[Treatment of New Daily Persistent Headache With Esketamine: Report of One Case].

New daily persistent headache (NDPH) is a kind of persistent headache that patients can identify the exact date of the sudden onset.It is one of the rare primary headaches difficult to be cured and may lead to disability,seriously affecting the daily life and work.The exact pathogenesis of NDPH remains unclear,which makes the treatment difficult.Here we report a case of refractory NDPH treated by intravenous injection of esketamine at a sub-anesthetic dose.

Anti-Toxoplasma gondii effect of tylosin in vitro and in vivo.

BACKGROUND: Toxoplasma gondii is an important protozoan pathogen with medical and veterinary importance worldwide. Drugs currently used for treatment of toxoplasmosis are less effective and sometimes cause serious side effects. There is an urgent need for the development of more effective drugs with relatively low toxicity. METHODS: The effect of tylosin on the viability of host cells was measured using CCK8 assays. To assess the inhibition of tylosin on T. gondii proliferation, a real-time PCR targeting the B1 gene was developed for T. gondii detection and quantification. Total RNA was extracted from parasites treated with tylosin and then subjected to transcriptome analysis by RNA sequencing (RNA-seq). Finally, murine infection models of toxoplasmosis were used to evaluate the protective efficacy of tylosin against T. gondii virulent RH strain or avirulent ME49 strain. RESULTS: We found that tylosin displayed low host toxicity, and its 50% inhibitory concentration was 175.3 µM. Tylsoin also inhibited intracellular T. gondii tachyzoite proliferation, with a 50% effective concentration of 9.759 µM. Transcriptome analysis showed that tylosin remarkably perturbed the gene expression of T. gondii, and genes involved in "ribosome biogenesis (GO:0042254)" and "ribosome (GO:0005840)" were significantly dys-regulated. In a murine model, tylosin treatment alone (100 mg/kg, i.p.) or in combination with sulfadiazine sodium (200 mg/kg, i.g.) significantly prolonged the survival time and raised the survival rate of animals infected with T. gondii virulent RH or avirulent ME49 strain. Meanwhile, treatment with tylosin significantly decreased the parasite burdens in multiple organs and decreased the spleen index of mice with acute toxoplasmosis. CONCLUSIONS: Our findings suggest that tylosin exhibited potency against T. gondii both in vitro and in vivo, which offers promise for treatment of human toxoplasmosis.

Enhancing Mechanical and Corrosion Properties of AISI 420 with Titanium-Nitride Reinforcement through High-Power-Density Selective Laser Melting Using Two-Stage Mixed TiN/AISI 420 Powder.

This study investigates the effect of laser volume energy density (VED) on the properties of AISI 420 stainless steel and TiN/AISI 420 composite manufactured by selective laser melting (SLM). The composite contained 1 wt.% TiN and the average diameters of AISI 420 and TiN powders were 45 µm and 1 µm, respectively. The powder for SLMing the TiN/AISI 420 composite was prepared using a novel two-stage mixing scheme. The morphology, mechanical, and corrosion properties of the specimens were analyzed, and their correlations with microstructures were investigated. The results showed that the surface roughness of both SLM samples decreases with increasing VED, while relative densities greater than 99% were achieved at VEDs higher than 160 J/mm3. The SLM AISI 420 specimen fabricated at a VED of 205 J/mm3 exhibited the highest density of 7.7 g/cm3, tensile strength (UTS) of 1270 MPa, and elongation of 3.86%. The SLM TiN/AISI 420 specimen at a VED of 285 J/mm3 had a density of 7.67 g/cm3, UTS of 1482 MPa, and elongation of 2.72%. The microstructure of the SLM TiN/AISI 420 composite displayed a ring-like micro-grain structure consisting of retained austenite on the grain boundary and martensite in the grain. The TiN particles strengthened the mechanical properties of the composite by accumulating along the grain boundary. The mean hardnesses of the SLM AISI 420 and TiN/AISI 420 specimens were 635 and 735 HV, respectively, which exceeded previously reported results. The SLM TiN/AISI 420 composite exhibited excellent corrosion resistance in both 3.5 wt.% NaCl and 6 wt.% FeCl3 solutions, with a resulting corrosion rate as low as 11 µm/year.

