+, Ca2+, Mg2+, SO42-, Cl−, dissolved inorganic carbon (DIC), dissolved organic carbon (DOC), and total dissolved nitrogen (TDN)—was caused by leaching of hillslope ash deposits during the first monsoon storms post-fire. Debris flow following the wildfire likely redistributed much of the ash-containing sediments along streams and valley bottoms. Sustained elevated solute concentrations observed in the surface waters throughout the post-fire period relative to pre-fire baselines is consistent with these soluble materials being periodically flushed from the soils during wet seasons, i.e., snowmelt and summer monsoons. As microbial mediated reactions and biotic uptake—due to plant regrowth—recover after fire, nutrient ion export (e.g., NO3-, Cl− and SO42-) steadily decreased toward the end of the post fire period, but remained above pre-fire levels, particularly for NO3- and SO42-. Surface water concentrations of polyvalent cations (e.g., Al and Fe) decreased significantly after the fire. Our observations suggest that changes in organic matter composition after fire (e.g., increased humification index—HIX) and the presence of pyrogenic carbon may not favor organo-metal complexation and transport. Finally, differences in burn severity among the three watersheds presented in this study, provide insights of the relative impact of solute exports and resilience. The catchments that experienced high burn severity exhibited greater solute fluxes than the less severely burn. Moreover, despite these differences, toward the end of the post-fire period these surface waters presented low and similar solute effluxes, indicating system recovery. Nonetheless, magnitudes and rebounds were solute and process specific. The results of this study highlight the importance of surface and near surface physical and biogeochemical processes on the long-lasting geochemical denudation of forested catchments following wildfires of varying intensities.

Genome-wide identification and expression analysis of metal tolerance protein (MTP) gene family in soybean (Glycine max) under heavy metal stress.

Plant metal tolerance proteins (MTPs) are plant membrane divalent cation transporters that specifically contribute to heavy metal stress resistance and mineral uptake. However, little is known about this family's molecular behaviors and biological activities in soybean. A total of 20 potential MTP candidate genes were identified and studied in the soybean genome for phylogenetic relationships, chromosomal distributions, gene structures, gene ontology, cis-elements, and previous gene expression. Furthermore, the expression of MTPs has been investigated under different heavy metals treatments. All identified soybean MTPs (GmaMTPs) contain a cation efflux domain or a ZT dimer and are further divided into three primary cation diffusion facilitator (CDF) groups: Mn-CDFs, Zn-CDFs, and Fe/Zn-CDFs. The developmental analysis reveals that segmental duplication contributes to the GmaMTP family's expansion. Tissue-specific expression profiling revealed comparative expression profiling in similar groups, although gene expression differed between groups. GmaMTP genes displayed biased responses in either plant leaves or roots when treated with heavy metal. In the leaves and roots, nine and ten GmaMTPs responded to at least one metal ion treatment. Furthermore, in most heavy metal treatments, GmaMTP1.1, GmaMTP1.2, GmaMTP3.1, GmaMTP3.2, GmaMTP4.1, and GmaMTP4.3 exhibited significant expression responses. Our findings provided insight into the evolution of MTPs in soybean. Overall, our findings shed light on the evolution of the MTP gene family in soybean and pave the path for further functional characterization of this gene family.

PfATP4 inhibitors in the Medicines for Malaria Venture Malaria Box and Pathogen Box block the schizont-to-ring transition by inhibiting egress rather than invasion.

The cation efflux pump Plasmodium falciparum ATPase 4 (PfATP4) maintains Na+ homeostasis in malaria parasites and has been implicated in the mechanism of action of many structurally diverse antimalarial agents, including >7% of the antimalarial compounds in the Medicines for Malaria Venture's 'Malaria Box' and 'Pathogen Box'. Recent screens of the 'Malaria Box' and 'Pathogen Box' revealed that many PfATP4 inhibitors prevent parasites from exiting their host red blood cell (egress) or entering new host cells (invasion), suggesting that these compounds may have additional molecular targets involved in egress or invasion. Here, we demonstrate that five PfATP4 inhibitors reduce egress but not invasion. These compounds appear to inhibit egress by blocking the activation of protein kinase G, an enzyme that, once stimulated, rapidly activates parasite egress. We establish a direct link between egress and PfATP4 function by showing that the inhibition of egress is attenuated in a Na+-depleted environment and in parasites with a mutation in pfatp4. Finally, we show that PfATP4 inhibitors induce host cell lysis when administered prior to the completion of parasite replication. Since host cell lysis mimics egress but is not followed by invasion, this phenomenon likely explains why several PfATP4 inhibitors were previously classified as invasion inhibitors. Collectively, our results confirm that PfATP4-mediated Na+ efflux is critical to the regulation of parasite egress.

