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Objectives: This study aims to investigate the expression patterns of mitochondrially encoded cytochrome c oxidase 1 (MT-CO1) in different organs and tissues of MRL/lpr mice aged six and 18 weeks. Materials and methods: Six-week-old female MRL/lpr mice (n=10) were considered young lupus model mice, and 18-week-old MRL/lpr mice (n=10) were considered old lupus model mice. Additionally, six-week-old (n=10) and 39-week-old (n=10) female Balb/c mice were used as the young and old controls, respectively. The messenger ribonucleic acid (mRNA) and protein expression levels of MT-CO1 in nine organs/tissues were detected via quantitative polymerase chain reaction (qPCR) and Western blot. Malondialdehyde (MDA) levels were determined with thiobarbituric acid colorimetry. The correlation coefficient of MT-CO1 mRNA levels and MDA levels in each organ/tissue at different ages was analyzed by Pearson correlation analysis. Results: The results showed that most non-immune organs/tissues (heart, lung, liver, kidneys, and intestines) showed increased MT-CO1 expression levels in younger MRL/lpr mice (pMRL/lpr mice. Lower mRNA expression and higher MDA levels were observed in the brains of MRL/lpr mice. However, all MRL/lpr mice showed higher MDA levels than Balb/c mice in every organ no matter younger or older MRL/lpr mice. Conclusion: Our study results suggest that lymphoid mitochondrial hyperfunction at organ level may be an important intrinsic pathogenesis in systemic lupus erythematosus activity, which may affect mitochondrial dysfunction in non-immune organs.

NASH is a chronic liver disease that affects 3%–6% of individuals and requires urgent therapeutic developments. Isolating the key cell types in the liver is a necessary step towards understanding their function and roles in disease pathogenesis. However, traditional isolation methods through gradient centrifugation can only collect one or a few cell types simultaneously and pose technical difficulties when applied to NASH livers. Taking advantage of identified cell surface markers from liver single‐cell RNAseq, here we established the combination of gradient centrifugation and antibody‐based cell sorting techniques to isolate five key liver cell types (hepatocytes, endothelial cells, stellate cells, macrophages and other immune cells) from a single mouse liver. This method yielded high purity of each cell type from healthy and NASH livers. Our five‐in‐one protocol simultaneously isolates key liver cell types with high purity under normal and NASH conditions, enabling for systematic and accurate exploratory experiments such as RNA sequencing.

We sought to analyse the expression characteristics of cytochrome C oxidase subunit I in mitochondrial of MRL/lpr lupus mice.The whole blood of MRL/lpr lupus mice was detected for whole mitochondrial genome sequencing performed by Illumina HiSeq PE150 instrument, compared with house mouse (NC_005089.1) and screened for the maximum difference gene, MT-CO1. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to detect the mRNA and protein expression of MT-CO1 in lupus mice and control mice. The total antioxidant capacities of lupus mice and control mice were measured using the rapid 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) method.The mitochondrial genome sequencing showed that five mitochondrial genes had base differences and MT-CO1 was the maximum difference gene, 31 in total. Among the 31 base difference sites, 2 were missense mutations and 29 were synonymous_variant. qRT-PCR test results showed that the MT-CO1 expression in lupus mouse blood was statistically lower than that in control mice blood (t=4.333; p=0.0003). Western blot test results revealed that the expression of MT-CO1 was lower in the lupus mice compared with the control mice at the protein level. Serum total antioxidant capacity testing showed that: the serum total antioxidant capacity of lupus mice was statistically lower than that of the control mice (t=9.957; p0.0001).High mutation rate and decreased expression of MT-CO1 in MRL/lpr lupus mice accompanied the decrease of antioxidant capacity, which indicated that abnormal MT-CO1 might be involved in the pathogenesis of SLE and the production of anti-dsDNA antibodies.

Regeneration plays an essential role in the restructuring of plant communities, with seedlings and sprouts representing two different regeneration strategies. As an important timber species, Cunnin...

