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Context No consensus exists about the optimal duration of the low-iodine diet (LID) in the preparation of 131I therapy in differentiated thyroid cancer (DTC) patients. Objective This work aimed to investigate if a LID of 4 days is enough to achieve adequate iodine depletion in preparation for 131I therapy. In addition, the nutritional status of the LID was evaluated. Methods In this prospective study, 65 DTC patients treated at 2 university medical centers were included between 2018 and 2021. The patients collected 24-hour urine on days 4 and 7 of the LID and kept a food diary before and during the LID. The primary outcome was the difference between the 24-hour urinary iodine excretion (UIE) on both days. Results The median 24-hour UIE on days 4 and 7 of the LID were not significantly different (36.1 mcg [interquartile range, 25.4-51.2 mcg] and 36.5 mcg [interquartile range, 23.9-47.7 mcg], respectively, P = .43). On day 4 of the LID, 72.1% of the DTC patients were adequately prepared (24-hour UIE Conclusion The 24-hour UIE on day 4 of the LID did not differ from day 7, and therefore shortening the LID from 7 to 4 days seems justified to prepare DTC patients for 131I therapy in areas with sufficient iodine intake and may be beneficial to maintain a sufficient nutritional intake during DTC treatment.

The sodium-iodide symporter (NIS, SLC5A5) is expressed at the basolateral membrane of the thyroid follicular cell, and facilitates the thyroidal iodide uptake required for thyroid hormone biosynthesis. Biallelic loss-of-function mutations in NIS are a rare cause of dyshormonogenic congenital hypothyroidism. Affected individuals typically exhibit a normally sited, often goitrous thyroid gland, with absent uptake of radioiodine in the thyroid and other NIS-expressing tissues. We report a novel homozygous NIS mutation (c.1067 C>T, p.S356F) in four siblings from a consanguineous Indian kindred, presenting with significant hypothyroidism. Functional characterization of the mutant protein demonstrated impaired plasma membrane localization and cellular iodide transport.

Monodirectional tissue P systems with promoters are natural inspired parallel computing paradigms, where only symport rules are permitted, and with the restriction of “monodirectionality”, objects for two given regions are transferred in one direction. In this article, a novel kind of P systems, monodirectional evolutional symport tissue P systems with promoters (MESTP P systems) is raised, where objects may be revised during the movement between two regions. The computational theory of MESTP P systems that rules are employed in a flat maximally parallel pattern is investigated. We prove that finite natural number sets are created by MESTP P systems applying one cell, at most 1 promoter and all evolutional symport rules having a maximal length 2 or with arbitrary number of cells, promoters and all evolutional symport rules having a maximal length 2. MESTP P systems are Turing universal when two cells, at most 1 promoter and all evolutional symport rules having a maximal length 2 are employed. In addition, with the help of cell division mechanism, monodirectional evolutional symport tissue P systems with promoters and cell division (MESTPD P systems) are employed to solve NP -complete (the SAT ) problem, where system uses at most 1 promoter and all evolutional symport rules having a maximal length 3. These results show that MESTP(D) P systems are still computationally powerful even if monodirectionality control mechanism is imposed, thereby developing membrane algorithms for MESTP(D) P systems is theoretically possible as well as potentially exploitable.

More than one third of proteins require metal ions to accomplish their functions, making them obligatory for the growth and survival of microorganisms in varying environmental niches. In prokaryotes, besides their involvement in various cellular and physiological processes, metal ions stimulate the uptake of citrate molecules. Citrate is a source of carbon and energy and is reported to be transported by secondary transporters. In Gram-positive bacteria, citrate molecules are transported in complex with divalent metal ions, whereas in Gram-negative bacteria they are translocated by Na+/citrate symporters. In this study, the presence of a novel divalent-metal-ion-complexed citrate-uptake system that belongs to the primary active ABC transporter superfamily is reported. For uptake, the metal-ion-complexed citrate molecules are sequestered by substrate-binding proteins (SBPs) and transferred to transmembrane domains for their transport. This study reports crystal structures of an Mg2+–citrate-binding protein (MctA) from the Gram-negative thermophilic bacterium Thermus thermophilus HB8 in both apo and holo forms in the resolution range 1.63–2.50 Å. Despite binding various divalent metal ions, MctA possesses the coordination geometry to bind its physiological metal ion, Mg2+. The results also suggest an extended subclassification of cluster D SBPs, which are known to bind and transport divalent-metal-ion-complexed citrate molecules. Comparative assessment of the open and closed conformations of the wild-type and mutant MctA proteins suggests a gating mechanism of ligand entry following an `asymmetric domain movement' of the N-terminal domain for substrate binding.

