A simplified herbal decoction attenuates myocardial infarction by regulating macrophage metabolic reprogramming and phenotypic differentiation via modulation of the HIF-1α/PDK1 axis.

BACKGROUND: Myocardial infarction (MI) poses a global public health challenge, often associated with elevated mortality rates and a grim prognosis. A crucial aspect of the inflammatory injury and healing process post-MI involves the dynamic differentiation of macrophages. A promising strategy to alleviate myocardial damage after MI is by modulating the inflammatory response and orchestrating the shift from pro-inflammatory (M1) to anti-inflammatory (M2) macrophages, aiming to achieve a reduced M1/M2 ratio. Nuanxinkang (NXK), a simplified herbal decoction, has demonstrated noteworthy cardioprotective, inflammation-regulating, and myocardial energy metabolism-regulating properties. METHODS: In this study, we constructed an MI model by ligating coronary arteries to investigate the efficacy of NXK in improving ventricular remodeling and cardiac function. Mice were administered NXK (1.65 g/kg/d) or an equivalent volume of regular saline via gavage for 28 consecutive days, commencing the day after surgery. Then, we conducted echocardiography to assess the cardiac function, Masson staining to illustrate the extent of myocardial fibrosis, TUNEL staining to reveal myocardial apoptosis, and flow cytometry to analyze the polarization of M1 and M2 macrophages in the hearts. Besides, a lipopolysaccharide (LPS)-induced pro-inflammatory macrophage (M1) polarization model was implemented in RAW264.7 cells to elucidate the underlying mechanism of NXK in regulating macrophage polarization. RAW264.7 cells were pre-treated with or without NXK-containing serum. Oxidative stress was detected by MitoSox staining, followed by Seahorse energy metabolism assay to evaluate alterations in mitochondrial metabolic patterns and ATP production. Both In vivo and in vitro, HIF-1α and PDK1 were detected by fluorescent quantitative PCR and Western blotting. RESULTS: In vivo, MI mice exhibited a decline in cardiac function, adverse ventricular remodeling, and an increase in glycolysis, coupled with M1-dominant polarization mediated by the HIF-1α/PDK1 axis. Notably, robust responses were evident with high-dose NXK treatment (1.65 g/kg/day), leading to a significant enhancement in cardiac function, inhibition of cardiac remodeling, and partial suppression of macrophage glycolysis and the inflammatory phenotype in MI mice. This effect was achieved through the modulation of the HIF-1α/PDK1 axis. In vitro, elevated levels of mitochondrial ROS production and glycolysis were observed in LPS-induced macrophages. Conversely, treatment with NXK notably reduced the oxidative stress damage induced by LPS and enhanced oxidative phosphorylation (OXPHOS). Furthermore, NXK demonstrated the ability to modify the energy metabolism and inflammatory characteristics of macrophages by modulating the HIF-1α/PDK1 axis. The influence of NXK on this axis was partially counteracted by the HIF-1α agonist DMOG. And NXK downregulated PDK1 expression, curtailed glycolysis, and reversed LPS-induced M1 polarization in macrophages, similar to the PDK1 inhibitor DCA. CONCLUSION: In conclusion, NXK protects against MI-induced cardiac remodeling by inducing metabolic reprogramming and phenotypic differentiation of macrophages, achieved through the modulation of the HIF-1α/PDK1 axis. This provides a novel and promising strategy for the treatment of MI.

Stochastic RNA editing of the Complexin C-terminus within single neurons regulates neurotransmitter release.