Alpine grasslands harbor diverse groups of flora and fauna, provide important ecosystem functions, and yield essential ecosystem goods and services, especially for the development of nature-based tourism. However, they are experiencing increasing anthropogenic perturbations such as tourist trampling. Although negative effects of tourist trampling on alpine vegetation have been frequently reported, previous studies have focused mainly on changes in taxonomic diversity after trampling, and rarely provide a mechanistic elucidation of trampling effects from a trait-based perspective. The present study evaluates the impacts of simulated trampling on taxonomic and functional diversity of a typical alpine grassland community in Shangri-La, China using a standardized protocol. The results showed that although taxonomic diversity was not statistically significantly affected by trampling, some functional attributes responded rapidly to trampling disturbance. Specifically, functional divergence decreased with an increase in trampling intensity, and characteristics of community-weighted mean trait values changed towards shorter species with reduced leaf area and lower leaf dry matter content. Such strong shifts in functional attributes may further affect ecosystem goods and services provided by alpine grasslands. Our inclusion of functional diversity in the analysis thus adds an important caution to previous studies predominantly focusing on taxonomic diversity, and it is urgent to keep alpine grasslands well managed and ecologically coherent so that their valuable functions and services can be safeguarded.

Summary Nonalcoholic fatty liver disease (NAFLD) is a global health-care problem with limited therapeutic options. To obtain a cellular resolution of pathogenesis, 82,168 single-cell transcriptomes (scRNA-seq) across different NAFLD stages were profiled, identifying hepatocytes and 12 other non-parenchymal cell (NPC) types. scRNA-seq revealed insights into the cellular and molecular mechanisms of the disease. We discovered a dual role for hepatic stellate cells in gene expression regulation and in the potential to trans-differentiate into myofibroblasts. We uncovered distinct expression profiles of Kupffer cells versus monocyte-derived macrophages during NAFLD progression. Kupffer cells showed stronger immune responses, while monocyte-derived macrophages demonstrated a capability for differentiation. Three chimeric NPCs were identified including endothelial-chimeric stellate cells, hepatocyte-chimeric endothelial cells, and endothelial-chimeric Kupffer cells. Our work identified unanticipated aspects of mouse with NAFLD at the single-cell level and advanced the understanding of cellular heterogeneity in NAFLD livers., Graphical abstract, Highlights • Of all, 82,168 single-cell transcriptomes across different NAFLD stages were profiled • Hepatocytes and 12 non-parenchymal cell types were identified in mouse liver • Three chimeric NPCs were identified in mouse liver, Animal physiology; Cell biology; Transcriptomics

The liver plays a critical role in maintaining ammonia homeostasis. Urea cycle defects, liver injury, or failure and glutamine synthetase (GS) deficiency result in hyperammonemia, serious clinical conditions, and lethality. In this study we used a mouse model with a defect in the urea cycle enzyme ornithine transcarbamylase (Otcspf-ash) to test the hypothesis that glucagon receptor inhibition using a monoclonal blocking antibody will reduce the hyperammonemia and associated lethality induced by a high-protein diet, which exacerbates disease. We found reduced expression of glutaminase, which degrades glutamine and increased expression of GS in livers of Otcspf-ash mice treated with the glucagon receptor blocking antibody. The gene expression changes favor ammonia consumption and were accompanied by increased circulating glutamine levels and diminished hyperammonemia. Otcspf-ash mice treated with the glucagon receptor-blocking antibody gained lean and body mass and had increased survival. These data suggest that glucagon receptor inhibition using a monoclonal antibody could reduce the risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle, liver injury, or failure and GS deficiency.

Significance The lobules are the functional units of the liver. They consist of 15–25 layers of hepatocytes with specialized metabolic functions and gene expression patterns relative to their position along the lobule, a phenomenon referred to as metabolic zonation. The Wnt/β-catenin pathway regulates hepatocyte function but how the zonation is controlled to meet the metabolic demands of the liver is unclear. Glucagon regulates hepatic function. We now demonstrate that glucagon contributes to liver zonation by interacting and opposing the actions of the Wnt/β-catenin pathway., Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal function. Wnt/β-catenin signaling controls metabolic zonation by activating genes in the perivenous hepatocytes, while suppressing genes in the periportal counterparts. We now demonstrate that glucagon opposes the actions of Wnt/β-catenin signaling on gene expression and metabolic zonation pattern. The effects were more pronounced in the periportal hepatocytes where 28% of all genes were activated by glucagon and inhibited by Wnt/β-catenin. The glucagon and Wnt/β-catenin receptors and their signaling pathways are uniformly distributed in periportal and perivenous hepatocytes and the expression is not regulated by the opposing signal. Collectively, our results show that glucagon controls gene expression and metabolic zonation in the liver through a counterplay with the Wnt/β-catenin signaling pathway.

