High levels of Mn2+ inhibit secretory pathway Ca2+/Mn2+‐ATPase (SPCA) activity and cause Golgi fragmentation in neurons and glia

Excess Mn²⁺ in humans causes a neurological disorder known as manganism, which shares symptoms with Parkinson's disease. However, the cellular mechanisms underlying Mn²⁺‐neurotoxicity and the involvement of Mn²⁺‐transporters in cellular homeostasis and repair are poorly understood and require further investigation. In this work, we have analyzed the effect of Mn²⁺ on neurons and glia from mice in primary cultures. Mn²⁺ overload compromised survival of both cell types, specifically affecting cellular integrity and Golgi organization, where the secretory pathway Ca²⁺/Mn²⁺‐ATPase is localized. This ATP‐driven Mn²⁺ transporter might take part in Mn²⁺ accumulation/detoxification at low loads of Mn²⁺, but its ATPase activity is inhibited at high concentration of Mn²⁺. Glial cells appear to be significantly more resistant to this toxicity than neurons and their presence in cocultures provided some protection to neurons against degeneration induced by Mn²⁺. Interestingly, the Mn²⁺ toxicity was partially reversed upon Mn²⁺ removal by wash out or by the addition of EDTA as a chelating agent, in particular in glial cells. These studies provide data on Mn²⁺ neurotoxicity and may contribute to explore new therapeutic approaches for reducing Mn²⁺ poisoning.     

The Shigella type III effector IpgD recodes Ca2+ signals during invasion of epithelial cells

The role of second messengers in the diversion of cellular processes by pathogens remains poorly studied despite their importance. Among these, Ca²⁺ virtually regulates all known cell processes, including cytoskeletal reorganization, inflammation, or cell death pathways. Under physiological conditions, cytosolic Ca²⁺ increases are transient and oscillatory, defining the so‐called Ca²⁺ code that links cell responses to specific Ca²⁺ oscillatory patterns. During cell invasion, Shigella induces atypical local and global Ca²⁺ signals. Here, we show that by hydrolyzing phosphatidylinositol‐(4,5)bisphosphate, the Shigella type III effector IpgD dampens inositol‐(1,4,5)trisphosphate (InsP₃) levels. By modifying InsP₃ dynamics and diffusion, IpgD favors the elicitation of long‐lasting local Ca²⁺ signals at Shigella invasion sites and converts Shigella‐induced global oscillatory responses into erratic responses with atypical dynamics and amplitude. Furthermore, IpgD eventually inhibits InsP₃‐dependent responses during prolonged infection kinetics. IpgD thus acts as a pathogen regulator of the Ca²⁺ code implicated in a versatility of cell functions. Consistent with this function, IpgD prevents the Ca²⁺‐dependent activation of calpain, thereby preserving the integrity of cell adhesion structures during the early stages of infection.     

Cellular localization of the K+‐dependent Na+–Ca2+ exchanger NCKX5 and the role of the cytoplasmic loop in its distribution in pigmented cells

NCKX5 is a bidirectional K⁺‐dependent Na⁺–Ca²⁺ exchanger, which belongs to the SLC24A gene family. In particular, the A111T mutation of NCKX5 has been associated with reduced pigmentation in European populations. In contrast to other NCKX isoforms, which function in the plasma membrane (PM), NCKX5 has been shown to localize either in the trans‐Golgi network (TGN) or in melanosomes. Moreover, sequences responsible for retaining its intracellular localization are unknown. This study addresses two major questions: (i) clarification of intracellular location of NCKX5 and (ii) identification of sequences that retain NCKX5 inside the cell. We designed a set of cDNA constructs representing NCKX5 loop deletion mutants and NCKX2–NCKX5 chimeras to address these two questions after expression in pigmented MNT1 cells. Our results show that NCKX5 is not a PM resident and is exclusively located in the TGN. Moreover, the large cytoplasmic loop is the determinant for retaining NCKX5 in the TGN.     