Genome-wide analyses of metal tolerance protein genes in apple (Malus domestica): Identification, characterization, expression and response to various metal ion stresses

The growth and production of fruit trees are adversely affected by heavy metal pollution in orchard lands. Plant metal homeostasis and resistance are regulated by metal tolerance proteins (MTP), which function as divalent cation transporters. To understand the possible role of MTPs in fruit trees, we identified 20 MdMTPs in the apple genome. Phylogenetic and collinear relationship analyses revealed that they belonged to Zn-MTP, Fe/Zn-MTP and Mn-MTP subfamilies, and have gone through two tandem and six segmental duplication events under purifying selection. All MdMTPs were predicted to form homodimer, with each monocase containing a conserved cation efflux domain (CED), the typical structural feature of MTP family, and one or two HXXXD or DXXXD residues. Further spatiotemporal expression analyses indicated that all MdMTP genes were ubiquitously expressed, and most of them were induced by at least three heavy metal ions, in roots, stems or leaves. Yeast and Arabidopsis mutant complementation experiments verified the function of MdMTP11.1 in response to heavy metal stress. Our findings provide an important genetic foundation for the functional elucidation of MdMTP genes, which can be used as the potential gene candidates for the breeding of new fruit tree cultivars with improved tolerance to heavy metal stress. • A total number of 20 metal tolerance protein (MTP) genes were identified in apple. • All MdMTP proteins contained a cation efflux domain to work for metal ion efflux. • Tandem and segmental duplication events occurred in MdMTPs under purifying selection. • Most of MdMTPs were induced by at least three heavy metal ions in various organs.

Microbial silver resistance mechanisms: recent developments.

In this mini-review, after a brief introduction into the widespread antimicrobial use of silver ions and nanoparticles against bacteria, fungi and viruses, the toxicity of silver compounds and the molecular mechanisms of microbial silver resistance are discussed, including recent studies on bacteria and fungi. The similarities and differences between silver ions and silver nanoparticles as antimicrobial agents are also mentioned. Regarding bacterial ionic silver resistance, the roles of the sil operon, silver cation efflux proteins, and copper-silver efflux systems are explained. The importance of bacterially produced exopolysaccharides as a physiological (biofilm) defense mechanism against silver nanoparticles is also emphasized. Regarding fungal silver resistance, the roles of metallothioneins, copper-transporting P-type ATPases and cell wall are discussed. Recent evolutionary engineering (adaptive laboratory evolution) studies are also discussed which revealed that silver resistance can evolve rapidly in bacteria and fungi. The cross-resistance observed between silver resistance and resistance to other heavy metals and antibiotics in bacteria and fungi is also explained as a clinically and environmentally important issue. The use of silver against bacterial and fungal biofilm formation is also discussed. Finally, the antiviral effects of silver and the use of silver nanoparticles against SARS-CoV-2 and other viruses are mentioned. To conclude, silver compounds are becoming increasingly important as antimicrobial agents, and their widespread use necessitates detailed understanding of microbial silver response and resistance mechanisms, as well as the ecological effects of silver compounds. Figure created with BioRender.com.

Genome-Wide Identification of Strawberry Metal Tolerance Proteins and Their Expression under Cadmium Toxicity