This study presented common lesions with systemic toxicities and uncommon adverse cutaneous reactions such as anaphylactic dermatitis in patients undergoing treatment with erlotinib for the benefit of practicing dermatologists and oncologists.Adverse cutaneous reactions associated with erlotinib were reported in 20 Chinese patients with cancer.Adverse cutaneous reactions reported included six cases of anaphylactic dermatitis, 12 cases of acneiform rash, nine cases of xerosis, five cases of nail changes and four cases of hair changes. One case of anaphylactic dermatitis manifested as erythema with swelling on the face and neck, and others as erosive and scaly erythema on the fold of skin, or red macules, papules, plaques and pigmentation on the whole body. Clinical details indicated anaphylactic reactions, including a high percentage of eosinophils in the peripheral blood, eosinophilic infiltration in the dermis layer and good response to antihistamines and topical steroids. Systemic toxicities accompanied by cutaneous reactions occurred in five patients including one case of anaphylactic dermatitis and four cases of acneiform rash. Elevated hepatic enzymes were observed among all the patients with grade-3 or grade-4 acneiform rashes. One patient with anaphylactic dermatitis and one with acneiform rash discontinued erlotinib administration due to severe lesions, high fever or severe elevation of hepatic enzymes.Anaphylactic cutaneous reactions caused by erlotinib are rarely described hitherto. Systemic toxicities should be emphasized especially in cases with severe skin disorders. Timely detection and appropriate early intervention in patients who develop severe cutaneous reaction while on erlotinib therapy should be considered clinically.

The resting membrane potential (Δψ) of the cell is negative on the cytosolic side and determined primarily by the plasma membrane’s selective permeability to K+. We show that lysosomal Δψ is set by lysosomal membrane permeabilities to Na+ and H+, but not K+, and is positive on the cytosolic side. An increase in juxta-lysosomal Ca2+ rapidly reversed lysosomal Δψ by activating a large voltage-dependent and K+-selective conductance (LysoKVCa). LysoKVCa is encoded molecularly by SLO1 proteins known for forming plasma membrane BK channels. Opening of single LysoKVCa channels is sufficient to cause the rapid, striking changes in lysosomal Δψ. Lysosomal Ca2+ stores may be refilled from endoplasmic reticulum (ER) Ca2+ via ER–lysosome membrane contact sites. We propose that LysoKVCa serves as the perilysosomal Ca2+ effector to prime lysosomes for the refilling process. Consistently, genetic ablation or pharmacological inhibition of LysoKVCa, or abolition of its Ca2+ sensitivity, blocks refilling and maintenance of lysosomal Ca2+ stores, resulting in lysosomal cholesterol accumulation and a lysosome storage phenotype.

Inactivating mutations in the insulin receptor results in extreme insulin resistance. The resulting hyperglycemia is very difficult to treat, and patients are at risk for early morbidity and mortality from complications of diabetes. We used the insulin receptor antagonist S961 to induce severe insulin resistance, hyperglycemia, and ketonemia in mice. Using this model, we show that glucagon receptor (GCGR) inhibition with a monoclonal antibody normalized blood glucose and β-hydroxybutyrate levels. Insulin receptor antagonism increased pancreatic β-cell mass threefold. Normalization of blood glucose levels with GCGR-blocking antibody unexpectedly doubled β-cell mass relative to that observed with S961 alone and 5.8-fold over control. GCGR antibody blockage expanded α-cell mass 5.7-fold, and S961 had no additional effects. Collectively, these data show that GCGR antibody inhibition represents a potential therapeutic option for treatment of patients with extreme insulin-resistance syndromes.

According to the hazardous characteristics of LPG storage tanks and their types and hazards of fire and explosion accidents, the impact evaluation method, the shock wave overpressure criterion and the heat flux criterion are applied to the standard of human injury. Fire shock wave and heat radiation generated by three different accident types were calculated. The quantitative estimation model of safety distance and LPG reserves was studied, which provided a supplementary basis for the personnel evacuation and firefighter command and decision on LPG fire scene.