Glutamate-Evoked Ca2+ Responses in the Rat Suprachiasmatic Nucleus: Involvement of Na+/K+-ATPase and Na+/Ca2+-Exchanger.

Glutamate mediates photic entrainment of the central clock in the suprachiasmatic nucleus (SCN) by evoking intracellular Ca2+ signaling mechanisms. However, the detailed mechanisms of glutamate-evoked Ca2+ signals are not entirely clear. Here, we used a ratiometric Ca2+ and Na+ imaging technique to investigate glutamate-evoked Ca2+ responses. The comparison of Ca2+ responses to glutamate (100 µM) and high (20 mM) K+ solution indicated slower Ca2+ clearance, along with rebound Ca2+ suppression for glutamate-evoked Ca2+ transients. Increasing the length of exposure time in glutamate, but not in 20 mM K+, slowed Ca2+ clearance and increased rebound Ca2+ suppression, a result correlated with glutamate-induced Na+ loads. The rebound Ca2+ suppression was abolished by ouabain, monensin, Na+-free solution, or nimodipine, suggesting an origin of activated Na+/K+-ATPase (NKA) by glutamate-induced Na+ loads. Ouabain or Na+-free solution also slowed Ca2+ clearance, apparently by retarding Na+/Ca2+-exchanger (NCX)-mediated Ca2+ extrusion. Together, our results indicated the involvement of glutamate-induced Na+ loads, NKA, and NCX in shaping the Ca2+ response to glutamate. Nevertheless, in the absence of external Na+ (NMDG substituted), Ca2+ clearance was still slower for the Ca2+ response to glutamate than for 20 mM K+, suggesting participation of additional Ca2+ handlers to the slower Ca2+ clearance under this condition.

Electrochemical skin conductance and heart rate variability in patients with non-dialysis chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is very common now and associates with high overall and cardiovascular mortality. Numerous studies have reported that Heart rate variability (HRV) could also be used to detect cardiovascular autonomic dysfunction (CAD). We investigated the association of electrochemical skin conductance (ESC) of EZSCAN results with HRV in non-dialysis CKD patients. METHODS: In a cross-sectional study, we enrolled 248 prevalent non-dialysis CKD patients. Patients underwent a 24-h Holter (CB-2302-A, Bio Instrument, China). A time domain analysis of HRV was performed, and the following parameters were obtained: SDNN, SDANN, rMSSD, pNN50. EZSCAN device (Impeto Medical, Paris, France) measures ESC values of each participants. Mean global skin conductance computed as 0.5 * (reflecting (right + left hand)/2 + (right and left foot)/2). Log transforms data into a normal distribution for statistical analysis. RESULTS: There were 142 males and 106 females included in the present study. Patients' age was 56.6±17.08 years. Logarithm(Log) (global ESC) was independently predicted by age (P<0.01), hypertension history, estimated Glomerular filtration rate (eGFR) and log SDNN (P<0.05). While log SDANN, rMSSD and pNN50 were not independent predictors for log (global ESC). CONCLUSION: Increased global ESC significantly associated with elevated HRV, specifically SDNN in non-dialysis CKD patients. This suggested that global ESC may appear to be an important predictor of CAD, and even could be used as a cardiovascular risk factor in non-dialysis CKD patients.

The α- and ß-Subunit Boundary at the Stem of the Mushroom-Like α3ß3-Type Oxygenase Component of Rieske Non-Heme Iron Oxygenases Is the Rieske-Type Ferredoxin-Binding Site.