Neurotransmitter release requires assembly of the SNARE complex fusion machinery, with multiple SNARE-binding proteins regulating when and where synaptic vesicle fusion occurs. The presynaptic protein Complexin (Cpx) controls spontaneous and evoked neurotransmitter release by modulating SNARE complex zippering. Although the central SNARE-binding helix is essential, post-translational modifications to Cpx's C-terminal membrane-binding amphipathic helix regulate its ability to control synaptic vesicle fusion. Here, we demonstrate that RNA editing of the Cpx C-terminus modifies its ability to clamp SNARE-mediated fusion and alters presynaptic output. RNA editing of Cpx across single neurons is stochastic, generating up to eight edit variants that fine tune neurotransmitter release by altering the subcellular localization and clamping properties of the protein. Similar stochastic editing rules for other synaptic genes were observed, indicating editing variability at single adenosines and across multiple mRNAs generates unique synaptic proteomes within the same population of neurons to fine tune presynaptic output.

Stochastic RNA editing of the Complexin C-terminus within single neurons regulates neurotransmitter release.

Neurotransmitter release requires assembly of the SNARE complex fusion machinery, with multiple SNARE-binding proteins regulating this process to control when and where synaptic vesicle fusion occurs. Complexin (Cpx) controls spontaneous and evoked neurotransmitter release by modulating SNARE complex zippering. Although the central SNARE-binding helix is essential, post-translational modifications to Cpx's C-terminal membrane-binding amphipathic helix modulate its activity. Here we demonstrate that RNA editing of the Cpx C-terminus regulates its ability to clamp SNARE-mediated fusion and alters presynaptic output. RNA editing of Cpx within single neurons is stochastic, generating up to eight edit variants that fine-tune neurotransmitter release by changing the subcellular localization and clamping properties of the protein. Similar editing rules for other synaptic genes were observed, indicating stochastic editing at single adenosines and across multiple mRNAs can generate unique synaptic proteomes within the same population of neurons to fine-tune presynaptic output.

Baicalin can enhance odonto/osteogenic differentiation of inflammatory dental pulp stem cells by inhibiting the NF-κB and ß-catenin/Wnt signaling pathways.

BACKGROUND: Scutellaria baicalensis Georgi is a famous traditional Chinese medicine, which is widely used in treating fever, upper respiratory tract infection and other diseases. Pharmacology study showed it can exhibit anti-bacterial, anti-inflammation and analgesic effects. In this study, we investigated the effect of baicalin on the odonto/osteogenic differentiation of inflammatory dental pulp stem cells (iDPSCs). METHODS AND RESULTS: iDPSCs were isolated from the inflamed pulps collected from pulpitis. The proliferation of iDPSCs was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,5-tetrazolium bromide (MTT) assay and flow cytometry. Alkaline phosphatase (ALP) activity assay, alizarin red staining, Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay were conducted to examine the differentiation potency along with the involvement of nuclear factor kappa B(NF-κB) and ß-catenin/Wnt signaling pathway. MTT assay and cell-cycle analysis demonstrated that baicalin had no influence on the proliferation of iDPSCs. ALP activity assay and alizarin red staining demonstrated that baicalin could obviously enhance ALP activity and calcified nodules formed in iDPSCs. RT-PCR and Western blot showed that the odonto/osteogenic markers were upregulated in baicalin-treated iDPSCs. Moreover, expression of cytoplastic phosphor-P65, nuclear P65, and ß-catenin in iDPSCs was significantly increased compared with DPSCs, but the expression in baicalin-treated iDPSCs was inhibited. In addition, 20 µM Baicalin could accelerate odonto/osteogenic differentiation of iDPSCs via inhibition of NF-κB and ß-catenin/Wnt signaling pathways. CONCLUSION: Baicalin can promote odonto/osteogenic differentiation of iDPSCs through inhibition of NF-κB and ß-catenin/Wnt pathways, thus providing direct evidence that baicalin may be effective in repairing pulp with early irreversible pulpitis.

Effects of Straw Return and Moisture Condition on Temporal Changes of DOM Composition and Cd Speciation in Polluted Farmland Soil.