The divalent anion sodium symporter (DASS) family of transporters (SLC13 family in humans) are key regulators of metabolic homeostasis, disruption of which results in protection from diabetes and obesity, and inhibition of liver cancer cell proliferation. Thus, DASS transporter inhibitors are attractive targets in the treatment of chronic, age-related metabolic diseases. The characterisation of several DASS transporters has revealed variation in the substrate selectivity and flexibility in the coupling ion used to power transport. Here, using the model DASS co-transporter, VcINDY from Vibrio cholerae, we have examined the interplay of the three major interactions that occur during transport: the coupling ion, the substrate, and the lipid environment. Using a series of high-throughput thermostability-based interaction assays, we have shown that substrate binding is Na+-dependent; a requirement that is orchestrated through a combination of electrostatic attraction and Na+-induced priming of the binding site architecture. We have identified novel DASS ligands and revealed that ligand binding is dominated by the requirement of two carboxylate groups in the ligand that are precisely distanced to satisfy carboxylate interaction regions of the substrate-binding site. We have also identified a complex relationship between substrate and lipid interactions, which suggests a dynamic, regulatory role for lipids in VcINDY's transport cycle.

Anaplastic thyroid cancer (ATC) is a rare but highly lethal disease. ATCs are resistant to standard therapies and are extremely difficult to manage. The stepwise cell dedifferentiation results in the impairment of the iodine-metabolizing machinery and the infeasibility of radioiodine treatment in ATC. Hence, reinducing iodine-metabolizing gene expression to restore radioiodine avidity is considered as a promising strategy to fight against ATC. In the present study, capsaicin (CAP), a natural potent transient receptor potential vanilloid type 1 (TRPV1) agonist, was discovered to reinduce ATC cell differentiation and to increase the expression of thyroid transcription factors (TTFs including TTF-1, TTF-2, and PAX8) and iodine-metabolizing proteins, including thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase, and sodium iodine symporter (NIS), in two ATC cell lines, 8505C and FRO. Strikingly, CAP treatment promoted NIS glycosylation and its membrane trafficking, resulting in a significant enhancement of radioiodine uptake of ATC cells in vitro. Mechanistically, CAP-activated TRPV1 channel and subsequently triggered Ca2+ influx, cyclic adenosine monophosphate (cAMP) generation, and cAMP-responsive element-binding protein (CREB) signal activation. Next, CREB recognized and bound to the promoter of SLC5A5 to facilitate its transcription. Moreover, the TRPV1 antagonist CPZ, the calcium chelator BAPTA, and the PKA inhibitor H-89 effectively alleviated the redifferentiation exerted by CAP, demonstrating that CAP might improve radioiodine avidity through the activation of the TRPV1‒Ca2+/cAMP/PKA/CREB signaling pathway. In addition, our study indicated that CAP might trigger a novel cascade to redifferentiate ATC cells and provide unprecedented opportunities for radioiodine therapy in ATC, bypassing canonical TSH‒TSHR pathway.

Symport/antiport P systems with membrane division are parallel computing models, which can solve NP-complete problems in polynomial time. In this paper, promoters are incorporated into symport/antiport P systems with membrane division, and a new kind of P systems, called symport/antiport P systems with promoters and membrane division (SAPD P systems, for short) is proposed. The computational efficiency of SAPD P systems is investigated. We show that a uniform solution to the QSAT problem is given by using only symport rules of length at most 2 and promoters of length at most 1 in a polynomial time (Note that the QSAT problem can be solved by symport/antiport P systems with membrane division using both symport rules and antiport rules of length at most 3 (Song et al. in BioSystems 130: 51–58, 2015)).

For the past 80 years, radioiodine (131I) has been used to ablate thyroid tissue not removed by surgery or to treat differentiated thyroid cancer that has metastasized to other parts of the body. However, the Na+/I− symporter (NIS), which mediates active iodide uptake into thyroid follicular cells, is also expressed in several non-thyroidal tissues. This NIS expression permits 131I accumulation and radiation damage in these non-target tissues, which accounts for the adverse effects of radioiodine therapy. We will review the data regarding the expression, function, and regulation of NIS in non-thyroidal tissues and explain the seemingly paradoxical adverse effects induced by 131I, the self-limited gastrointestinal adverse effects in contrast to the permanent salivary dysfunction that is seen after 131I therapy. We propose that prospective studies are needed to uncover the time-course of pathological processes underlying development and progression or ultimate resolution of 131I-induced salivary ductal obstruction and nasolacrimal duct obstruction. Finally, preventive measures and early therapeutic interventions that can be applied potentially to eliminate or alleviate long-term radioiodine adverse effects will be discussed.

The divalent anion sodium symporter (DASS) family contains both sodium-driven anion co-transporters and anion/anion exchangers. The family belongs to a broader Ion Transporter Superfamily (ITS), which comprises 24 families of transporters, including those of AbgT antibiotic efflux transporters. The human proteins in the DASS family play major physiological roles and are drug targets. We recently determined multiple structures of the human sodium-dependent citrate transporter (NaCT) and the succinate/dicarboxylate transporter from Lactobacillus acidophilus (LaINDY). Structures of both proteins show high degrees of structural similarity to the previously-determined VcINDY fold. Conservation between these DASS protein structures and those from the AbgT familiy indicates that the VcINDY fold represents the overall protein structure for the entire ITS superfamily. The new structures of NaCT and LaINDY are captured in the inward- or outward-facing conformations, respectively. The domain arrangements in these structures agree with a rigid-body elevator-type transport mechanism for substrate translocation across the membrane. Two separate NaCT structures in complex with a substrate or an inhibitor allowed us to explain the inhibition mechanism and propose a detailed classification scheme for grouping disease-causing mutations in the human protein. Structural understanding of multiple kinetic states of DASS proteins is a first step towards the detailed characterization of their entire transport cycle.