STIM1 activation of adenylyl cyclase 6 connects Ca2+ and cAMP signaling during melanogenesis

Endoplasmic reticulum (ER)–plasma membrane (PM) junctions form functionally active microdomains that connect intracellular and extracellular environments. While the key role of these interfaces in maintenance of intracellular Ca²⁺ levels has been uncovered in recent years, the functional significance of ER‐PM junctions in non‐excitable cells has remained unclear. Here, we show that the ER calcium sensor protein STIM1 (stromal interaction molecule 1) interacts with the plasma membrane‐localized adenylyl cyclase 6 (ADCY6) to govern melanogenesis. The physiological stimulus α‐melanocyte‐stimulating hormone (αMSH) depletes ER Ca²⁺ stores, thus recruiting STIM1 to ER‐PM junctions, which in turn activates ADCY6. Using zebrafish as a model system, we further established STIM1's significance in regulating pigmentation in vivo. STIM1 domain deletion studies reveal the importance of Ser/Pro‐rich C‐terminal region in this interaction. This mechanism of cAMP generation creates a positive feedback loop, controlling the output of the classical αMSH‐cAMP‐MITF axis in melanocytes. Our study thus delineates a signaling module that couples two fundamental secondary messengers to drive pigmentation. Given the central role of calcium and cAMP signaling pathways, this module may be operative during various other physiological processes and pathological conditions.     

Linking waterlogging tolerance with Mn2+ toxicity: a case study for barley

Vast agricultural areas are affected by flooding, causing up to 80% yield reduction and resulting in multibillion dollar losses. Up to now, the focus of plant breeders was predominantly on detrimental effects of anoxia, while other (potentially equally important) traits were essentially neglected; one of these is soil elemental toxicity. Excess water triggers a progressive decrease in soil redox potential, thus increasing the concentration of Mn²⁺ that can be toxic to plants if above a specific threshold. This work aimed to quantify the relative contribution of Mn²⁺ toxicity to waterlogging stress tolerance, using barley as a case study. Twenty barley (Hordeum vulgare) genotypes contrasting in waterlogging stress tolerance were studied for their ability to cope with toxic (1 mm ) amounts of Mn²⁺ in the root rhizosphere. Under Mn²⁺ toxicity, chlorophyll content of most waterlogging‐tolerant genotypes (TX9425, Yerong, CPI‐71284‐48 and CM72) remained above 60% of the control value, whereas sensitive genotypes (Franklin and Naso Nijo) had 35% less chlorophyll than 35% of controls. Manganese concentration in leaves was not related to visual Mn²⁺ toxicity symptoms, suggesting that various Mn²⁺ tolerance mechanisms might operate in different tolerant genotypes, i.e. avoidance versus tissue tolerance. The overall significant (r = 0.60) correlation between tolerance to Mn²⁺ toxicity and waterlogging in barley suggests that plant breeding for tolerance to waterlogging traits may be advanced by targeting mechanisms conferring tolerance to Mn²⁺ toxicity, at least in this species.     

Boron deficiency alters cytosolic Ca2+ concentration and affects the cell wall components of pollen tubes in Malus domestica