Metal tolerance proteins (MTPs) are divalent cation transporters, known to upkeep the mineral nutrition of plants and heavy metal transport at cell, tissue, or whole plant levels. However, information related to evolutionary relationships and biological functions of MTP genes in strawberry (Fragaria vesca L.) remain elusive. Herein, we identified 12 MTP genes from the strawberry genome and divided them into three main groups (i.e., Zn-MTP, Fe/Zn MTP, and Mn-MTP), which is similar to MTP grouping in Arabidopsis and rice. The strawberry MTPs (FvMTPs) are predicted to be localized in the vacuole, while open reading frame (ORF) length ranged from 1113 to 2589 bp with 370 to 862 amino acids, and possess 4 to 6 transmembrane domains (TMDs), except for FvMTP12 that possessed 16 TMDs. All the FvMTP genes had putative cation efflux and cation diffusion facilitator domains along with a zinc dimerization (ZT-dimer) domain in Mn-MTPs. The collinear analysis suggested their conservation between strawberry and Arabidopsis MTPs. Promoter analysis also demonstrated that some of them might possibly be regulated by hormones and abiotic stress factors. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis proposed that FvMTP genes are involved in cation transport and homeostasis. The expression analysis showed that FvMTP1, FvMTP1.1, and FvMTP4 were significantly induced in leaf samples, while FvMTP1.1 and FvMTP4 were significantly regulated in roots of cadmium (Cd)-treated strawberry plants during progressive stress duration. The findings of Cd accumulation depicted that Cd contents were significantly higher in root tissues than that of leaf tissues of strawberry. These results are indicative of their response during the specific duration in Cd detoxification, while further functional studies can accurately verify their specific role.

Changes in Astroglial K+ upon Brief Periods of Energy Deprivation in the Mouse Neocortex.

Malfunction of astrocytic K+ regulation contributes to the breakdown of extracellular K+ homeostasis during ischemia and spreading depolarization events. Studying astroglial K+ changes is, however, hampered by a lack of suitable techniques. Here, we combined results from fluorescence imaging, ion-selective microelectrodes, and patch-clamp recordings in murine neocortical slices with the calculation of astrocytic [K+]. Brief chemical ischemia caused a reversible ATP reduction and a transient depolarization of astrocytes. Moreover, astrocytic [Na+] increased by 24 mM and extracellular [Na+] decreased. Extracellular [K+] increased, followed by an undershoot during recovery. Feeding these data into the Goldman–Hodgkin–Katz equation revealed a baseline astroglial [K+] of 146 mM, an initial K+ loss by 43 mM upon chemical ischemia, and a transient K+ overshoot of 16 mM during recovery. It also disclosed a biphasic mismatch in astrocytic Na+/K+ balance, which was initially ameliorated, but later aggravated by accompanying changes in pH and bicarbonate, respectively. Altogether, our study predicts a loss of K+ from astrocytes upon chemical ischemia followed by a net gain. The overshooting K+ uptake will promote low extracellular K+ during recovery, likely exerting a neuroprotective effect. The resulting late cation/anion imbalance requires additional efflux of cations and/or influx of anions, the latter eventually driving delayed astrocyte swelling.

Changes in physiological responses and MTP (metal tolerance protein) transcripts in soybean (Glycine max) exposed to differential iron availability

Members of MTP (metal tolerance protein) family are potential metal ion transporters, but little is known about how their responses and expression are altered in response to the deficiency and excess of Fe in soybean. In this study, root and shoot length and biomass in addition to leaf chlorophyll score, PSII efficiency and photosynthetic performance index were adversely affected by Fe-deficiency and excess Fe. Fe and S concentrations in the root and shoot, as well as the increased root FCR activity, consistently decreased and increased, respectively, accompanied by elevated Zn levels under Fe deficiency and Fe toxicity. This implies that Fe-uptake of plants subjected to differential Fe availability are likely determined by S and Zn nutritional status. In qPCR analysis, GmMTP5, GmMTP7, GmMTP8, and GmMTP10 genes showed downregulation under Fe shortage, whereas GmMTP6 and GmMTP11 were significantly upregulated due to Fe-toxicity. Further, GmMTP1, GmMTP3, GmMTP6, GmMTP7, and GmMTP10 were significantly induced in response to Fe toxicity, indicating their potential role in metal tolerance. Bioinformatics analysis showed that soybean MTP genes possessed a close relationship with certain Arabidopsis genes (i.e. ZAT, MTPB1) involved in solute transport and metal sequestration. Furthermore, top five motifs of soybean MTP protein correspond to the cation efflux family exhibited strong amino acid and evolutionary similarities with Arabidopsisthaliana. These findings shed light on Fe homeostasis mechanisms in soybean and could be used to regulate Fe uptake through breeding or transgenic manipulations of MTP genes.