In the CNS, amino acid (AA) neurotransmitters and neurotransmitter precursors are subject to tight homeostatic control mediated by blood-brain barrier (BBB) solute carrier amino acid transporters (AATs). Since the BBB is composed of multiple closely apposed cell types and opportunities for human in vivo studies are limited, we used in vitro and computational approaches to investigate human BBB AAT activity and regulation. Quantitative real-time PCR (qPCR) of the human BBB endothelial cell model hCMEC/D3 (D3) was used to determine expression of selected AAT, tight junction (TJ), and signal transduction (ST) genes under various culture conditions. L-leucine uptake data were interrogated with a computational model developed by our group for calculating AAT activity in complex cell cultures. This approach is potentially applicable to in vitro cell culture drug studies where multiple “receptors” may mediate observed responses. Of 7 Leu AAT genes expressed by D3 only the activity of SLC7A5-SLC3A2/LAT1-4F2HC (LAT1), SLC43A2/LAT4 (LAT4) and sodium-dependent AATs, SLC6A15/B0AT2 (B0AT2), and SLC7A7/y+LAT1 (y+LAT1) were calculated to be required for Leu uptake. Therefore, D3 Leu transport may be mediated by a potentially physiologically relevant functional cooperation between the known BBB AAT, LAT1 and obligatory exchange (y+LAT1), facilitative diffusion (LAT4), and sodium symporter (B0AT2) transporters.

Riboflavin transporter deficiency 2 (RTD2) is a rare neurological disorder caused by mutations in the Solute carrier family 52 member 2 (Slc52a2) gene encoding human riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed and mediates tissue distribution of riboflavin, a water-soluble vitamin that, after conversion into FMN and FAD, plays pivotal roles in carbohydrate, protein, and lipid metabolism. The 3D structure of RFVT2 has been constructed by homology modeling using three different templates that are equilibrative nucleoside transporter 1 (ENT1), Fucose: proton symporter, and glucose transporter type 5 (GLUT5). The structure has been validated by several approaches. All known point mutations of RFVT2, associated with RTD2, have been localized in the protein 3D model. Six of these mutations have been introduced in the recombinant protein for functional characterization. The mutants W31S, S52F, S128L, L312P, C325G, and M423V have been expressed in E. coli, purified, and reconstituted into proteoliposomes for transport assay. All the mutants showed impairment of function. The Km for riboflavin of the mutants increased from about 3 to 9 times with respect to that of WT, whereas Vmax was only marginally affected. This agrees with the improved outcome of most RTD2 patients after administration of high doses of riboflavin.

Nitrates are natural compounds present in soil and water; however, the intense use of fertilizers has increased their presence in groundwater with deleterious effects on human health. There is evidence of nitrates acting as endocrine disruptors; however, the underlying molecular mechanisms have not been fully described. Here, we investigated the effect of subchronic exposure to different concentrations of sodium nitrate in female Wistar rats, evaluating thyroid hormonal parameters, such as Nis transporter (Na+ /I- symporter, Slc5a5) and Tsh-R receptor protein expression, as well as transcription of the Tpo (thyroperoxidase), Tg (tiroglobulin), Duox2 (dual oxidase 2), Pds (pendrin), and Mct8 (Mct8 transporter, Slc16a2) genes. Hematological and histochemical changes in the liver and thyroid were also explored. Significant differences were found in platelet and leukocyte counts; although a significant increase in the weight of the thyroid gland was observed, no differences were found in the levels of the hormones Tsh, T3, and T4, but a modulation of the mRNA expression of the Tg, Tpo, Duox2, Mct8, and Pds genes was observed. Morphological changes were also found in liver and thyroid tissue according to the exposure doses. In conclusion, subchronic exposure to sodium nitrate induces leukocytosis consistent with an inflammatory response and upregulation of Sod2 in the liver and increases the expression of genes involved in the synthesis of thyroid hormones, keeping thyroid hormone levels stable. Histological changes in the thyroid gland suggest a goitrogenic effect.

Sodium-coupled neurotransmitter transporters play a fundamental role in the termination of synaptic neurotransmission, which makes them a major drug target. The reconstitution of these secondary active transporters into liposomes has shed light on their molecular transport mechanisms. From the earliest days of the reconstitution technique up to today’s single-molecule studies, insights from live functioning transporters have been indispensable for our understanding of their physiological impact. The two classes of sodium-coupled neurotransmitter transporters, the neurotransmitter: sodium symporters and the excitatory amino acid transporters, have vastly different molecular structures, but complementary proteoliposome studies have sought to unravel their ion-dependence and transport kinetics. Furthermore, reconstitution experiments have been used on both protein classes to investigate the role of e.g. the lipid environment, of posttranslational modifications, and of specific amino acid residues in transport. Techniques that allow the detection of transport at a single-vesicle resolution have been developed, and single-molecule studies have started to reveal single transporter kinetics, which will expand our understanding of how transport across the membrane is facilitated at protein level. Here, we review a selection of the results and applications where the reconstitution of the two classes of neurotransmitter transporters has been instrumental.

Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A., Guan and Hariharan report two crystal structures of melibiose transporter MelB in complex with substrate analogs, nitrophenyl-galactoside, and dodecyl-melibioside. Both structures revealed similar specific site for sugar recognition and resolved the cation-binding pocket, advancing the understanding of MelB and related transporters.

The secondary-active Na-K-Cl cotransporter 1 (NKCC1), member of the cation-chloride cotransporter (CCC) family, ensures the electroneutral movement of Cl-, Na+, and K+ ions across cellular membranes. NKCC1 regulates Cl- homeostasis and cell volume, handling a pivotal role in transepithelial water transport and neuronal excitability. Aberrant NKCC1 transport is hence implicated in a variety of human diseases (hypertension, renal disorders, neuropathies, and cancer). Building on the newly resolved NKCC1 cryo-EM structure, all-atom enhanced sampling simulations unprecedentedly unlock the mechanism of NKCC1-mediated ion transport, assessing the order and the molecular basis of its interdependent ion translocation. Our outcomes strikingly advance the understanding of the physiological mechanism of CCCs and disclose a key role of CCC-conserved asparagine residues, whose side-chain promiscuity ensures the transport of both negatively and positively charged ions along the same translocation route. This study sets a conceptual basis to devise NKCC-selective inhibitors to treat diseases linked to Cl- dishomeostasis.

BACKGROUND Volatile anesthetic exposure during development leads to long-term cognitive deficits in rats which are dependent on age and sex. Female rats are protected relative to male rats for the same exposure on postnatal day 7. Here we test our hypothesis that androgens can modulate chloride cotransporter expression to alter the susceptibility to neurotoxicity from GABAergic drugs using female rats with exogenous testosterone exposure. METHODS Female rats were injected with testosterone (100 μg/animal) or vehicle on postnatal days 1 to 6. On postnatal day 7, the animals were randomized to either isoflurane exposure or sham. Spatial memory was assessed with the Barnes maze starting on postnatal day 41. Western blots were run from testosterone treated postnatal day 7 animals to measure levels of chloride cotransporters sodium-potassium-chloride symporter (NKCC1) and chloride-potassium symporter 5 (KCC2). RESULTS Exogenous testosterone modulated isoflurane anesthetic neurotoxicity in female rats based on poor performance in the probe trial of the Barnes Maze. By contrast, females with vehicle and isoflurane exposure were able to differentiate the goal position. These behavioral differences corresponded to differences in the protein levels of NKCC1 and KCC2 after exogenous testosterone exposure, with NKCC1 increasing (P

Na+, Cl− and K+ are the most abundant electrolytes present in biological fluids that are essential to the regulation of pH homeostasis, membrane potential and cell volume in living organisms. Herein, we report synthetic crown ether-thiourea conjugates as a cation/anion symporter, which can transport both Na+ and Cl− across lipid bilayers with relatively high transport activity. Surprisingly, the ion transport activities were diminished when high concentrations of K+ ions were present outside the vesicles. This unusual behavior resulted from the strong affinity of the transporters for K+ ions, which led to predominant partitioning of the transporters as the K+ complexes in the aqueous phase preventing the transporter incorporation into the membrane. Synthetic membrane transporters with Na+, Cl− and K+ transport capabilities may have potential biological and medicinal applications. [Figure not available: see fulltext.]

Acute kidney injury (AKI) diagnosis relies on plasma creatinine concentration (Crpl), a relatively insensitive, surrogate biomarker of glomerular filtration rate that increases only after significant damage befalls. However, damage in different renal structures may occur without increments in Crpl, a condition known as subclinical AKI. Thus, detection of alterations in other aspects of renal function different from glomerular filtration rate must be included in an integral diagnosis of AKI. With this aim, we adapted to and validated in rats (for preclinical research) the furosemide stress test (FST), a tubular function test hitherto performed only in humans. We also tested its sensitivity in detecting subclinical tubular alterations. In particular, we predisposed rats to AKI with 3 mg/kg cisplatin and subsequently subjected them to a triggering insult (ie, 50 mg/kg/d gentamicin for 6 days) that had no effect on nonpredisposed animals but caused an overt AKI in predisposed rats. The FST was performed immediately before adding the triggering insult. Predisposed animals showed a reduced response to the FST (namely, reduced furosemide-induced diuresis and K+ excretion), whereas nonpredisposed animals showed no alteration, compared to the controls. Computational modeling of epithelial transport of solutes and water along the nephrons applied to experimental data suggested that proximal tubule transport was only minimally reduced, the sodium-chloride symporter was upregulated by 50%, and the renal outer medullary potassium channel was downregulated by 85% in predisposed animals. In conclusion, serial coupling of the FST and computational modeling may be used to detect and localize subclinical tubular alterations.

Baruch Kanner and this author have had parallel careers investigating neurotransmitter transporters. At multiple times during their careers, they have found themselves collaborating or competing, but always learning from each other. This commentary elaborates on the interactions between the Kanner and Rudnick laboratories, with a focus on transporters in the Neurotransmitter: Sodium Symporter (NSS) family of amino acid and amine transporters. A key focus of these interactions is the mechanism by which chloride ions activate and drive transport.