Cumene dioxygenase (CumDO) is an initial enzyme in the cumene degradation pathway of Pseudomonas fluorescens IP01 and is a Rieske non-heme iron oxygenase (RO) that comprises two electron transfer components (reductase [CumDO-R] and Rieske-type ferredoxin [CumDO-F]) and one catalytic component (α3ß3-type oxygenase [CumDO-O]). Catalysis is triggered by electrons that are transferred from NAD(P)H to CumDO-O by CumDO-R and CumDO-F. To investigate the binding mode between CumDO-F and CumDO-O and to identify the key CumDO-O amino acid residues for binding, we simulated docking between the CumDO-O crystal structure and predicted model of CumDO-F and identified two potential binding sites: one is at the side-wise site and the other is at the top-wise site in mushroom-like CumDO-O. Then, we performed alanine mutagenesis of 16 surface amino acid residues at two potential binding sites. The results of reduction efficiency analyses using the purified components indicated that CumDO-F bound at the side-wise site of CumDO-O, and K117 of the α-subunit and R65 of the ß-subunit were critical for the interaction. Moreover, these two positively charged residues are well conserved in α3ß3-type oxygenase components of ROs whose electron donors are Rieske-type ferredoxins. Given that these residues were not conserved if the electron donors were different types of ferredoxins or reductases, the side-wise site of the mushroom-like structure is thought to be the common binding site between Rieske-type ferredoxin and α3ß3-type oxygenase components in ROs. IMPORTANCE We clarified the critical amino acid residues of the oxygenase component (Oxy) of Rieske non-heme iron oxygenase (RO) for binding with Rieske-type ferredoxin (Fd). Our results showed that Rieske-type Fd-binding site is commonly located at the stem (side-wise site) of the mushroom-like α3ß3 quaternary structure in many ROs. The resultant binding site was totally different from those reported at the top-wise site of the doughnut-like α3-type Oxy, although α3-type Oxys correspond to the cap (α3 subunit part) of the mushroom-like α3ß3-type Oxys. Critical amino acid residues detected in this study were not conserved if the electron donors of Oxys were different types of Fds or reductases. Altogether, we can suggest that unique binding modes between Oxys and electron donors have evolved, depending on the nature of the electron donors, despite Oxy molecules having shared α3ß3 quaternary structures.

Glomerular filtration Rate, urine Albumin/ creatinine ratio and current perception threshold in patients with diabetic kidney disease.

BACKGROUND: Diabetic microvascular complications, including diabetic kidney disease (DKD), retinopathy (DR), and neuropathy (DN), were major causes of morbidity and mortality in diabetic patients worldwide. It has been suggested that urinary albumin-to-creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) were not the only indicators of renal function impairment in DKD and that they were also associated with diabetic peripheral neuropathy (DPN) which might affect nerve conduction velocity (NCV). As 30-40% of DPN patients had no subjective symptoms, while current perception threshold (CPT) could detect sensory nerve damage at an early stage. As a result, we aimed to investigate correlation between UACR, eGFR and CPT in DKD patients. METHODS: A total of 273 DKD patients from the First Affiliated Hospital of Kunming Medical University from January 2018 to June 2020 were enrolled to complete the CPT test. CPT values of the bilateral median nerve and superficial and deep peroneal nerves at 2000 Hz, 250 Hz, and 5 Hz were collected. RESULTS: In normoesthesia and hypaesthesia patients with DKD, MDRD-eGFR correlated negatively with TC (r = -0.135, P = 0.037), left superficial peroneal and deep peroneal nerve 2000 Hz CPT (r = -0.205, P = 0.001) and right superficial peroneal and deep peroneal nerve 2000 Hz CPT (r = -0.154, P = 0.017). Besides, left and right superficial peroneal and deep peroneal nerve 2000 Hz CPT correlated with CKD-EPI-eGFR and UACR. Multivariate logistic regression analysis found left superficial peroneal and deep peroneal nerve 2000 Hz CPT was independently associated with both MDRD-eGFR and CKD-EPI-eGFR. CONCLUSION: Decreased MDRD-eGFR and CKD-EPI-eGFR were expected to be a predictor of peripheral nerve injury in normoesthesia and hypaesthesia patients with DKD.

Propofol induces apoptosis and ameliorates 5­fluorouracil resistance in OSCC cells by reducing the expression and secretion of amphiregulin.