• Boron (B) is essential for normal plant growth, including pollen tube growth. B deficiency influences various physiological and metabolic processes in plants. However, the underlying mechanism of B deficiency in pollen tube growth is not sufficiently understood. In the present research, the influence of B deficiency on apple (Malus domestica) pollen tube growth was studied and the possible regulatory mechanism evaluated.
• Apple pollen grains were cultured under different concentrations of B. Scanning ion‐selective electrode technique, fluorescence labelling and Fourier‐transform infrared (FTIR) analysis were used to detect calcium ion flux, cytosolic Ca²⁺ concentration ([Ca²⁺]cyt), actin filaments and cell wall components of pollen tubes.
• B deficiency inhibited apple pollen germination and induced retardation of tube growth. B deficiency increased extracellular Ca²⁺ influx and thus led to increased [Ca²⁺]cyt in the pollen tube tip. In addition, B deficiency modified actin filament arrangement at the pollen tube apex. B deficiency also altered the deposition of pollen tube wall components. Clear differences were not observed in the distribution patterns of cellulose and callose between control and B deficiency treated pollen tubes. However, B deficiency affected distribution patterns of pectin and arabinogalactan proteins (AGP). Clear ring‐like signals of pectins and AGP on control pollen tubes varied according to B deficiency. B deficiency further decreased acid pectins, esterified pectins and AGP content at the tip of the pollen tube, which were supported by changes in chemical composition of the tube walls.
• B appears to have an active role in pollen tube growth by affecting [Ca²⁺]cyt, actin filament assembly and pectin and AGP deposition in the pollen tube. These findings provide valuable information that enhances our current understanding of the mechanism regulating pollen tube growth.

     

Identification of low Ca2+ stress‐induced embryo apoptosis response genes in Arachis hypogaea by SSH‐associated library lift (SSHaLL)

Calcium is a universal signal in the regulation of wide aspects in biology, but few are known about the function of calcium in the control of early embryo development. Ca²⁺ deficiency in soil induces early embryo abortion in peanut, producing empty pods, which is a general problem; however, the underlying mechanism remains unclear. In this study, embryo abortion was characterized to be caused by apoptosis marked with cell wall degradation. Using a method of SSH cDNA libraries associated with library lift (SSHaLL), 62 differentially expressed genes were isolated from young peanut embryos. These genes were classified to be stress responses, catabolic process, carbohydrate and lipid metabolism, embryo morphogenesis, regulation, etc. The cell retardation with cell wall degradation was caused by up‐regulated cell wall hydrolases and down‐regulated cellular synthases genes. HsfA4a, which was characterized to be important to embryo development, was significantly down‐regulated under Ca²⁺‐deficient conditions from 15 days after pegging (DAP) to 30 DAP. Two AhCYP707A4 genes, encoding abscisic acid (ABA) 8′‐hydroxylases, key enzymes for ABA catabolism, were up‐regulated by 21‐fold under Ca²⁺‐deficient conditions upstream of HsfA4a, reducing the ABA level in early embryos. Over‐expression of AhCYP707A4 in Nicotiana benthamiana showed a phenotype of low ABA content with high numbers of aborted embryos, small pods and less seeds, which confirms that AhCYP707A4 is a key player in regulation of Ca²⁺ deficiency‐induced embryo abortion via ABA‐mediated apoptosis. The results elucidated the mechanism of low Ca²⁺‐induced embryo abortion and described the method for other fields of study.     

Differential regulation of CaMKIIα interactions with mGluR5 and NMDA receptors by Ca2+ in neurons

Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca²⁺/calmodulin‐dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse‐enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state‐dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca²⁺/calmodulin binding and activation) loses its affinity for the receptor. Ca²⁺ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα‐binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca²⁺‐dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα‐sensitive site. Together, the long intracellular C‐terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5‐dependent Ca²⁺ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross‐talk between the two receptors.

     

