FUS Regulates Activity of MicroRNA-Mediated Gene Silencing.

MicroRNA-mediated gene silencing is a fundamental mechanism in the regulation of gene expression. It remains unclear how the efficiency of RNA silencing could be influenced by RNA-binding proteins associated with the microRNA-induced silencing complex (miRISC). Here we report that fused in sarcoma (FUS), an RNA-binding protein linked to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), interacts with the core miRISC component AGO2 and is required for optimal microRNA-mediated gene silencing. FUS promotes gene silencing by binding to microRNA and mRNA targets, as illustrated by its action on miR-200c and its target ZEB1. A truncated mutant form of FUS that leads its carriers to an aggressive form of ALS, R495X, impairs microRNA-mediated gene silencing. The C. elegans homolog fust-1 also shares a conserved role in regulating the microRNA pathway. Collectively, our results suggest a role for FUS in regulating the activity of microRNA-mediated silencing.

A C9orf72-CARM1 axis regulates lipid metabolism under glucose starvation-induced nutrient stress.

Cells undergo metabolic adaptation during environmental changes by using evolutionarily conserved stress response programs. This metabolic homeostasis is exquisitely regulated, and its imbalance could underlie human pathological conditions. We report here that C9orf72, which is linked to the most common forms of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is a key regulator of lipid metabolism under stress. Loss of C9orf72 leads to an overactivation of starvation-induced lipid metabolism that is mediated by dysregulated autophagic digestion of lipids and increased de novo fatty acid synthesis. C9orf72 acts by promoting the lysosomal degradation of coactivator-associated arginine methyltransferase 1 (CARM1), which in turn regulates autophagy-lysosomal functions and lipid metabolism. In ALS/FTD patient-derived neurons or tissues, a reduction in C9orf72 function is associated with dysregulation in the levels of CARM1, fatty acids, and NADPH oxidase NOX2. These results reveal a C9orf72-CARM1 axis in the control of stress-induced lipid metabolism and implicates epigenetic dysregulation in relevant human diseases.

G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA.

The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadruplex conformation of G-rich C9orf72 repeat RNA, we find that G-quadruplex motifs are abundant and conserved features of NEAT1. Furthermore, we determine that NONO binds NEAT1 G-quadruplexes with structural specificity and provide evidence that G-quadruplex motifs mediate NONO-NEAT1 association, with NONO binding sites on NEAT1 corresponding largely to G-quadruplex motifs, and treatment with a G-quadruplex-disrupting small molecule causing dissociation of native NONO-NEAT1 complexes. Together, these findings position G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1 and provide a framework for further investigation into the role of G-quadruplexes in paraspeckle formation and function.

A Helicase Unwinds Hexanucleotide Repeat RNA G-Quadruplexes and Facilitates Repeat-Associated Non-AUG Translation.

The expansion of a hexanucleotide repeat GGGGCC (G4C2) in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 expansion leads to repeat-associated non-AUG (RAN) translation and the production of toxic dipeptide repeat (DPR) proteins, but the mechanisms of RAN translation remain enigmatic. Here, we report that the RNA helicase DHX36 is a robust positive regulator of C9orf72 RAN translation. DHX36 has a high affinity for the G4C2 repeat RNA, preferentially binds to the repeat RNA's G-quadruplex conformation, and efficiently unwinds the G4C2 G-quadruplex structures. Native DHX36 interacts with the G4C2 repeat RNA and is essential for effective RAN translation in the cell. In induced pluripotent stem cells and differentiated motor neurons derived from C9orf72-linked ALS patients, reducing DHX36 significantly decreased the levels of endogenous DPR proteins. DHX36 is also aberrantly upregulated in tissues of C9orf72-linked ALS patients. These results indicate that DHX36 facilitates C9orf72 RAN translation by resolving repeat RNA G-quadruplex structures and may be a potential target for therapeutic intervention.

[Determination of lipophilic marine biotoxins in shellfish foods by ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry].