Climate comfort is an important index for evaluating the liveability of a region, and it is also the basis for building a healthy living destination. Quantitative analysis of tourism climate comfort index was built in this paper through average temperature, relative humidity, wind speed and other climatic factors in Kunming, using Fuzzy Analytic Hierarchy Process to evaluate the tourism comfort of Kunming. The results show that Kunming has a pleasant perennial climate, a long tourism cycle and a great advantage in tourism climate comfort. The conclusions can provide scientific basis for the related work of healthy living destination.

To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca(2+)-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P(2), a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P(2)-TRPML1-ALG-2-dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation.

BACKGROUND: Elucidation of the genetic factors underlying chronic liver disease may reveal new therapeutic targets. METHODS: We used exome sequence data and electronic health records from 46,544 participants in the DiscovEHR human genetics study to identify genetic variants associated with serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Variants that were replicated in three additional cohorts (12,527 persons) were evaluated for association with clinical diagnoses of chronic liver disease in DiscovEHR study participants and two independent cohorts (total of 37,173 persons) and with histopathological severity of liver disease in 2391 human liver samples. RESULTS: A splice variant (rs72613567:TA) in HSD17B13, encoding the hepatic lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13, was found to be associated with reduced levels of ALT (P=4.20×10(−12)) and AST (P=6.2×10(−10)). Among DiscovEHR study participants, this variant was found to be associated with a reduced risk of alcoholic liver disease (by 42% [95% confidence interval {CI}, 20 to 58] among heterozygotes and by 53% [95% CI, 3 to 77] among homozygotes), nonalcoholic liver disease (by 17% [95% CI, 8 to 25] among heterozygotes and by 30% [95% CI, 13 to 43] among homozygotes), alcoholic cirrhosis (by 42% [95% CI, 14 to 61] among heterozygotes and by 73% [95% CI, 15 to 91] among homozygotes), and nonalcoholic cirrhosis (by 26% [95% CI, 7 to 40] among heterozygotes and by 49% [95% CI, 15 to 69] among homozygotes). Associations were confirmed in two independent cohorts. The rs72613567:TA variant was associated with a reduced risk of nonalcoholic steatohepatitis, but not steatosis, in human liver samples. The rs72613567:TA variant mitigated liver injury associated with the risk-increasing PNPLA3 p.I148M allele and resulted in an unstable and truncated protein with reduced enzymatic activity. CONCLUSIONS: A loss-of-function variant in HSD17B13 is associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.)

The Pettitt abrupt change test method based on ArcGIS was used to undertake change-point analysis on climatic (precipitation and potential evapotranspiration; 39 meteorological stations) and runoff data (27 hydrological stations) from 1954–2015 in the Nenjiang basin. The hydrological sensitivity analysis method was also used to calculate the influential component of climate change upstream, mid-stream, and downstream of the Nenjiang basin, as well as the effect of anthropogenic activities on runoff. Our results show that the upstream area has the highest contribution rate of climate change, followed by the mid-stream area; the downstream area has the lowest contribution rate. Studying climate change contribution rates in various sites in the Nenjiang basin, in addition to anthropogenic activities affecting runoff, can provide the foundation for the protection and utilization of basin water resources, as well as the conservation and restoration of wetlands.

Neuronal polarization is pivotal for neural network formation during brain development. Axon differentiation is a hallmark of initial neuronal polarization. Here, we report that the leucine-rich repeat-containing protein netrin-G ligand-2 (NGL-2) as a polarity regulator that localizes asymmetrically in rat hippocampal neurons and is required for differentiation of the future axon. NGL-2 was associated with PAR complex, and this interaction resulted in local stabilization of axonal microtubules. Further study showed that the C terminal of NGL-2 binds to the PDZ domain of PAR6, and NGL-2 interacts with PAR3 and atypical PKCζ (aPKCζ), with PAR6 acting as a bridge or modifier. Then, NGL-2 regulates the local stabilization of microtubules and promotes axon differentiation by the aPKCζ/microtubule affinity-regulating kinase 2 pathway. These findings reveal the critical role of NGL-2 in regulating axon differentiation in rat hippocampal neurons and reveal a novel partner of the PAR complex.