Among the different types of oral cancer, >90% of cases are oral squamous cell carcinoma (OSCC). 5­fluorouracil (5­FU) is a commonly used treatment for OSCC, but cells typically display resistance to the drug. Propofol, an intravenous anesthetic agent, exhibits certain anticancer effects, including the inhibition of cancer cell proliferation, migration and invasion. Secreted proteins, such as growth factors and cytokines are involved in cancer development and progression, but the effect of propofol on secreted proteins in OSCC is not completely understood. An MTT assay, flow cytometry and western blotting were performed to determine the anticancer effects of propofol. The secretion profile of OSCC was determined using an antibody array, and clinical importance was assessed using the Gene Expression Profiling Interactive Analysis database. The results were verified by performing reverse transcription­quantitative PCR (RT­qPCR) and western blotting. 5­FU­resistant cells were established to determine the role of the gene of interest in drug resistance. The results demonstrated that propofol decreased cell viability and promoted cell apoptosis. The antibody array results showed that propofol attenuated the secretion of multiple growth factors. The bioinformatics results indicated that amphiregulin (AREG) was expressed at significantly higher levels in cancer tissues, which was also related to poor prognosis. The results of RT­qPCR and western blotting revealed that propofol decreased AREG expression. Pretreatment with exogenous recombinant AREG increased EGFR activation and conferred propofol resistance. Moreover, the results indicated that the expression and activation of AREG was also related to 5­FU resistance, but propofol ameliorated 5­FU drug resistance. Therefore, the present study suggested that propofol combination therapy may serve as an effective treatment strategy for OSCC.

Exposure assessment of PM2.5 using smart spatial interpolation on regulatory air quality stations with clustering of densely-deployed microsensors.

Accurate mapping of air pollutants is essential for epidemiological studies and environmental risk assessments. Concentrations measured by air quality monitoring stations (AQMS) have primarily been used to assess the exposure of PM2.5. However, the low coverage and amount of monitoring stations affect the errors of spatial interpolation or geostatistical estimates. In contrast to other integrated approaches developed for improved air pollution estimates, this study utilizes data from low-cost microsensors densely deployed in Taiwan to improve the popular spatial interpolation approach called inverse distance weighting (IDW). A large dataset from thousands of low-cost sensors could improve spatial interpolation by describing the distribution of PM2.5 in detail. Therefore, this study presents a clustering-based method to assess the distribution of PM2.5. Then, a smarter IDW is performed based on correlated observations from the selected air quality stations. The publicly available data chosen for this investigation pertained to Taiwan, which has deployed 74 monitoring stations and more than 11,000 low-cost sensors since December 2020. The results of leave-one-out cross-validation indicate that there are fewer PM2.5 estimation errors in the developed approach than in estimations that use kriging across almost all of the months and sampled dates of 2019 and 2020, particularly those with higher PM2.5 spatial heterogeneities. Spatial heterogeneities could result in more significant estimation errors in mainstream approaches. The root mean square error of the monthly average estimate for PM2.5 ranged from 1.17 to 3.86 µg/m3. We also found that the clustering of one month characterizing the pattern of PM2.5 distribution could perform well in spatial interpolations based on historical data from monitoring stations. According to the information on the openaq platform, low-cost sensors are in demand in cities and areas. This trend might pave the way for the application of the proposed approach in other areas for superior exposure assessments.

Effects of Laser Spot Size on the Mechanical Properties of AISI 420 Stainless Steel Fabricated by Selective Laser Melting.