Straw return can improve soil quality and change the mobility and bioavailability of pollutants in soil. Elevated cadmium (Cd) contents in farmland soils were often reported. However, the impacts of straw-derived dissolved organic matter (DOM) on Cd speciation in soil remain poorly understood. In this study, the effects of straw return and moisture condition on temporal changes of DOM composition and Cd speciation in farmland soils were explored through a laboratory incubation experiment. The humified components of DOM were negatively correlated with exchangeable, carbonate-bound, and Fe-Mn oxide-bound Cd (p < 0.01), while its protein-like component was negatively correlated with residual Cd (p < 0.01). It was found that selected fluorescence parameters could be used to predict temporal changes of Cd geochemical fractions. Straw addition led to increases in soil DOM content during the first three days of the incubation. Flooding should be avoided in the first three days following the straw application to reduce the risk of DOM-facilitated Cd mobilization.

Occurrence of antibiotic resistome in farmland soils near phosphorus chemical industrial area.

Heavy metal pollution of soil surrounding phosphorus chemical industry has been a long-concerned problem; however, the occurrence of antibiotic resistance genes (ARGs) in farmland soils in its vicinity remains unexplored. In this study, variations of heavy metals, ARGs, mobile genetic elements (MGEs), and microbiome in surface soils of farmland along the prevailing downwind direction of a phosphorus chemical industrial zone were investigated. Cadmium (Cd) contents in soils close to the industrial zone (≤ 500 m away) were statistically higher than those at greater distances (1000 to 4000 m). A comparable ARG diversity was observed across soils, while the relative abundance of ARGs decreased markedly with increasing distance in the range of 1000 m. The soil in closest proximity to the industrial zone (20 m away) exhibited divergent compositions of ARGs, MGEs, and bacterial community from the other soils at farther locations (500 to 4000 m away). Variation partitioning analysis revealed that Cd and MGEs levels were the primary factors controlling ARG distribution. Structural equation modeling further indicated that the direct effect of Cd on ARG abundance was stronger than its indirect effect via affecting MGEs and microbiome. The observed prevalence of ARGs in farmland soils highlights the necessity of including resistome in the framework for environmental risk assessment of phosphorus chemical manufacturing.

Two new pyrrolidine alkaloids from the red alga Acanthophora spicifera.

Two new pyrrolidine alkaloids, acanthophoraines B (1) and C (2), together with five known ones (3-7) were isolated from the red alga Acanthophora spicifera. Their structures were elucidated by extensive spectroscopic methods and single-crystal X-ray diffraction analysis. The absolute configuration of 2 was established by ECD calculation. The antibacterial activities of 1-7 were also evaluated.

Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle release and replenishment in a dosage-dependent manner.

Synchronous neurotransmitter release is triggered by Ca2+ binding to the synaptic vesicle protein Synaptotagmin 1, while asynchronous fusion and short-term facilitation is hypothesized to be mediated by plasma membrane-localized Synaptotagmin 7 (SYT7). We generated mutations in Drosophila Syt7 to determine if it plays a conserved role as the Ca2+ sensor for these processes. Electrophysiology and quantal imaging revealed evoked release was elevated 2-fold. Syt7 mutants also had a larger pool of readily-releasable vesicles, faster recovery following stimulation, and intact facilitation. Syt1/Syt7 double mutants displayed more release than Syt1 mutants alone, indicating SYT7 does not mediate the residual asynchronous release remaining in the absence of SYT1. SYT7 localizes to an internal membrane tubular network within the peri-active zone, but does not enrich at active zones. These findings indicate the two Ca2+ sensor model of SYT1 and SYT7 mediating all phases of neurotransmitter release and facilitation is not applicable at Drosophila synapses.

A new thiodiketopiperzaine from the marine sponge Tedania sp.

A new thiodiketopiperzaine, tedanizaine A (1), together with six known ones, were isolated from the marine sponge Tedania sp. Their structures were determined by spectroscopic analyses. The absolute configuration of 1 was established by ECD calculation. Compound 1 was the second example of thiodiketopiperazine bearing a thiazolidine unit. Cytotoxic activities of 1 were also evaluated.