The integrity of the plasma membrane is maintained through an active repair process, especially in skeletal and cardiac muscle cells, in which contraction-induced mechanical damage frequently occurs in vivo. Muscular dystrophies (MDs) are a group of muscle diseases characterized by skeletal muscle wasting and weakness. An important cause of these group of diseases is defective repair of sarcolemmal injuries, which normally requires Ca(2+) sensor proteins and Ca(2+)-dependent delivery of intracellular vesicles to the sites of injury. MCOLN1 (also known as TRPML1, ML1) is an endosomal and lysosomal Ca(2+) channel whose human mutations cause mucolipidosis IV (ML4), a neurodegenerative disease with motor disabilities. Here we report that ML1-null mice develop a primary, early-onset MD independent of neural degeneration. Although the dystrophin-glycoprotein complex and the known membrane repair proteins are expressed normally, membrane resealing was defective in ML1-null muscle fibers and also upon acute and pharmacological inhibition of ML1 channel activity or vesicular Ca(2+) release. Injury facilitated the trafficking and exocytosis of vesicles by upmodulating ML1 channel activity. In the dystrophic mdx mouse model, overexpression of ML1 decreased muscle pathology. Collectively, our data have identified an intracellular Ca(2+) channel that regulates membrane repair in skeletal muscle via Ca(2+)-dependent vesicle exocytosis.

We analyzed basal area (BA) growth using growth data obtained from permanent plots over 4 years for five major tree species in Anhui Province, eastern China. The studied species were dominant conifers (Pinus massoniana and Cunninghamia lanceolata) and co-dominant broad-leaved species (Castanopsis eyrei, Castanopsis sclerophylla, and Loropetalum chinense). A hierarchical Bayesian approach was used to estimate species-specific parameters and to quantify a spatially autocorrelated random effect. We selected a model that included only the following relevant predictor variables: initial size, asymmetric competition, spatially autocorrelated random effect, and random effect associated with plots. For all species analyzed, the model accounted for significant proportions of the variation (R 2 = 70–98 %) in BA growth rates. The initial slope of the relationship between BA growth rate and the initial BA tended to be steeper for P. massoniana than for C. lanceolata. The BA growth rate increased from an initial low value and then leveled off, with a lower maximum BA growth rate for C. lanceolata than for P. massoniana. The BA growth rate of P. massoniana was significantly affected by asymmetric competition with neighbors. The results of our analyses were used to predict to what extent thinning neighboring trees at different intensities would reduce competition impacts on BA growth of P. massoniana and C. lanceolata. Our results also helped to clarify the ecological characteristics of the species analyzed, as well as the spatial distribution of unknown factors influencing tree growth.

Ion-dependent channels and transporters have been identified in lysosomes, including the V-ATPase H+ pump and transient receptor potential mucolipin channels (TRPMLs), the principle Ca2+ release channels in the lysosome, but much less is understood about the roles of Na+ and K+ in lysosomal physiology. Wang et al. describe a voltage-sensitive, Ca2+-activated K+ current in the lysosome (LysoKVCa) and show that LysoKVCa regulates lysosomal membrane potential and refilling of lysosomal Ca2+ stores., The resting membrane potential (Δψ) of the cell is negative on the cytosolic side and determined primarily by the plasma membrane’s selective permeability to K+. We show that lysosomal Δψ is set by lysosomal membrane permeabilities to Na+ and H+, but not K+, and is positive on the cytosolic side. An increase in juxta-lysosomal Ca2+ rapidly reversed lysosomal Δψ by activating a large voltage-dependent and K+-selective conductance (LysoKVCa). LysoKVCa is encoded molecularly by SLO1 proteins known for forming plasma membrane BK channels. Opening of single LysoKVCa channels is sufficient to cause the rapid, striking changes in lysosomal Δψ. Lysosomal Ca2+ stores may be refilled from endoplasmic reticulum (ER) Ca2+ via ER–lysosome membrane contact sites. We propose that LysoKVCa serves as the perilysosomal Ca2+ effector to prime lysosomes for the refilling process. Consistently, genetic ablation or pharmacological inhibition of LysoKVCa, or abolition of its Ca2+ sensitivity, blocks refilling and maintenance of lysosomal Ca2+ stores, resulting in lysosomal cholesterol accumulation and a lysosome storage phenotype.