The purpose of this study is to investigate the effects of laser spot size on the mechanical properties of AISI 420 stainless steel, fabricated by selective laser melting (SLM), process. Tensile specimens were built directly via the SLM process, using various laser spot diameters, namely 0.1, 0.2, 0.3, and 0.4 mm. The corresponding volumetric energy density (EV) is 80, 40, 26.7, and 20 J/mm3, respectively. Experimental results indicate that laser spot size is an important process parameter and has significant effects on the surface roughness, hardness, density, tensile strength, and microstructure of the SLM AISI 420 builds. A large laser spot with low volumetric energy density results in balling, un-overlapped defects, a large re-heated zone, and a large sub-grain size. As a result, SLM specimens fabricated by the largest laser spot diameter of 0.4 mm exhibit the roughest surface, lowest densification, and lowest ultimate tensile strength. To ensure complete melting of the powder and melt pool stability, EV of 80 J/mm3 proves to be a suitable laser energy density value for the given SLM processing and material system.

Mutational Signature Analysis Reveals Widespread Contribution of Pyrrolizidine Alkaloid Exposure to Human Liver Cancer.

BACKGROUND AND AIMS: Mutational signature analyses are an effective tool in identifying cancer etiology. Humans are frequently exposed to pyrrolizidine alkaloids (PAs), the most common carcinogenic phytotoxins widely distributed in herbal remedies and foods. However, due to the lack of human epidemiological data, PAs are classified as group II hepatocarcinogens by the World Health Organization. This study identified a PA mutational signature as the biomarker to investigate the association of PA exposure with human liver cancer. APPROACH AND RESULTS: Pyrrole-protein adducts (PPAs), the PA exposure biomarker, were measured and found in 32% of surgically resected specimens from 34 patients with liver cancer in Hong Kong. Next, we delineated the mode of mutagenic and tumorigenic actions of retrorsine, a representative PA, in mice and human hepatocytes (HepaRG). Retrorsine induced DNA adduction, DNA damage, and activation of tumorigenic hepatic progenitor cells, which initiated hepatocarcinogenesis. PA mutational signature, as the unique molecular fingerprint of PA-induced mutation, was derived from exome mutations in retrorsine-exposed mice and HepaRG cells. Notably, PA mutational signature was validated in genomes of patients with PPA-positive liver cancer but not patients with PPA-negative liver cancer, confirming the specificity of this biomarker in revealing PA-associated liver cancers. Furthermore, we examined the established PA mutational signature in 1,513 liver cancer genomes and found that PA-associated liver cancers were potentially prevalent in Asia (Mainland China [48%], Hong Kong [44%], Japan [22%], South Korea [6%], Southeast Asia [25%]) but minor in Western countries (North America [3%] and Europe [5%]). CONCLUSIONS: This study provides a clinical indication of PA-associated liver cancer. We discovered an unexpectedly extensive implication of PA exposure in patients with liver cancer, laying the scientific basis for precautionary approaches and prevention of PA-associated human liver cancers.

An integrated strategy toward comprehensive characterization and quantification of multiple components from herbal medicine: An application study in Gelsemium elegans / 中草药·英文版

Objective: To develop a powerful integrated strategy based on liquid chromatography coupled with mass spectrometry (LC-MS) systems for the comprehensive characterization and quantification of multiple components of herbal medicines. Methods: Firstly, different mobile phase additives, analysis time, and MS acquisition modes were orthogonally tested with liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) in order to detect as many components of Gelsemium elegans as possible with high peak intensity. Secondly, several data mining strategies, including database searching, diagnostic ion filtering and neutral loss filtering, were utilized to perform chemical profiling. Subsequently, this study focused on the quantification and validation of the performance of a liquid chromatography-triple mass spectrometry (LC-QqQ/MS) assay based on derivative multiple reaction monitoring (DeMRM). Results: A total of 147 components from G. elegans were characterized, among them 116 nontarget components were reported for the first time. A sensitive and reproducible LC-QqQ/MS method was successfully developed and validated for the simultaneous relative quantification of 41 components of G. elegans. This LC-QqQ/MS method was then applied to compare the contents of components in the roots, stems and leaves. Conclusion: The present integrated strategy would significantly contribute to chemical studies on herbal medicine, and its utility could be extended to other research fields, such as metabolomics, quality control, and pharmacokinetics.

Effects of Build Direction on the Mechanical Properties of a Martensitic Stainless Steel Fabricated by Selective Laser Melting.