OBJECTIVE: An ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry(UPLC-QqLIT-MS) was established for determination of lipophilic marine biotoxins in shellfish. And the 12 lipophilic marine biotoxins in shellfish were surveyed. METHODS: The lipophilic marine biotoxins in homogenized shellfish were ultrasonically extracted by methanol in super-sonic instrument, and cleaned up by solid phase extraction of Strata-X column, and eluted with methanol(containing 0.3% ammonia water). The elution was diluted with water, and cleaned by 0.22 µm millipore filter. The filtrate was separated on a Waters ACQUITY UPLC BEH C_(18) column(150 mm×2.1 mm, 1.7 µm)by gradient elution in 12 minutes with acetronitrile-water(containing 0.01%(V/V) ammonia and 2 mmol/L ammonium formate) as mobile phase, and detected by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), identified by electrospray ionization(ESI) in simultaneous scanning mode of positive and negative ions using multiple reaction monitoring, and quantified with external standards. Information dependent acquisition scan function(IDA) combined with enhanced production scan(EPI) was used to confirm the 12 lipophilic marine biotoxins. RESULTS: The calibration curves of 12 lipophilic marine biotoxins showed good linearity in the range of 0.5-50 µg/L with correlation coefficients were 0.9984-0.9999.The detection limits of the method were 0.15-0.29 µg/kg. The recoveries of three spiking levels ranged from 80.0% to 116.0%, and the relative standard deviation(RSD) were 0.6%-6.4%(n=7). CONCLUSION: The method for determination of 12 lipophilic marine biotoxins in shellfish by UPLC-QqLIT-MS was of operation convenience, less interference from impurities and good accuracy, which could meet the requirements for the determination of 12 lipophilic marine biotoxins residues in sea foods.

Transmission of dynamic supercoiling in linear and multi-way branched DNAs and its regulation revealed by a fluorescent G-quadruplex torsion sensor.

DNA supercoiling is an important regulator of gene activity. The transmission of transcription-generated supercoiling wave along a DNA helix provides a way for a gene being transcribed to communicate with and regulate its neighboring genes. Currently, the dynamic behavior of supercoiling transmission remains unclear owing to the lack of a suitable tool for detecting the dynamics of supercoiling transmission. In this work, we established a torsion sensor that quantitatively monitors supercoiling transmission in real time in DNA. Using this sensor, we studied the transmission of transcriptionally generated negative supercoiling in linear and multi-way DNA duplexes. We found that transcription-generated dynamic supercoiling not only transmits along linear DNA duplex but also equally diverges at and proceeds through multi-way DNA junctions. We also show that such a process is regulated by DNA-protein interactions and non-canonical DNA structures in the path of supercoiling transmission. These results imply a transcription-coupled mechanism of dynamic supercoiling-mediated intra- and inter-chromosomal signal transduction pathway and their regulation in DNA.

Guanine-vacancy-bearing G-quadruplexes responsive to guanine derivatives.

G-quadruplex structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and nanodevices. G-quadruplexes are normally composed of four Gn (n ≥ 3) tracts assembled into a core of multiple stacked G-quartet layers. By dimethyl sulfate footprinting, circular dichroism spectroscopy, thermal melting, and photo-cross-linking, here we describe a unique type of intramolecular G-quadruplex that forms with one G2 and three G3 tracts and bears a guanine vacancy (G-vacancy) in one of the G-quartet layers. The G-vacancy can be filled up by a guanine base from GTP or GMP to complete an intact G-quartet by Hoogsteen hydrogen bonding, resulting in significant G-quadruplex stabilization that can effectively alter DNA replication in vitro at physiological concentration of GTP and Mg(2+). A bioinformatic survey shows motifs of such G-quadruplexes are evolutionally selected in genes with unique distribution pattern in both eukaryotic and prokaryotic organisms, implying such G-vacancy-bearing G-quadruplexes are present and play a role in gene regulation. Because guanine derivatives are natural metabolites in cells, the formation of such G-quadruplexes and guanine fill-in (G-fill-in) may grant an environment-responsive regulation in cellular processes. Our findings thus not only expand the sequence definition of G-quadruplex formation, but more importantly, reveal a structural and functional property not seen in the standard canonical G-quadruplexes.

Simultaneous determination of urinary parabens, bisphenol A, triclosan, and 8-hydroxy-2'-deoxyguanosine by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A simple and fast method was developed for the simultaneous determination of five parabens, bisphenol A (BPA), triclosan (TCS), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in human urine using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The solid-phase extraction (SPE) procedure, chromatographic conditions, and MS/MS parameters were optimized to achieve maximum sensitivity and accuracy for the analytes. The validation results showed that the correlation coefficients (R (2)) and recoveries ranged from 0.999 to 1 and 83.9 to 109.9 %, respectively, and the intra-day and inter-day precisions (relative standard deviation, RSD) were within the range of 1.3-8.5 % and 1.3-9.0 %, respectively. The limits of detection for the analytes ranged from 0.001 to 0.05 µg/L. The method was successfully employed to determine parabens, BPA, TCS, and 8-OHdG in urine samples from school students in Guangzhou, China. The results showed that methyl, ethyl, n-propyl parabens, BPA, TCS, and 8-OHdG were frequently detected in urine samples. n-Butyl and benzyl parabens were only detected in a part of the samples due to their low concentrations in urine.