Context Iodide transport defect (ITD) (Online Mendelian Inheritance in Man No. 274400) is an uncommon cause of dyshormonogenic congenital hypothyroidism due to loss-of-function variants in the SLC5A5 gene, which encodes the sodium/iodide symporter (NIS), causing deficient iodide accumulation in thyroid follicular cells. Objective This work aims to determine the molecular basis of a patient’s ITD clinical phenotype. Methods The propositus was diagnosed with dyshormonogenic congenital hypothyroidism with minimal 99mTc-pertechnetate accumulation in a eutopic thyroid gland. The propositus SLC5A5 gene was sequenced. Functional in vitro characterization of the novel NIS variant was performed. Results Sanger sequencing revealed a novel homozygous missense p.G561E NIS variant. Mechanistically, the G561E substitution reduces iodide uptake, because targeting of G561E NIS to the plasma membrane is reduced. Biochemical analyses revealed that G561E impairs the recognition of an adjacent tryptophan-acidic motif by the kinesin-1 subunit kinesin light chain 2 (KLC2), interfering with NIS maturation beyond the endoplasmic reticulum, and reducing iodide accumulation. Structural bioinformatic analysis suggests that G561E shifts the equilibrium of the unstructured tryptophan-acidic motif toward a more structured conformation unrecognizable to KLC2. Consistently, knockdown of Klc2 causes defective NIS maturation and consequently decreases iodide accumulation in rat thyroid cells. Morpholino knockdown of klc2 reduces thyroid hormone synthesis in zebrafish larvae leading to a hypothyroid state as revealed by expression profiling of key genes related to the hypothalamic-pituitary-thyroid axis. Conclusion We report a novel NIS pathogenic variant associated with dyshormonogenic congenital hypothyroidism. Detailed molecular characterization of G561E NIS uncovered the significance of KLC2 in thyroid physiology.

The SLC25A22 (Solute Carrier Family 25, Member 22) gene encodes for a mitochondrial glutamate/H+ symporter and is involved in the mitochondrial transport of metabolites across the mitochondrial membrane. We hereby report a 12-year-old girl presenting with early-onset epileptic encephalopathy, hypotonia, and global developmental delay. Whole exome sequencing identified a novel homozygous missense mutation in SLC25A22 gene (c.97A>G; p.Lys33Glu), as the likely cause of the disease. The phenotype of our patient and EEG recordings do not completely overlap with the phenotypes previously described, leading to a new and more complex form of disease associated with SLC25A22 variants, characterized by dyskinetic movements and oculogyric crisis.

The Picea glauca genome contains a locus that encodes for a nucleobase cation symporter 1 (PgNCS1). As a gymnosperm, P. glauca belongs to a key taxonomic position for an ongoing evolution-function analysis of viridiplantae nucleobase cation symporter 1 proteins (NCS1). Here the solute transport and binding properties for PgNCS1 are determined through heterologous expression in Saccharomyces cerevisiae strains deficient in functional NCS1 loci. PgNCS1 displays a broad, yet unique, solute specificity profile –common with other plant NCS1. Yeast containing PgNCS1 transport adenine, guanine, hypoxanthine, xanthine and uracil and are sensitive to growth on 8-azaadenine. Neither cytosine nor 5 flourocytosine are transported by PgNCS1 but along with caffeine and uric acid, act as competitive inhibitors of [3H]-adenine and [3H]-hypoxanthine uptake. This transporter displays high affinity for adenine (Km = 2.67 μM), guanine (Ki = 1.71 μM) and hypoxanthine (Ki = 1.82 μM) but lesser affinity for xanthine (Ki = 5.36 μM). Arabidopsis plants that are deficient in their endogenous NCS1, yet carry PgNCS1, show significant uptake of [3H]-adenine. The results support previous studies and together confirm a broad nucleobase transport and binding pattern for plant NCS1 across the viridiplantae. PgNCS1 displays a broad yet unique nucleosbase transport and binding profile. It transports adenine, guanine, hypoxanthine, xanthine and uracil but only binds cytosine, caffeine, and uric acid.

P-type H+ ATPases mediate active H+ efflux from plant cells. They generate a proton motive force across the plasma membrane, providing the free energy to drive the transport of other solutes, partly by coupling to H+ influx. Wegner & Shabala (2020) recently suggested that passive H+ influx can exceed pump-driven efflux due to 'active buffering', that is, cytosolic H+ scavenging and apoplastic H+ generation by metabolism ('biochemical pH clamp'). Charge balance is provided by K+ efflux or anion influx. Here, this hypothesis is extended to net H+ efflux: even though H+ pumping is faster than backflow via symporters and antiporters, a progressive increase in the transmembrane pH gradient is avoided. Cytosolic H+ release is associated with bicarbonate formation from CO2 . Bicarbonate serves as substrate for the PEPCase, catalyzing the reaction from phosphoenolpyruvate to oxaloacetate, which is subsequently reduced to malate. Organic anions such as malate and citrate are released across the plasma membrane and are (partly) protonated in the apoplast, thus limiting pump-induced acidification. Moreover, a 'biophysical pH clamp' is introduced, that is, adjustment of apoplastic/cytosolic pH involving net H+ fluxes across the plasma membrane, while the gradient between compartments is maintained. The clamps are not mutually exclusive but are likely to coexist.