[Relationship Between the Vertical Distribution of Nutrients and Bacterial Community Structures in Sediment Interstitial Waters of Stratified Reservoirs with Different Water Temperatures].

In order to study the relationship between the vertical distribution of nutrients and bacterial community structures in sediment interstitial waters of stratified reservoirs with different water temperatures, MiSeq high-throughput sequencing was used to analyze and compare the structural characteristics of sediment bacterial communities after reservoirs were built. Additionally, redundancy analysis (RDA) was used to assess the bacterial communities and environmental factors with Cannoco software. The results showed that the temperature difference between the surface and bottom layer of the Xiaowan Reservoir was 3.3℃, and the maximum thermal gradient was 0.2℃·m-1; thus, it was a typical stratified reservoir. The temperature difference between the surface and bottom layer of the Manwan Reservoir was 0.1℃; thus, it was a typical mixed reservoir. The average concentrations of NH4+-N and NO3--N in sediment interstitial waters of the Xiaowan Reservoir were 2.233 mg·L-1 and 0.030 mg·L-1, while those of Manwan were 2.569 mg·L-1 and 0.016 mg·L-1, respectively. In the different reservoirs, the concentrations of NH4+-N showed upward trends, and while variation of NO3--N was not obvious, the content of NO3--N reached a minimum value in the deep layer. In comparisons between reservoirs, only NO3--N showed a significant difference, in which Xiaowan had obviously higher concentrations than Manwan. The bacterial community structures in the Xiaowan and Manwan reservoir sediments had the same dominant bacteria at the phylum, class, and genus levels. The differences of water temperature stratification had no significant effect on nutrients and microorganisms in the sediments. Under the influence of other factors, the denitrifying bacteria in the Manwan Reservoir sediments were more abundant than those in the Xiaowan Reservoir, and the nitrifying bacteria and anammox bacteria in the Xiaowan Reservoir sediments were more abundant than those in the Manwan Reservoir. In the same reservoir, the denitrifying bacteria in the bottom of the sediments were more abundant, and the organic degradation bacteria, nitrifying bacteria, anammox bacteria, and phosphate-solubilizing bacteria were less abundant in this zone. These trends contributed to the differences of nutrients vertically in the different reservoirs.

[Effects of Biochar Application and Ageing on the Adsorption of Antibiotics in Purple Soil].

Effects of biochar application (at rates of 0%, 1%, and 5%) and field ageing (for 1 a) on the sorption of three antibiotics (sulfadiazine, sulfamethazine, and florfenicol) were studied by batch equilibrium tests for a calcareous purple soil of sloping cropland, which is widely distributed in hilly areas of the upper Yangtze River. A soil-water ratio of 1 g:1 mL, which was close to the actual field soil water content condition, was adopted in the experiment, and comparisons were made to the commonly used ratio of 1 g:10 mL. The results showed that adsorption isotherm data of the studied antibiotics were fitted well by the linear and Freundlich model, and the free energy (ΔGθ) ranged from -0.39 to 11.53 kJ·mol-1, thus indicating that the adsorption of the three antibiotics on the control soils and biochar amended soils was mainly physical adsorption. Application of fresh biochar was found to markedly enhance the adsorption of antibiotics, especially for sulfamethazine and florfenicol of low Kow values, while such an effect was strongly weakened after ageing. The fluorescence peak response intensity of the adsorption equilibrium solution of the aged biochar amended soil was lower than that of the fresh biochar amended soil, so the difference in adsorption performance between fresh and aged biochar amended soils may have been due to the decrease of unstable and soluble organic matter in the latter's biochar after aging.

Identification of a new SYT2 variant validates an unusual distal motor neuropathy phenotype.