[Determination of 3-nitropropionic acid in poisoning samples by ultra-performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: An ultra-performance liquid chromatography-tandem mass spectrometric method was established for determination of 3-nitropropionic acid of sugarcane, sugarcane bagasse, vomit, serum and urine. METHODS: The 3-nitropropionic acid in poisoning samples was extracted by acetonitrile in super-sonic instrument. The supernatant was cleaned up with PSA column and eluted with 10% ammonia water-methanol (10: 90, V/V), then the purified solution was concentrated by nitrogen, dissolved with water (containing 0.4% formic acid) and cleaned by 0.22 µm millipore filter. The filtrate was detected by ultra-performance liquid chromatography-tandem mass spectrometry, identified by electrospray ionization (ESI) in negative mode using multiple reaction monitoring, and quantified with external standards of sample matrix matching. The sample extract was separated on an acquity BEH C18 column (2.1 mm x 150 mm x 1.7 µm) by gradient elution in 10 minutes with acetronitrile-water as mobile phase. RESULTS: The calibration curves of 3-nitropropionic acid residues showed good linearity in the range of 1.0 - 50 µg/kg with correlation coefficient of 0.9993 or 0.9998. The detection limits of the method were from 0.06 µg/kg to 0.30 µg/kg, and limits of quantitation ranged from 0.20 µg/kg to 1.0 µg/kg. The recoveries of three spiking levels (1.0, 10.0 and 100.0 µg/kg) ranged from 86.9% to 102.0%, and the relative standard deviations of 1.80%- 4.19% were obtained. CONCLUSION: The method for determination of 3-nitropropionic acid in poisoning samples by UPLC-MS/MS is of operation convenience, less interference from impurities and good accuracy, which could provide evidence and treatment for mouldy sugarcane poisoning.

DNA G-quadruplex formation in response to remote downstream transcription activity: long-range sensing and signal transducing in DNA double helix.

G-quadruplexes, four-stranded structures formed by Guanine-rich nucleic acids, are implicated in many physiological and pathological processes. G-quadruplex-forming sequences are abundant in genomic DNA, and G-quadruplexes have recently been shown to exist in the genome of mammalian cells. However, how G-quadruplexes are formed in the genomes remains largely unclear. Here, we show that G-quadruplex formation can be remotely induced by downstream transcription events that are thousands of base pairs away. The induced G-quadruplexes alter protein recognition and cause transcription termination at the local region. These results suggest that a G-quadruplex-forming sequence can serve as a sensor or receiver to sense remote DNA tracking activity in response to the propagation of mechanical torsion in a DNA double helix. We propose that the G-quadruplex formation may provide a mean for long-range sensing and communication between distal genomic locations to coordinate regulatory transactions in genomic DNA.

[Determination of 7 penicilins and penicilloic acids in milk products by high performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: A high performance liquid chromatography-tandem mass spectrometric method was established for determination of 7 penicilins (cloxacillin, nafcillin, oxacillin, penicillin V, amoxicillin, penicillin G, ampicillin) and their penicilloic acids (cloxacilloic acid, nafcilloic acid, oxacilloic acid, penicilloic acid V, amoxicilloic acid, penicilloic acid G and ampicilloic acid) in milk products. And the 7 penicilins and penicilloic acids in milk products were surveyed. METHODS: The 7 penicilins and penicilloic acids in milk products were extracted by water in super-sonic instrument , precipitated proteins by acetonitrile and degreased fat by n-hexane with liquid-liquid extraction, then the purified solution was concentrated by nitrogen, dissolved with acetonitrile-water (10 + 90, V/V) and cleaned by 0.22 µm millipore filter. The filtrate was detected by high performance liquid chromatography-tandem mass spectrometry, identified by electrospray ionization (ESI) in positive mode using multiple reaction monitoring, and quantified with external standards. RESULTS: The calibration curves of 7 penicilins and penicilloic acids showed good linearity in the range of 1.0-200 µg/kg with correlation coefficients were above 0.9992. The detection limits of the method were from 0.03 µg/kg to 0.15 µg/kg. The recoveries of three spiking levels ranged from 80.0% to 110.0%, and RSDs of 7.06% or less were obtained. CONCLUSION: The method for determination of 7 penicilins and penicilloic acids in milk products by HPLC-MS/MS was of operation convenience, less interference from impurities and good accuracy, which could meet the requirements for the determination of 7 penicilins and penicilloic acids residues in milk products.