Obligate biotrophic pathogens like the pea powdery mildew© (PM) Erysiphe pisi establish long-term feeding relationships with their host, during which they siphon sugars from host cells through haustoria. Plants in turn deploy sugar transporters to restrict carbon allocation toward pathogens, as a defense mechanism. Studies in Arabidopsis have shown that sugar transport protein 13 (STP13), a proton-hexose symporter involved in apoplasmic hexose retrieval, contributes to bacterial and necrotrophic fungal resistance by limiting sugar flux toward these pathogens. By contrast, expression of Lr67res,a transport-deficient wheat STP13 variant harboring two amino acid substitutions (G144R and V387L), conferred resistance against biotrophic fungi in wheat and barley, indicating its broad applicability in disease management. Here, we investigated the role of STP13 and STP13G144R in legume–PM interactions. We show that Medicago truncatula STP13.1 is a proton-hexose symporter involved in basal resistance against PM and indirectly show that Lr67res-mediated PM resistance, so far reported only in monocots, is transferable to legumes. Among the 30 MtSTPs, STP13.1 exhibited the highest fold induction in PM-challenged leaves and was also responsive to chitosan, ABA and sugar treatment. Functional assays in yeast showed that introduction of the G144R mutation but not V388L abolished MtSTP13.1’s hexose uptake ability. Virus-induced gene silencing of MtSTP13 repressed pathogenesis-related (PR) gene expression and enhanced PM susceptibility in M. truncatula whereas transient overexpression of MtSTP13.1 or MtSTP13.1G144R in pea induced PR and isoflavonoid pathway genes and enhanced PM resistance. We propose a model in which STP13.1-mediated sugar signaling triggers defense responses against PM in legumes.

BACKGROUND: Decreased Natrium iodide symporter (NIS) expression levels or diminished NIS targeting thyroid cancer cells’ plasma membrane leads to radioiodine-refractory disease. AIM: The aim of this study was to analyze the NIS expression in thyroid tumors. MATERIALS AND METHODS: The samples were thyroid tissues of patients who underwent surgery for a thyroid tumor. The tissues were processed for NIS protein expressions by immunohistochemistry (IHC) and Western blot (WB). Graves’ disease samples were used as positive controls. The samples were incubated without the primary antibody, and they were used as negative controls for IHC examination. Na+/K+ ATPase was a plasma membrane protein marker in the WB procedure. RESULTS: Twenty-nine samples were assessed for NIS protein. All of them showed the expression in the cytoplasm with intensity 1+ to 3+ with Allred score 3-8. Fourteen out of 29 cases (48.2%) showed NIS cytoplasm staining intensity ≥2+ consist of 10 papillary thyroid cancer (PTC), three follicular thyroid cancer, and one adenoma. Membrane staining was found in 2 samples of PTC (6.9%). Six samples (adenoma 1 sample, PTC 5 samples) showed NIS expression at membrane very weak (1+); they were considered as negative. NIS protein has several bands of ~ 80 kDa, ~ 62 kDa, and ~ 49 kDa. CONCLUSION: NIS expression in thyroid cancer mostly expresses in the cytoplasm instead of the membrane. NIS will play a functional role in the membrane to bring iodine across the membrane against the concentration. It can be the main reason for the lack of response of radioiodine in some differentiated thyroid cancers.

Iodine transportation is an important step in thyroid hormone biosynthesis. Uptake of iodine into the thyroid follicle is mediated mainly by the basolateral sodium-iodide symporter (NIS or solute carrier family 5 member 5: SLC5A5), and iodine efflux across the apical membrane into the follicular lumen is mediated by pendrin (SLC26A4). In addition to these transporters, SLC26A7, which has recently been identified as a causative gene for congenital hypothyroidism, was found to encode a novel apical iodine transporter in the thyroid. Although SLC5A5 and SLC26A4 have been well-characterized, little is known about SLC26A7, including its regulation by TSH, the central hormone regulator of thyroid function. Using rat thyroid FRTL-5 cells, we showed that the mRNA levels of Slc26a7 and Slc26a4, two apical iodine transporters responsible for iodine efflux, were suppressed by TSH, whereas the mRNA level of Slc5a5 was induced. Forskolin and dibutyryl cAMP (dbcAMP) had the same effect as that of TSH on the mRNA levels of these transporters. TSH, forskolin and dbcAMP also had suppressive effects on SLC26A7 promoter activity, as assessed by luciferase reporter gene assays, and protein levels, as determined by Western blot analysis. TSH, forskolin and dbcAMP also induced strong localization of Slc26a7 to the cell membrane according to immunofluorescence staining and confocal laser scanning microscopy. Together, these results suggest that TSH suppresses the expression level of Slc26a7 but induces its accumulation at the cell membrane, where it functions as an iodine transporter.

The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site–associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.