Live Tissue Imaging Reveals Distinct Transcellular Pathways for Organic Cations and Anions at the Blood-Cerebrospinal Fluid Barrier

Formed by the choroid plexus epithelial (CPE) cells, the blood-cerebrospinal fluid barrier (BCSFB) plays an active role in removing drugs, toxins, and metabolic wastes from the brain. Several organic cation and anion transporters are expressed in the CPE cells, but how they functionally mediate transepithelial transport of organic cations and anions remain unclear. In this study, we visualized the transcellular transport of fluorescent organic cation and organic anion probes using live tissue imaging in freshly isolated mouse choroid plexuses (CPs). The cationic probe, 4-[4-(dimethylamino)phenyl]-1-methylpyridinium iodide (IDT307) was transported into CPE cells at the apical membrane and highly accumulated in mitochondria. Consistent with the lack of expression of organic cation efflux transporters, there was little efflux of IDT307 into the blood capillary space. Furthermore, IDT307 uptake and intracellular accumulation was attenuated by approximately 70% in CP tissues from mice with targeted deletion of the plasma membrane monoamine transporter (Pmat). In contrast, the anionic probe fluorescein-methotrexate (FL-MTX) was rapidly transported across the CPE cells into the capillary space with little intracellular accumulation. Rifampicin, an inhibitor of organic anion transporting polypeptides (OATPs), completely blocked FL-MTX uptake into the CPE cells whereas MK-571, a pan-inhibitor of multidrug resistance associated proteins (MRPs), abolished basolateral efflux of FL-MTX. In summary, our results suggest distinct transcellular transport pathways for organic cations and anions at the BCSFB and reveal a pivotal role of PMAT, OATP and MRP transporters in organic cation and anion transport at the blood-cerebrospinal fluid interface. SIGNIFICANCE STATEMENT: Live tissue imaging revealed that while organic cations are transported from the cerebrospinal fluid (CSF) into the choroid plexus epithelial cells by plasma membrane monoamine transporter without efflux into the blood, amphipathic anions in the CSF are efficiently transported across the BCSFB through the collaborated function of apical organic anion transporting polypeptides and basolateral multidrug resistance associated proteins. These findings contribute to a mechanistic understanding of the molecular and cellular pathways for choroid plexus clearance of solutes from the brain.

Plant Trans-Golgi Network/Early Endosome pH regulation requires Cation Chloride Cotransporter (CCC1).

Plant cells maintain a low luminal pH in the trans-Golgi-network/early endosome (TGN/EE), the organelle in which the secretory and endocytic pathways intersect. Impaired TGN/EE pH regulation translates into severe plant growth defects. The identity of the proton pump and proton/ion antiporters that regulate TGN/EE pH have been determined, but an essential component required to complete the TGN/EE membrane transport circuit remains unidentified - a pathway for cation and anion efflux. Here, we have used complementation, genetically encoded fluorescent sensors, and pharmacological treatments to demonstrate that Arabidopsis cation chloride cotransporter (CCC1) is this missing component necessary for regulating TGN/EE pH and function. Loss of CCC1 function leads to alterations in TGN/EE-mediated processes including endocytic trafficking, exocytosis, and response to abiotic stress, consistent with the multitude of phenotypic defects observed in ccc1 knockout plants. This discovery places CCC1 as a central component of plant cellular function.

Insights into the response of mangrove sediment microbiomes to heavy metal pollution: Ecological risk assessment and metagenomics perspectives