Mammalian two-pore channel proteins (TPC1, TPC2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double-knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P(2) and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na(+), not K(+), as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes and may explain the specificity of PI(3,5)P(2) in regulating the fusogenic potential of intracellular organelles.

Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes ‘host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca2+-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca2+ release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell., Reactive oxygen species (ROS) damage cell components, necessitating their clearance through autophagy. Here, the authors show that ROS can induce autophagy by triggering TRPML1 to release Ca2+ from the lysosomal lumen, in turn activating the autophagy and lysosomal biogenesis regulator TFEB.

Resealing allows cells to mend damaged membranes rapidly when plasma membrane (PM) disruptions occur. Models of PM repair mechanisms include the "lipid-patch", "endocytic removal", and "macro-vesicle shedding" models, all of which postulate a dependence on local increases in intracellular Ca(2+) at injury sites. Multiple calcium sensors, including synaptotagmin (Syt) VII, dysferlin, and apoptosis-linked gene-2 (ALG-2), are involved in PM resealing, suggesting that Ca(2+) may regulate multiple steps of the repair process. Although earlier studies focused exclusively on external Ca(2+), recent studies suggest that Ca(2+) release from intracellular stores may also be important for PM resealing. Hence, depending on injury size and the type of injury, multiple sources of Ca(2+) may be recruited to trigger and orchestrate repair processes. In this review, we discuss the mechanisms by which the resealing process is promoted by vesicular Ca(2+) channels and Ca(2+) sensors that accumulate at damage sites.

The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1(V432P)) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1(V432P)-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca(2+)-dependent, constitutive Ca(2+) permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca(2+) release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca(2+) and Fe(2+)/Mn(2+) dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML1 proteins at the PM.

miR-101 has been suggested as a tumor suppressor, but the promoter methylation and loss of heterozygosity didn’t contribute to its low expression in astrocytoma. We investigated the role of a new long non-coding RNA CASC2c binding with miR-101. High CASC2c was positively correlated with astrocytoma progression, and an unfavorable prognosis factor for patients. Knockdown CASC2c inhibited proliferation and tumorgenesis. Overexpression of CASC2c promotes the malignant characteristic of astrocytoma cells.CASC2c directly bound miR-101 and mediated pre-miR-101 processing into mature miR-101, and functions as a competitor of miR-101 target genes such as CPEB1. Patients who possessed both low CASC2c and high miR-101 had a longer survival than those of low CASC2c alone or high miR-101 alone. In summary, CASC2c plays the onco-RNA role in the tumorgenesis of astrocytoma by acting as a decoy miR-101 sponge. Combination of low expression of CASC2c and high expression of miR-101 has an important referential significance to evaluate the prognosis of patients.

The integrity of the plasma membrane is maintained through an active repair process, especially in skeletal and cardiac muscle cells, in which contraction-induced mechanical damage frequently occurs in vivo. Muscular dystrophies (MDs) are a group of muscle diseases characterized by skeletal muscle wasting and weakness. An important cause of these group of diseases is defective repair of sarcolemmal injuries, which normally requires Ca(2+) sensor proteins and Ca(2+)-dependent delivery of intracellular vesicles to the sites of injury. MCOLN1 (also known as TRPML1, ML1) is an endosomal and lysosomal Ca(2+) channel whose human mutations cause mucolipidosis IV (ML4), a neurodegenerative disease with motor disabilities. Here we report that ML1-null mice develop a primary, early-onset MD independent of neural degeneration. Although the dystrophin-glycoprotein complex and the known membrane repair proteins are expressed normally, membrane resealing was defective in ML1-null muscle fibers and also upon acute and pharmacological inhibition of ML1 channel activity or vesicular Ca(2+) release. Injury facilitated the trafficking and exocytosis of vesicles by upmodulating ML1 channel activity. In the dystrophic mdx mouse model, overexpression of ML1 decreased muscle pathology. Collectively, our data have identified an intracellular Ca(2+) channel that regulates membrane repair in skeletal muscle via Ca(2+)-dependent vesicle exocytosis.

SummaryPhagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.