K(+) and NO(3)(−) are the major forms of potassium and nitrogen that are absorbed by the roots of most terrestrial plants. In this study, we observed that a close relationship between NO(3)(−) and K(+) in Arabidopsis (Arabidopsis thaliana) is mediated by NITRATE TRANSPORTER1.1 (NRT1.1). The nrt1.1 knockout mutants showed disturbed K(+) uptake and root-to-shoot allocation, and were characterized by growth arrest under K(+)-limiting conditions. The K(+) uptake and root-to-shoot allocation of these mutants were partially recovered by expressing NRT1.1 in the root epidermis-cortex and central vasculature using SULFATE TRANSPORTER1;2 and PHOSPHATE1 promoters, respectively. Two-way analysis of variance based on the K(+) contents in nrt1.1-1/K(+) transporter1, nrt1.1-1/high-affinity K(+) transporter5-3, nrt1.1-1/K(+) uptake permease7, and nrt1.1-1/stelar K(+) outward rectifier-2 double mutants and the corresponding single mutants and wild-type plants revealed physiological interactions between NRT1.1 and K(+) channels/transporters located in the root epidermis–cortex and central vasculature. Further study revealed that these K(+) uptake-related interactions are dependent on an H(+)-consuming mechanism associated with the H(+)/NO(3)(−) symport mediated by NRT1.1. Collectively, these data indicate that patterns of NRT1.1 expression in the root epidermis–cortex and central vasculature are coordinated with K(+) channels/transporters to improve K(+) uptake and root-to-shoot allocation, respectively, which in turn ensures better growth under K(+)-limiting conditions.

Gut microbiome (GM) might be associated with radioiodine (RAI)-refractory papillary thyroid carcinoma (PTC) through different mechanisms related to sodium/iodide (Na+/I–) symporter (NIS) regulation. However, whether thyroid carcinoma (TC), especially RAI-refractory PTC, causes dysbiosis, or vice versa, is still unknown. Further studies are needed to investigate the mechanism between GM and RAI-refractory PTC.

Obesity is a major health problem worldwide, given its growing incidence and its association with a variety of comorbidities. Weight gain results from an increase in energy intake without a concomitant increase in energy expenditure. To combat the obesity epidemic, many studies have focused on the pathways underlying satiety and hunger signaling, while other studies have concentrated on the mechanisms involved in energy expenditure, most notably adaptive thermogenesis. Hypothyroidism in humans is typically associated with a decreased basal metabolic rate, lower energy expenditure, and weight gain. However, hypothyroid mouse models have been reported to have a leaner phenotype than euthyroid controls. To elucidate the mechanism underlying this phenomenon, we used a drug-free mouse model of hypothyroidism: mice lacking the sodium/iodide symporter (NIS), the plasma membrane protein that mediates active iodide uptake in the thyroid. In addition to being leaner than euthyroid mice, owing in part to reduced food intake, these hypothyroid mice show signs of compensatory up-regulation of the skeletal-muscle adaptive thermogenic marker sarcolipin, with an associated increase in fatty acid oxidation (FAO). Neither catecholamines nor thyroid-stimulating hormone (TSH) are responsible for sarcolipin expression or FAO stimulation; rather, thyroid hormones are likely to negatively regulate both processes in skeletal muscle. Our findings indicate that hypothyroidism in mice results in a variety of metabolic changes, which collectively lead to a leaner phenotype. A deeper understanding of these changes may make it possible to develop new strategies against obesity.

Sodium glucose transporters (SGLTs) belong to the mammalian solute carrier family SLC5. This family includes 12 different members in human that mediate the transport of sugars, vitamins, amino acids, or smaller organic ions such as choline. The SLC5 family belongs to the sodium symporter family (SSS), which encompasses transporters from all kingdoms of life. It furthermore shares similarity to the structural fold of the APC (amino acid-polyamine-organocation) transporter family. Three decades after the first molecular identification of the intestinal Na+-glucose cotransporter SGLT1 by expression cloning, many new discoveries have evolved, from mechanistic analysis to molecular genetics, structural biology, drug discovery, and clinical applications. All of these advances have greatly influenced physiology and medicine. While SGLT1 is essential for fast absorption of glucose and galactose in the intestine, the expression of SGLT2 is largely confined to the early part of the kidney proximal tubules, where it reabsorbs the bulk part of filtered glucose. SGLT2 has been successfully exploited by the pharmaceutical industry to develop effective new drugs for the treatment of diabetic patients. These SGLT2 inhibitors, termed gliflozins, also exhibit favorable nephroprotective effects and likely also cardioprotective effects. In addition, given the recent finding that SGLT2 is also expressed in tumors of pancreas and prostate and in glioblastoma, this opens the door to potential new therapeutic strategies for cancer treatment by specifically targeting SGLT2. Likewise, further discoveries related to the functional association of other SGLTs of the SLC5 family to human pathologies will open the door to potential new therapeutic strategies. We furthermore hope that the herein summarized information about the physiological roles of SGLTs and the therapeutic benefits of the gliflozins will be useful for our readers to better understand the molecular basis of the beneficial effects of these inhibitors, also in the context of the tubuloglomerular feedback (TGF), and the renin-angiotensin system (RAS). The detailed mechanisms underlying the clinical benefits of SGLT2 inhibition by gliflozins still warrant further investigation that may serve as a basis for future drug development.