OsVIL2 functions with PRC2 to induce flowering by repressing OsLFL1 in rice

Flowering is exquisitely regulated by both promotive and inhibitory factors. Molecular genetic studies with Arabidopsis have verified several epigenetic repressors that regulate flowering time. However, the roles of chromatin remodeling factors in developmental processes have not been well explored in Oryza sativa (rice). We identified a chromatin remodeling factor OsVIL2 (O. sativa VIN3‐LIKE 2) that promotes flowering. OsVIL2 contains a plant homeodomain (PHD) finger, which is a conserved motif of histone binding proteins. Insertion mutations in OsVIL2 caused late flowering under both long and short days. In osvil2 mutants OsLFL1 expression was increased, but that of Ehd1, Hd3a and RFT1 was reduced. We demonstrated that OsVIL2 is bound to native histone H3 in vitro. Chromatin immunoprecipitation analyses showed that OsVIL2 was directly associated with OsLFL1 chromatin. We also observed that H3K27me3 was significantly enriched by OsLFL1 chromatin in the wild type, but that this enrichment was diminished in the osvil2 mutants. These results indicated that OsVIL2 epigenetically represses OsLFL1 expression. We showed that OsVIL2 physically interacts with OsEMF2b, a component of polycomb repression complex 2. As observed from osvil2, a null mutation of OsEMF2b caused late flowering by increasing OsLFL1 expression and decreasing Ehd1 expression. Thus, we conclude that OsVIL2 functions together with PRC2 to induce flowering by repressing OsLFL1.     

Vertically Aligned Sn4+ Preintercalated Ti2CTX MXene Sphere with Enhanced Zn Ion Transportation and Superior Cycle Lifespan

While 2D MXenes have been widely used in energy storage systems, surface barriers induced by restacking of nanosheets and the limited kinetics resulting from insufficient interlayer spacing are two unresolved issues. Here an Sn⁴⁺ preintercalated Ti₂CT_X with effectively enlarged interlayer spacing is synthesized. The preintercalated Ti₂CT_X is aligned on a carbon sphere to further enhance ion transportation by shortening the ion diffusion path and enhancing the reaction kinetics. As a result, when paired with a Zn anode, 12 500 cycles, which equals 2 800 h cycle time, and 5% capacity fluctuation are obtained, surpassing all reported MXene‐based aqueous electrodes. At 0.1 A g^‐1, the capacity reaches 138 mAh g^‐1, and 92 mAh g^‐1 remains even at 5 A g^‐1. In addition, the low anti‐self‐discharge rate of 0.989 mV h^‐1 associated with a high capacity retention of 80.5% over 548 h is obtained. Moreover, the fabricated quasi‐solid capacitor based on a hydrogel film electrolyte exhibits good mechanical deformation and weather resistance. This work employs both preintercalation and alignment to MXene and achieves enhanced ion diffusion kinetics in an aqueous zinc ion capacitors (ZICs) system, which may be applied to other MXene batteries for enhanced performance.     

Selective degeneration of dopaminergic neurons by MPP+ and its rescue by D2 autoreceptors in Drosophila primary culture

Drosophila melanogaster is widely used to study genetic factors causing Parkinson's disease (PD) largely because of the use of sophisticated genetic approaches and the presence of a high conservation of gene sequence/function between Drosophila and mammals. However, in Drosophila, little has been done to study the environmental factors which cause over 90% of PD cases. We used Drosophila primary neuronal culture to study degenerative effects of a well‐known PD toxin MPP⁺. Dopaminergic (DA) neurons were selectively degenerated by MPP⁺, whereas cholinergic and GABAergic neurons were not affected. This DA neuronal loss was because of post‐mitotic degeneration, not by inhibition of DA neuronal differentiation. We also found that MPP⁺‐mediated neurodegeneration was rescued by D2 agonists quinpirole and bromocriptine. This rescue was through activation of Drosophila D2 receptor DD2R, as D2 agonists failed to rescue MPP⁺‐toxicity in neuronal cultures prepared from both a DD2R deficiency line and a transgenic line pan‐neuronally expressing DD2R RNAi. Furthermore, DD2R autoreceptors in DA neurons played a critical role in the rescue. When DD2R RNAi was expressed only in DA neurons, MPP⁺ toxicity was not rescued by D2 agonists. Our study also showed that rescue of DA neurodegeneration by Drosophila DD2R activation was mediated through suppression of action potentials in DA neurons.     