OBJECTIVE: To report a new SYT2 missense mutation causing distal hereditary motor neuropathy and presynaptic neuromuscular junction (NMJ) transmission dysfunction. METHODS: We report a multigenerational family with a new missense mutation, c. 1112T>A (p. Ile371Lys), in the C2B domain of SYT2, describe the clinical and electrophysiologic phenotype associated with this variant, and validate its pathogenicity in a Drosophila model. RESULTS: Both proband and her mother present a similar clinical phenotype characterized by a slowly progressive, predominantly motor neuropathy and clear evidence of presynaptic NMJ dysfunction on nerve conduction studies. Validation of this new variant was accomplished by characterization of the mutation homologous to the human c. 1112T>A variant in Drosophila, confirming its dominant-negative effect on neurotransmitter release. CONCLUSIONS: This report provides further confirmation of the role of SYT2 in human disease and corroborates the resultant unique clinical phenotype consistent with heriditary distal motor neuropathy. SYT2-related motor neuropathy is a rare disease but should be suspected in patients presenting with a combination of presynaptic NMJ dysfunction (resembling Lambert-Eaton myasthenic syndrome) and a predominantly motor neuropathy, especially in the context of a positive family history.

Surfactant-enhanced flushing enhances colloid transport and alters macroporosity in diesel-contaminated soil.

Soil contamination by diesel has been often reported as a result of accidental spillage, leakage and inappropriate use. Surfactant-enhanced soil flushing is a common remediation technique for soils contaminated by hydrophobic organic chemicals. In this study, soil flushing with linear alkylbenzene sulfonates (LAS, an anionic surfactant) was conducted for intact columns (15cm in diameter and 12cm in length) of diesel-contaminated farmland purple soil aged for one year in the field. Dynamics of colloid concentration in column outflow during flushing, diesel removal rate and resulting soil macroporosity change by flushing were analyzed. Removal rate of n-alkanes (representing the diesel) varied with the depth of the topsoil in the range of 14%-96% while the n-alkanes present at low concentrations in the subsoil were completely removed by LAS-enhanced flushing. Much higher colloid concentrations and larger colloid sizes were observed during LAS flushing in column outflow compared to water flushing. The X-ray micro-computed tomography analysis of flushed and unflushed soil cores showed that the proportion of fine macropores (30-250µm in diameter) was reduced significantly by LAS flushing treatment. This phenomenon can be attributed to enhanced clogging of fine macropores by colloids which exhibited higher concentration due to better dispersion by LAS. It can be inferred from this study that the application of LAS-enhanced flushing technique in the purple soil region should be cautious regarding the possibility of rapid colloid-associated contaminant transport via preferential pathways in the subsurface and the clogging of water-conducting soil pores.

Concentration and spectroscopic characteristics of DOM in surface runoff and fracture flow in a cropland plot of a loamy soil.

Being crucial for predicting the impact of source inputs on a watershed in rainfall events, an understanding of the dynamics and characteristics of dissolved organic matter (DOM) export from the soil under particular land use types, particularly those associated with underground flows is still largely lacking. A field study was carried out using a 1500m2 slope farmland plot in the hilly area of Sichuan Basin, Southwest China. The discharge of surface runoff and fracture flow was recorded and samples were collected in four representative rainfall events. For DOM characterization, concentration of dissolved organic carbon (DOC) and absorbance/excitation-emission matrix (EEM) fluorescence were analyzed. Soil water potential was also determined using tensiometers for understanding the runoff generation mechanisms. The DOC values for both surface and fracture flow showed significant responses to rainfall, with hydrological path being the primary factor in determining DOM dynamics. EEM-PARAFAC analyses indicated that the soil DOM mainly consisted of two terrestrial humic-like components with peaks located at Ex/Em 270(380)/480nm (C1) and 250(320)/410nm (C2), respectively. Concentrations of these components also responded strongly to rainfall, fluctuating in good agreement with the corresponding DOCs. Although there was no change in the presence of the components themselves, their relative distributions varied during precipitation, with the C1/C2 ratio increasing with the proportion of soil pre-event water. As the dynamic changes of soil DOM characteristics can be successfully captured using spectroscopic techniques, they may serve as a tracer for understanding hydrological paths based on their potential correlations with water source differences during rains.