Mechanical properties and microstructure are investigated for a martensitic stainless steel (AISI 420) fabricated by selective laser melting (SLM) in three build directions. The tensile specimens built by SLM are classified into three groups. Group A is horizontally built in the thickness direction, Group B is horizontally built in the width direction, and Group C is vertically built in the length direction. The loading direction in tensile test is parallel to the build direction of Group C, but perpendicular to that of Groups A and B. Experimental results indicate build direction has significant effects on the residual stress, hardness, and tensile properties of SLM builds. Microstructural analyses indicate the as-fabricated SLM AISI 420 builds exhibit elongated cells and acicular structures which are composed of martensite and retained austenite phases growing along the build direction. Such anisotropy in the microstructure leads to anisotropic mechanical properties as Group C specimens (length direction) exhibit greater yield stress, ultimate tensile stress, and elongation than the specimens of Groups A (thickness direction) and B (width direction). The residual compressive stress in the gauge section also contributes to the superior tensile properties of Group C (length direction), as compared to Groups A (thickness direction) and B (width direction), which exhibit residual tensile stress in the gauge section.

Linear Displacement Calibration System Integrated with a Novel Auto-Alignment Module for Optical Axes.

The quality of processed workpieces is affected directly by the precision of the linear stage. Therefore, the linear displacement calibration of machine tools must be implemented before delivery and after employment for a period of time. How to perform a precise calibration with high inspection efficiency is a critical issue in the precision mechanical engineering industry. In this study, the self-developed system integrated by the measurement module based on the common path Fabry-Pérot interferometer for linear displacement and the auto-alignment module for optical axes was proposed to realize the automatic linear displacement calibration of the linear stages. The measurement performance of the developed structure was verified experimentally. With the auto-alignment module, the cosine error was reduced to 0.36 nm and the entire procedure accomplished within 75 s without the limitation of the perceived resolution of the human eye, operational experience, and the risk of misalignment and broken cable. According to the comparison of experimental results for the linear displacement, the repeatability of the proposed measurement module was less than 0.171 µm. After the compensation procedure according to the linear displacement calibration, the systematic positional deviation, repeatability, and accuracy of the linear axis could be improved to 4 µm, 1 µm, and 5 µm respectively. Hence, the calibration efficiency can be improved by 80% with the proposed compact system, which is beneficial for the linear displacement calibration of machine tools in the precision mechanical engineering industry.

Absorption difference between hepatotoxic pyrrolizidine alkaloids and their N-oxides - Mechanism and its potential toxic impact.

ETHNOPHARMACOLOGICAL RELEVANCE: Pyrrolizidine alkaloids (PAs) are a group of phytotoxins widely present in about 3% of flowering plants. Many PA-containing herbal plants can cause liver injury. Our previous studies demonstrated that PA N-oxides are also hepatotoxic, with toxic potency much lower than the corresponding PAs, due to significant differences in their toxicokinetic fates. AIM OF STUDY: This study aimed to investigate the oral absorption of PAs and PA N-oxides for better understanding of their significant differences in toxicokinetics and toxic potency. MATERIALS AND METHODS: The oral absorption of PAs and PA N-oxides in rats and in rat in situ single pass intestine perfusion model was investigated. The intestinal permeability and absorption mechanisms of five pairs of PAs and PA N-oxides were evaluated by using Caco-2 monolayer model. RESULTS: The plasma concentrations of total PAs and PA N-oxides within 0-60 min were significantly lower in rats orally treated with a PA N-oxide-containing herbal alkaloid extract than with a PA-containing herbal alkaloid extract at the same dose, indicating that the absorption of PA N-oxides was lower than that of PAs. Using the rat in situ single pass intestine perfusion model, less cumulative amounts of retrorsine N-oxide in mesenteric blood were observed compared to that of retrorsine. In Caco-2 monolayer model, all five PAs showed absorption with Papp AtoB values [(1.43-16.26) × 10-6 cm/s] higher than those of corresponding N-oxides with Papp AtoB values lower than 1.35 × 10-6 cm/s. A further mechanistic study demonstrated that except for senecionine N-oxide, retrorsine N-oxide, and lycopsamine N-oxide, all PAs and PA N-oxides investigated were absorbed via passive diffusion. While, for these 3 PA N-oxides, in addition to passive diffusion as their primary transportation, efflux transporter-mediated active transportation was also involved but to a less extent with the efflux ratio of 2.31-3.41. Furthermore, a good correlation between lipophilicity and permeability of retronecine-type PAs and their N-oxides with absorption via passive diffusion was observed, demonstrating that PAs have a better oral absorbability than that of the corresponding PA N-oxides. CONCLUSION: We discovered that among many contributors, the lower intestinal absorption of PA N-oxides was the initiating contributor that caused differences in toxicokinetics and toxic potency between PAs and PA N-oxides.