Recruitment of CCR6+ Foxp3+ regulatory gastric infiltrating lymphocytes in Helicobacter pylori gastritis

Helicobacter pylori (H. pylori) infection is associated with an inflammatory response in the gastric mucosa, leading to chronic gastritis, peptic ulcers, and gastric cancer. Increased T‐cell infiltration is found at sites of H. pylori infection. The CCR6⁺ subset of CD4⁺ regulatory T cells (Tregs), a newly characterized subset of Tregs, has been reported to contribute to local immune inhibition. However, whether CCR6⁺ Tregs are present in H. pylori gastritis, and what their relationship is to disease prognosis, remains to be elucidated. In this study, gastric infiltrating lymphocytes were isolated from endoscopic biopsy specimens of H. pylori gastritis patients and analyzed. We found that in gastric infiltrating lymphocytes, CCR6⁺ CD4⁺ CD25^high Tregs, which express high levels of CD45RO, are positively associated with more severe inflammation in gastric mucosa during H. pylori infection. Furthermore, the frequency of CCR6⁺ Tregs in gastric infiltrating lymphocytes, but not CCR6^? Tregs, is significantly increased in inflamed gastric tissues, which is inversely correlated with significantly lower expression of IFN‐γ⁺ CD8⁺ T cells. We also found that the frequency of CCR6⁺ Tregs is positively correlated with the frequency of CD4⁺ IFN‐γ⁺ T cells. In addition, the frequency of CCR6⁺ Tregs, but not that of CCR6^? Tregs, is significantly correlated with increased inflammation in H. pylori gastritis. This study demonstrates that immunosuppression in H. pylori gastritis might be related to the activity of CCR6⁺ Tregs, which could influence disease prognosis.     

Methylation of ELOVL2 gene as a new epigenetic marker of age

The discovery of biomarkers able to predict biological age of individuals is a crucial goal in aging research. Recently, researchers' attention has turn toward epigenetic markers of aging. Using the Illumina Infinium HumanMethylation450 BeadChip on whole blood DNA from a small cohort of 64 subjects of different ages, we identified 3 regions, the CpG islands of ELOVL2, FHL2, and PENK genes, whose methylation level strongly correlates with age. These results were confirmed by the Sequenom's EpiTYPER assay on a larger cohort of 501 subjects from 9 to 99 years, including 7 cord blood samples. Among the 3 genes, ELOVL2 shows a progressive increase in methylation that begins since the very first stage of life (Spearman's correlation coefficient = 0.92) and appears to be a very promising biomarker of aging.     

The mitochondrial NAD+ transporter (NDT1) plays important roles in cellular NAD+ homeostasis in Arabidopsis thaliana

Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme required for all living organisms. In eukaryotic cells, the final step of NAD⁺ biosynthesis is exclusively cytosolic. Hence, NAD⁺ must be imported into organelles to support their metabolic functions. Three NAD⁺ transporters belonging to the mitochondrial carrier family (MCF) have been biochemically characterized in plants. AtNDT1 (At2g47490), focus of the current study, AtNDT2 (At1g25380), targeted to the inner mitochondrial membrane, and AtPXN (At2g39970), located in the peroxisomal membrane. Although AtNDT1 was presumed to reside in the chloroplast membrane, subcellular localization experiments with green fluorescent protein (GFP) fusions revealed that AtNDT1 locates exclusively in the mitochondrial membrane in stably transformed Arabidopsis plants. To understand the biological function of AtNDT1 in Arabidopsis, three transgenic lines containing an antisense construct of AtNDT1 under the control of the 35S promoter alongside a T‐DNA insertional line were evaluated. Plants with reduced AtNDT1 expression displayed lower pollen viability, silique length, and higher rate of seed abortion. Furthermore, these plants also exhibited an increased leaf number and leaf area concomitant with higher photosynthetic rates and higher levels of sucrose and starch. Therefore, lower expression of AtNDT1 was associated with enhanced vegetative growth but severe impairment of the reproductive stage. These results are discussed in the context of the mitochondrial localization of AtNDT1 and its important role in the cellular NAD⁺ homeostasis for both metabolic and developmental processes in plants.