A synaptotagmin suppressor screen indicates SNARE binding controls the timing and Ca2+ cooperativity of vesicle fusion.

The synaptic vesicle Ca2+ sensor Synaptotagmin binds Ca2+ through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, we took advantage of observations that mutations in the C2B domain Ca2+-binding pocket dominantly disrupt release from invertebrates to humans. We performed an intragenic screen for suppressors of lethality induced by expression of Synaptotagmin C2B Ca2+-binding mutants in Drosophila. This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca2+ cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, we propose a sequence of molecular interactions mediated by Synaptotagmin that promote Ca2+ activation of the synaptic vesicle fusion machinery.

A review of source tracking techniques for fine sediment within a catchment.

Excessive transport of fine sediment, and its associated pollutants, can cause detrimental impacts in aquatic environments. It is therefore important to perform accurate sediment source apportionment to identify hot spots of soil erosion. Various tracers have been adopted, often in combination, to identify sediment source type and its spatial origin; these include fallout radionuclides, geochemical tracers, mineral magnetic properties and bulk and compound-specific stable isotopes. In this review, the applicability of these techniques to particular settings and their advantages and limitations are reviewed. By synthesizing existing approaches, that make use of multiple tracers in combination with measured changes of channel geomorphological attributes, an integrated analysis of tracer profiles in deposited sediments in lakes and reservoirs can be made. Through a multi-scale approach for fine sediment tracking, temporal changes in soil erosion and sediment load can be reconstructed and the consequences of changing catchment practices evaluated. We recommend that long-term, as well as short-term, monitoring of riverine fine sediment and corresponding surface and subsurface sources at nested sites within a catchment are essential. Such monitoring will inform the development and validation of models for predicting dynamics of fine sediment transport as a function of hydro-climatic and geomorphological controls. We highlight that the need for monitoring is particularly important for hilly catchments with complex and changing land use. We recommend that research should be prioritized for sloping farmland-dominated catchments.

Adsorption of linear alkylbenzene sulfonates on carboxyl modified multi-walled carbon nanotubes.

Understanding of the adsorption behavior of organic pollutants on carbon nanotubes (CNTs) and its governing factors are crucial for the assessment of transport and fate of organic pollutants. This study explored adsorption characteristics of linear alkylbenzene sulfonates (LAS) on carboxyl modified multi-walled carbon nanotubes (CMMWCNTs) and the effect of solution chemistry and temperature on LAS sorption. Results indicted LAS adsorption isotherms to display five distinct regions of sorption at 25°C and 60°C. Regardless of temperature, the adsorption isotherm of LAS on the CMMWCNTs was well described using the Freundlich equation. This result indicated heterogeneous distribution of adsorption sites on the CMMWCNT surface. At low initial LAS concentrations, below the critical micelle concentration, (2, 10 and 50mgL-1) LAS adsorption on the CMMWCNTs followed pseudo second-order kinetics. The highest LAS adsorption was observed at ionic strengths of 1.0molL-1 for NaCl; and 0.2molL-1 for both CaCl2 and MgCl2. However, LAS sorption was greatest in the presence of sodium-divalent anion salts and at higher temperatures. These findings are of relevance to the fate and environmental risk of LAS in the presence of CMMWCNTs in high salinity wastewaters or effluents and brackish receiving surface water bodies (e.g., at estuaries).

Biochar increased water holding capacity but accelerated organic carbon leaching from a sloping farmland soil in China.