Whole-genome sequencing and analysis of the Chinese herbal plant

Background@# () (2n = 2x = 16) is genus of flowering plants belonging to the Gelsemicaeae family.@*Method@#Here, a high-quality genome assembly using the Oxford Nanopore Technologies (ONT) platform and high-throughput chromosome conformation capture techniques (Hi-C) were used.@*Results@#A total of 56.11 Gb of raw GridION X5 platform ONT reads (6.23 Gb per cell) were generated. After filtering, 53.45 Gb of clean reads were obtained, giving 160 × coverage depth. The genome assemblies 335.13 Mb, close to the 338 Mb estimated by k-mer analysis, was generated with contig N50 of 10.23 Mb. The vast majority (99.2%) of the assembled sequence was anchored onto 8 pseudo-chromosomes. The genome completeness was then evaluated and 1338 of the 1440 conserved genes (92.9%) could be found in the assembly. Genome annotation revealed that 43.16% of the genome is composed of repetitive elements and 23.9% is composed of long terminal repeat elements. We predicted 26,768 protein-coding genes, of which 84.56% were functionally annotated.@*Conclusion@#The genomic sequences of could be a valuable source for comparative genomic analysis in the Gelsemicaeae family and will be useful for understanding the phylogenetic relationships of the indole alkaloid metabolism.

Role of Intracellular Na+ in the Regulation of [Ca2+]i in the Rat Suprachiasmatic Nucleus Neurons.

Transmembrane Ca2+ influx is essential to the proper functioning of the central clock in the suprachiasmatic nucleus (SCN). In the rat SCN neurons, the clearance of somatic Ca2+ following depolarization-induced Ca2+ transients involves Ca2+ extrusion via Na+/Ca2+ exchanger (NCX) and mitochondrial Ca2+ buffering. Here we show an important role of intracellular Na+ in the regulation of [Ca2+]i in these neurons. The effect of Na+ loading on [Ca2+]i was determined with the Na+ ionophore monensin and the cardiac glycoside ouabain to block Na+/K+-ATPase (NKA). Ratiometric Na+ and Ca2+ imaging was used to measure the change in [Na+]i and [Ca2+]i, and cell-attached recordings to investigate the effects of monensin and ouabain on spontaneous firing. Our results show that in spite of opposite effects on spontaneous firing and basal [Ca2+], both monensin and ouabain induced Na+ loading, and increased the peak amplitude, slowed the fast decay rate, and enhanced the slow decay phase of 20 mM K+-evoked Ca2+ transients. Furthermore, both ouabain and monensin preferentially enhanced nimodipine-insensitive Ca2+ transients. Together, our results indicate that in the SCN neurons the NKA plays an important role in regulating [Ca2+]i, in particular, associated with nimodipine-insensitive Ca2+ channels.