BACKGROUND Elobixibat, a novel inhibitor of apical sodium-dependent bile acid transporter for treating chronic constipation, increases colonic bile acid concentrations, stimulating bowel function. However, it is not clear which bile acids are altered, or whether altered gut microbiota are associated with functional effects that may alter bowel function. AIMS To investigate the effects of elobixibat on changes in the faecal concentrations of total and individual bile acids and in faecal microbiota. METHODS This was a prospective, single-centre study. After baseline period, patients received 10 mg daily of elobixibat for 2 weeks. We evaluated the effects on bowel function, changes in faecal bile acid concentrations and composition of gut bacteria, before and after elobixibat administration. RESULTS In the 30 patients analysed, the frequency of pre- and post-treatment bowel movements per fortnight was 7 and 10 (P

Four cellulose membranes were grafted with glycine, L-alanine, L-serine, and L-threonine amino acids. The grafted membranes were characterized by different physicochemical techniques (ATR-IR, gravi...

Mediator complex subunit 16 (MED16) is a component of the mediator complex and functions as a coactivator in transcriptional events at almost all RNA polymerase II–dependent genes. In this study, we report that the expression of MED16 is markedly decreased in papillary thyroid cancer (PTC) tumors compared with normal thyroid tissues. In vitro, MED16 overexpression in PTC cells significantly inhibited cell migration, enhanced sodium/iodide symporter expression and iodine uptake, and decreased resistance to radioactive (131)I (RAI). Conversely, PTC cells in which MED16 had been further knocked down (MED16(KD)) exhibited enhanced cell migration, epithelial–mesenchymal transition, and RAI resistance, accompanied by decreased sodium/iodide symporter levels. Moreover, cell signaling through transforming growth factor β (TGF-β) was highly activated after the MED16 knockdown. Similar results were obtained in MED12(KD) PTC cells, and a co-immunoprecipitation experiment verified interactions between MED16 and MED12 and between MED16 and TGF-βR2. Of note, the application of LY2157299, a potent inhibitor of TGF-β signaling, significantly attenuated MED16(KD)-induced RAI resistance both in vitro and in vivo. In conclusion, our findings indicate that MED16 reduction in PTC contributes to tumor progression and RAI resistance via the activation of the TGF-β pathway.

Erwinia amylovora causes fire blight, one of the more serious diseases for apple and pear cultivation. Previous studies generated E. amylovora mutants in purine metabolic pathway that still retain the ability to grow on host tissue and produce limited disease symptoms. Here we show that the E. amylovora genome has a locus that encodes for a xanthine permease belonging to the nucleobase cation symporter 2 (NCS2) family and the encoded protein displays a high level of amino acid sequence similarity to the Escherichia coli XanP. Our hypothesis is to investigate if the lack of a xanthine transporter has an effect upon disease progression. Heterologous expression of EaXanP in nucleobase transporter-deficient E. coli strains, coupled with radiolabeled nucleobase uptake studies determined that EaXanP is a high affinity xanthine transporter with a Km of 1.2 μM + 0.1 μM that confers sensitivity to growth on caffeine (1,3,7-trimethylxanthine). An E. amylovora ΔxanP::Camr mutant shows resistance to growth on caffeine, while over expression of EaXanP increases growth sensitivity to caffeine. While the EaXanP gene is expressed in infected immature pear fruitlets, an E. amylovora xanthine transport mutant is still able to grow and cause disease symptoms on immature pears and apple fruitlets.

The Na+/I- symporter (NIS), the plasma membrane protein that actively transports I- (stoichiometry 2Na+:1I-) in thyroid physiology and radioiodide-based thyroid cancer treatment, also transports the environmental pollutant perchlorate (stoichiometry 1Na+:1ClO4-), which competes with I- for transport. Until now, the mechanism by which NIS transports different anion substrates with different stoichiometries has remained unelucidated. We carried out transport measurements and analyzed these using a statistical thermodynamics-based equation and electrophysiological experiments to show that the different stoichiometry of ClO4- transport is due to ClO4- binding to a high-affinity non-transport allosteric site that prevents Na+ from binding to one of its two sites. Furthermore, low concentrations of ClO4- inhibit I- transport not only by competition but also, critically, by changing the stoichiometry of I- transport to 1:1, which greatly reduces the driving force. The data reveal that ClO4- pollution in drinking water is more dangerous than previously thought.

Neurochemical research 47(1), 9-22 (2022). doi:10.1007/s11064-021-03252-x special issue: "Special Issue in Honor of Baruch Kanner", Published by Springer Science + Business Media B.V, Dordrecht [u.a.]

The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure-function relationships of NSS family proteins. With hydrogen-deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperature (25°C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 – 95°C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45°C and 60°C). We have further compared the conformational impact of binding the efficiently transported substrate, alanine (Ala) to the much slower transported leucine (Leu). At elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25°C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. Furthermore, comparing the HDX signature of Ala vs. Leu we observe distinct differences that could correspond to the faster transport rate (kcat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of LeuT in the presence of Ala. Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.