Rapid urbanisation and ensuing anthropogenic pollution lead to an escalated occurrence of heavy metals and metal-resistant bacteria in the soil ecosystem. Mangrove ecosystems are particularly vulnerable to heavy metal bioaccumulation and often act as metal sinks of the coastal areas. As a consequence, the microbial population in mangrove sediments develop multifarious metal tolerance mechanisms to combat metal toxicity. In this context, metagenomic investigation of two mangroves, viz. Mangalavanam and Puthuvypin from the heavily populated metropolitan city, Cochin (Central Kerala, India) was undertaken to discern the metal resistance functions and taxonomic diversity of the microbial consortia. Estimation of heavy metal content using Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-MS) identified the abundance of zinc, chromium, nickel copper, lead, arsenic, and cadmium in the mangrove sediments. Ecological risk index values indicated high cadmium contamination of the two estuarine samples. Whole metagenome shotgun sequencing of the Central Kerala mangroves and comparative analysis with mangrove metal resistomes from other geographical regions revealed the prevalence of cobalt-zinc-cadmium resistance and preponderance of Proteobacteria in all the datasets. Cation efflux system protein CusA constituted the majority of the reads at the function level. Comparative analysis of taxonomy identified the dominance of Anaeromyxobacter, Geobacter, Pseudomonas, Candidatus Solibacter, and Pelobacter in the mangrove datasets. Non-metric multidimensional scaling analysis of the metal resistance genes depicted strong geographical clustering of the function and composition of metal resistant bacteria, suggesting a strong innate resilience of microbiome towards anthropogenic perturbations. More robust studies with intensive sampling will enhance our understanding of the occurrence, interactions, and functions of microbial heavy metal resistome in mangrove ecosystems.

Computational characterization and expression profile of MTP1 gene associated with zinc homeostasis across dicot plant species

This study characterizes different MTP1 (metal tolerance protein)/ZAT (zinc transporter) homologs involved in zinc (Zn) homeostasis in plants. BLAST analysis of AtMTP1 protein against 15 plant species showed 21 MTP1 homologs. These MTP1 protein homologs generally contain ~400 residues, six transmembrane helices and cation transmembrane transporter activity (GO:0008324), which can be utilized in predicting Zn uptake and tolerance mechanisms. These MTP1 genes having 1 exon are located on chromosomes 2, 7, and 14. Motifs contain conserved sequences of 41–50 residues belonging to the cation efflux family, which may help target binding sites and transcription factors. Further, AtMTP1 shows close similarities with Glycine max and Medicago trunculata. Interactome analysis suggests the association of AtMTP1 with cadmium/zinc-transporting ATPase and ZIP metal ion transporter. The AtMTP1 network is predominantly connected to cadmium/zinc-transporting ATPase (heavy metal ATPase 2, HMA2; heavy metal ATPase 3, HMA3; heavy metal ATPase 4, HMA4), cation efflux protein (MTP11), and metal tolerance protein C3 (AT4G58060). The Genevestigator predicts the high expression potential of AtMTP1 in the apical root during senescence, seedling, and bolting stages in an association with 11 co-expressed genes, mainly linked to estradiol toxicity and heat stress. Besides, AtMTP1 protein homologs possess conserved N-glyco motifs and physicochemical properties. The similarity and interactions of AtMTP1 gene with other genes suggest that Zn homeostasis in plants is associated with the regulation of different genes. These findings may advance our understanding to further develop plants capable of maintaining Zn homeostasis under adverse conditions.

Transcriptome analysis of Escherichia coli K1 after therapy with hesperidin conjugated with silver nanoparticles

BackgroundsEscherichia coli K1 causes neonatal meningitis. Transcriptome studies are indispensable to comprehend the pathology and biology of these bacteria. Recently, we showed that nanoparticles loaded with Hesperidin are potential novel antibacterial agents against E. coli K1. Here, bacteria were treated with and without Hesperidin conjugated with silver nanoparticles, and silver alone, and 50% minimum inhibitory concentration was determined. Differential gene expression analysis using RNA-seq, was performed using Degust software and a set of genes involved in cell stress response and metabolism were selected for the study.Results50% minimum inhibitory concentration with silver-conjugated Hesperidin was achieved with 0.5 μg/ml of Hesperidin conjugated with silver nanoparticles at 1 h. Differential genetic analysis revealed the expression of 122 genes (≥ 2-log FC, P< 0.01) in both E. coli K1 treated with Hesperidin conjugated silver nanoparticles and E. coli K1 treated with silver alone, compared to untreated E. coli K1. Of note, the expression levels of cation efflux genes (cusA and copA) and translocation of ions, across the membrane genes (rsxB) were found to increase 2.6, 3.1, and 3.3- log FC, respectively. Significant regulation was observed for metabolic genes and several genes involved in the coordination of flagella.ConclusionsThe antibacterial mechanism of nanoparticles maybe due to disruption of the cell membrane, oxidative stress, and metabolism in E. coli K1. Further studies will lead to a better understanding of the genetic mechanisms underlying treatment with nanoparticles and identification of much needed novel antimicrobial drug candidates.