Intestinal and hepatic biotransformation of pyrrolizidine alkaloid N-oxides to toxic pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) are among the most significant groups of phytotoxins present in more than 6000 plants in the world. Hepatotoxic retronecine-type PAs and their corresponding N-oxides usually co-exist in plants. Although PA-induced hepatotoxicity is known for a long time and has been extensively studied, the toxicity of PA N-oxide is rarely investigated. Recently, we reported PA N-oxide-induced hepatotoxicity in humans and rodents and also suggested the association of such toxicity with metabolic conversion of PA N-oxides to the corresponding toxic PAs. However, the detailed biochemical mechanism of PA N-oxide-induced hepatotoxicity is largely unknown. The present study investigated biotransformation of four representative cyclic retronecine-type PA N-oxides to their corresponding PAs in both gastrointestinal tract and liver. The results demonstrated that biotransformation of PA N-oxides to PAs was mediated by both intestinal microbiota and hepatic cytochrome P450 monooxygenases (CYPs), in particular CYP1A2 and CYP2D6. Subsequently, the formed PAs were metabolically activated predominantly by hepatic CYPs to form reactive metabolites exerting hepatotoxicity. Our findings delineated, for the first time, that the metabolism-mediated mechanism of PA N-oxide intoxication involved metabolic reduction of PA N-oxides to their corresponding PAs in both intestine and liver followed by oxidative bioactivation of the resultant PAs in the liver to generate reactive metabolites which interact with cellular proteins leading to hepatotoxicity. In addition, our results raised a public concern and also encouraged further investigations on potentially remarkable variations in PA N-oxide-induced hepatotoxicity caused by significantly altered intestinal microbiota due to individual differences in diets, life styles, and medications.

The Na+/H+-Exchanger NHE1 Regulates Extra- and Intracellular pH and Nimodipine-sensitive [Ca2+]i in the Suprachiasmatic Nucleus.

The central clock in the suprachiasmatic nucleus (SCN) has higher metabolic activity than extra-SCN areas in the anterior hypothalamus. Here we investigated whether the Na+/H+ exchanger (NHE) may regulate extracellular pH (pHe), intracellular pH (pHi) and [Ca2+]i in the SCN. In hypothalamic slices bathed in HEPES-buffered solution a standing acidification of ~0.3 pH units was recorded with pH-sensitive microelectrodes in the SCN but not extra-SCN areas. The NHE blocker amiloride alkalinised the pHe. RT-PCR revealed mRNA for plasmalemmal-type NHE1, NHE4, and NHE5 isoforms, whereas the NHE1-specific antagonist cariporide alkalinised the pHe. Real-time PCR and western blotting failed to detect day-night variation in NHE1 mRNA and protein levels. Cariporide induced intracellular acidosis, increased basal [Ca2+]i, and decreased depolarisation-induced Ca2+ rise, with the latter two effects being abolished with nimodipine blocking the L-type Ca2+ channels. Immunofluorescent staining revealed high levels of punctate colocalisation of NHE1 with serotonin transporter (SERT) or CaV1.2, as well as triple staining of NHE1, CaV1.2, and SERT or the presynaptic marker Bassoon. Our results indicate that NHE1 actively extrudes H+ to regulate pHi and nimodipine-sensitive [Ca2+]i in the soma, and along with CaV1.2 may also regulate presynaptic Ca2+ levels and, perhaps at least serotonergic, neurotransmission in the SCN.

Serum endocan and circadian heart rate variability in non-dialysis stage 5 chronic kidney disease patients.

BACKGROUND: Chronic kidney disease (CKD) is very common now and is associated with high overall and cardiovascular mortality. Numerous studies have reported that elevated heart rate (HR) is a risk factor for cardiovascular mortality. We investigated the link between serum endocan and circadian heart rate variability in non-dialysis stage 5 CKD patients. METHODS: In a cross-sectional study, we enrolled 54 prevalent n non-dialysis stage 5 CKD patients (32 males, aged 48.2 ± 14.92 years). HR was measured with an automatic system. Serum endocan level was analyzed by ELISA. RESULTS: Night/day HR ratio was independently predicted by serum endocan level (P < 0.01) and hypertension history (P < 0.05). Adjusted R2 of the model was 0.222. CONCLUSION: Increased serum endocan is significantly associated with circadian heart rate variability in non-dialysis stage 5 CKD patients. Further investigation is needed to explore the potential benefits of serum endocan lowering therapy in this patient group.