A hydrologically contained field study, to assess biochar (produced from mixed crop straws) influence upon soil hydraulic properties and dissolved organic carbon (DOC) leaching, was conducted on a loamy soil (entisol). The soil, noted for its low plant-available water and low soil organic matter, is the most important arable soil type in the upper reaches of the Yangtze River catchment, China. Pore size distribution characterization (by N2 adsorption, mercury intrusion, and water retention) showed that the biochar had a tri-modal pore size distribution. This included pores with diameters in the range of 0.1-10 µm that can retain plant-available water. Comparison of soil water retention curves between the control (0) and the biochar plots (16 t ha(-1) on dry weight basis) demonstrated biochar amendment to increase soil water holding capacity. However, significant increases in DOC concentration of soil pore water in both the plough layer and the undisturbed subsoil layer were observed in the biochar-amended plots. An increased loss of DOC relative to the control was observed upon rainfall events. Measurements of excitation-emission matrix (EEM) fluorescence indicated the DOC increment originated primarily from the organic carbon pool in the soil that became more soluble following biochar incorporation.

Synaptotagmin 2 mutations cause an autosomal-dominant form of lambert-eaton myasthenic syndrome and nonprogressive motor neuropathy.

Synaptotagmin 2 is a synaptic vesicle protein that functions as a calcium sensor for neurotransmission but has not been previously associated with human disease. Via whole-exome sequencing, we identified heterozygous missense mutations in the C2B calcium-binding domain of the gene encoding Synaptotagmin 2 in two multigenerational families presenting with peripheral motor neuron syndromes. An essential calcium-binding aspartate residue, Asp307Ala, was disrupted by a c.920A>C change in one family that presented with an autosomal-dominant presynaptic neuromuscular junction disorder resembling Lambert-Eaton myasthenic syndrome. A c.923C>T variant affecting an adjacent residue (p.Pro308Leu) produced a presynaptic neuromuscular junction defect and a dominant hereditary motor neuropathy in a second family. Characterization of the mutation homologous to the human c.920A>C variant in Drosophila Synaptotagmin revealed a dominant disruption of synaptic vesicle exocytosis using this transgenic model. These findings indicate that Synaptotagmin 2 regulates neurotransmitter release at human peripheral motor nerve terminals. In addition, mutations in the Synaptotagmin 2 C2B domain represent an important cause of presynaptic congenital myasthenic syndromes and link them with hereditary motor axonopathies.

Genetic analysis of synaptotagmin C2 domain specificity in regulating spontaneous and evoked neurotransmitter release.

Synaptic vesicle fusion mediates communication between neurons and is triggered by rapid influx of Ca(2+). The Ca(2+)-triggering step for fusion is regulated by the synaptic vesicle transmembrane protein Synaptotagmin 1 (Syt1). Syt1 contains two cytoplasmic C2 domains, termed C2A and C2B, which coordinate Ca(2+) binding. Although C2A and C2B share similar topology, binding of Ca(2+) ions to the C2B domain has been suggested as the only critical trigger for evoked vesicle release. If and how C2A domain function is coordinated with C2B remain unclear. In this study, we generated a panel of Syt1 chimeric constructs in Drosophila to delineate the unique and shared functions of each C2 domain in regulation of synaptic vesicle fusion. Expression of Syt 1 transgenes containing only individual C2 domains, or dual C2A-C2A or C2B-C2B chimeras, failed to restore Syt1 function in a syt1(-/-) null mutant background, indicating both C2A and C2B are specifically required to support fast synchronous release. Mutations that disrupted Ca(2+) binding to both C2 domains failed to rescue evoked release, but supported synaptic vesicle docking and endocytosis, indicating that these functions of Syt1 are Ca(2+)-independent. The dual C2 domain Ca(2+)-binding mutant also enhanced spontaneous fusion while dramatically increasing evoked release when coexpressed with native Syt1. Together, these data indicate that synaptic transmission can be regulated by Syt1 multimerization and that both C2 domains of Syt1 are uniquely required for modulating Ca(2+)-independent spontaneous fusion and Ca(2+)-dependent synchronous release.