Neuropeptide Y is expressed in neurons of the hypothalamic arcuate nucleus and has been ascribed a role as a stimulant of food intake. Neuropeptide Y Y1 and Y2 receptors are also localised in the arcuate nucleus, and it has been suggested that the Y1 receptor mediates part of the effect of neuropeptide Y on feeding behaviour. In the present study, immunohistochemistry and in situ hybridization were used to investigate the effect of food deprivation on the expression of Y1 and Y2 receptors in the arcuate nucleus of the rat. Fasting for 48 h induced a decrease in the number and area of Y1 receptor immunoreactive neurons in the arcuate nucleus. Furthermore, arcuate Y1 receptor mRNA levels also decreased after food deprivation. The decrease in the number of the Y1 receptor immunoreactive neurons was partially attenuated by supplementing the drinking water with 10% glucose. In contrast, fasting did not significantly change Y2 receptor mRNA levels in the arcuate nucleus. These results support the view that Y1 receptors in the arcuate nucleus play a role in the feeding pattern induced by neuropeptide Y.

To detect the heterogeneity of mitochondrial DNA (mtDNA) in black and white hair of patients with type 2 diabetes.MtDNA was extracted from the hair shaft of the patients to amplify two target DNA fragment from mtDNA coding region and control region using PCR. The differences in the heterogeneity in the target DNA fragment was analyzed between diabetic patients and the control group with denaturing high-performance liquid chromatography (DHPLC).In the control subjects and diabetic patients, the mtDNA heterogeneity in the black hair was 3% and 10% in 20-45 year-old groups and 9% and 17% in 45-70 year-old groups, as compared to 9%, 20%, 21%, and 40% in the white hair, respectively. The mtDNA heterogeneity in the black and white hair was both higher in the diabetic patients than in the control subjects of the same age group, and was also higher in older age subgroups in both control and diabetic groups (P0.05). The white hair mtDNA showed a significantly higher heterogeneity than the black hair mtDNA in the two age groups of diabetic patients and in 45-70 year-old control group (P0.05).The mtDNA heterogeneity in the hair increases in type 2 diabetic patients and show an association with aging.

Lysosomes, traditionally believed to be the terminal “recycle center” for biological “garbage”, are now known to play indispensable roles in membrane trafficking and intracellular signaling pathways. The multiple functions require the establishment of lysosomal acidic lumen, and this pH regulation has been shown to be tightly coupled with ionic (H+, Ca2+, Mg2+, Na+, K+, and Cl−) flux/homeostasis of the lysosome. Moreover, recent studies have suggested that the ion fluxes may directly regulate lysosomal dynamics. PI(3,5)P2 is an endolysosome-specific phosphoinositide that regulates ion homeostasis and membrane trafficking of endolysosomes via poorly understood mechanisms; human mutations of PI(3,5)P2-metabolizing enzymes cause muscle and neurodegenerative diseases. Here we measure that sodium is the predominant cation in the lysosome using atomic absorption, indicating a large Na+ concentration gradient is present across the lysosomal membrane. By the lysosome patch-clamp technique, we report that PI(3,5)P2 specifically activates two novel Na+-selective channels in the lysosome. Their identification provides insight into divalent cation selectivity, as well as to the mechanisms used by membranous lipids to directly regulate ion flux resulting in rapid changes in membrane potential and the fusogenic potential of intracellular organelles. We are currently investigating their physiological functions in detail.

Global disruption of transient receptor potential-melastatin-like 7 ( Trpm7 ) in mice results in embryonic lethality before embryonic day 7. Using tamoxifen-inducible disruption of Trpm7 and multiple Cre recombinase lines, we show that Trpm7 deletion before and during organogenesis results in severe tissue-specific developmental defects. We find that Trpm7 is essential for kidney development from metanephric mesenchyme but not ureteric bud. Disruption of neural crest Trpm7 at early stages results in loss of pigment cells and dorsal root ganglion neurons. In contrast, late disruption of brain-specific Trpm7 after embryonic day 10.5 does not alter normal brain development. We developed induced pluripotent stem cells and neural stem (NS) cells in which Trpm7 disruption could be induced. Trpm7 −/− NS cells retained the capacities of self-renewal and differentiation into neurons and astrocytes. During in vitro differentiation of induced pluripotent stem cells to NS cells, Trpm7 disruption prevents the formation of the NS cell monolayer. The in vivo and in vitro results demonstrate a temporal requirement for the Trpm7 channel kinase during embryogenesis.