[High performance liquid chromatography coupled with electrospray tandem mass spectrometry for the analysis of dicyandiamide and melamine residues in milk and milk products].

OBJECTIVE: To establish a sensitive method for the analysis of dicyanodiamide and melamine residue in milk and milk products by, hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry. METHODS: Samples were extracted with 2.5% (V/V) formic acid solution. Acetonitrile was used to precipitate proteins. The separation was carried on Acquity UPLC BEH Amide column (100 mm x 2.1 mm x 1.7 mm) by gradient elution with acetonitrile - water as mobile phase. Identification was achieved by electrospray ionization (ESI) in positive mode using multiple reaction monitoring, and quantified with external standards. RESULTS: The dicyanodiamide and melamine were linear in the range of 5.0 - 1000.0 µg/L with correlation coefficient of 0.9995 for dicyanodiamide and 0.9997 for melamine. The detection limit of the method were 10.0 µg/kg for dicyanodiamide and melamine. The spiked at three levels ranged 86.0% - 100.0% (0.050 mg/kg), 90.0% - 104.0% (1.0 mg/kg) and 90.0% - 100.1% (10.0 mg/kg). And the relative standard derivations were lower than 1.06% -7.77%. The within-day precisions were 2.35% (dicyanodiamide) and 3.44% (melamine), and the inter-day precision 3.87% (dicyanodiamide) and 5.39% (melamine). CONCLUSION: The method was sensitive, accurate and precise. It can be used in monitoring quality of milk production and daily analysis of milk and milk products.

[Efficiency evaluation of capsaicinoids to discriminate bio-waste oils from edible vegetable oils].

OBJECTIVE: To evaluate the efficiency of capsaicinoids to discriminate bio-waste oil from edible vegetable oil. METHODS: 14 raw vegetable oils, 24 fried waste oils, 34 kitchen-waste oils, 32 edible non-peanut vegetable oil, 32 edible peanuts oil, 16 edible oil add flavorand and 11 refined bio-waste oils were prepared and examined for capsaicinoids including capsaicin, dihydrocapsaicin and nonylic acid vanillylamide. The detection results of the above samples were statistically tested based on sample category to assessment identify the effectiveness of the bio-waste oils with capsaicinoids. RESULTS: As a indicator, capsaincin was possessed of high detection sensitivity and has the highest efficiency to discern kitchen-waste oils and refined bio-waste oils samples from edible non-peanut vegetable oil correctly. The accuracy rate of identification were 100% and 90.1% respectively. There is the background in peanut oil. CONCLUSION Capsaicin added in cooking process can be retained in the refining process and hardly be removed in the refining process. In the case of fully eliminating the background interference, capsaicinoids can effectively identify bio-waste oils and edible vegetable oil in combination.

[Determination of hexabromocyclododecanes diastereoisomers in human breast milk by HPLC-MS/MS].

OBJECTIVE: A method for the determination of ac, 3 and γ-hexabromocyclododecanes (HBCDs) in human breast milk was developed by HPLC-MS/MS. METHODS: 3 -5 g human breast milk powder was spiked with '3C-HBCDs and then been extracted using Soxhlet extraction. The extract was dried and dissolved in 6ml of cyclohexane/ethyl acetate (1:1 ), then purified by gel permeation chromatography (GPC). The effluent was concentrated with rotary evaporation and then re-dissolved in hexane. 2ml of sulphuric acid was added to remove the fat for further clean-up. After drying under nitrogen, the supernatant was dissolved in 100 µl of methanol and finally determined by HPLC-MS/MS. RESULTS: The linear range for the three diastereoisomers of HBCDs was in 1 - 50 µg/L, with correlation coefficients ranging from 0. 9997 to 0.998. The detection limits of the three diastereoisomers ranged from 0. 12 to 0. 22 µg/L. The recoveries for three spiking levels ranged from 82. 80% to 110. 60% . The intra-day and inter-day relative standard deviations (RSD) were all less than 9. 4%. CONCLUSION: The developed method was simple, convenient and sensitive. It was suitable for the determination of or, P3 and y-HBCDs in breast milk and other matrix in the future.

CRISPR-Cas13d targeting suppresses repeat-associated non-AUG translation of C9orf72 hexanucleotide repeat RNA.

A hexanucleotide GGGGCC repeat expansion in the non-coding region of C9orf72 gene is the most common genetic mutation identified in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The resulting repeat RNA and dipeptide repeat proteins from non-conventional repeat translation have been recognized as important markers associated with the diseases. CRISPR-Cas13d, a powerful RNA targeting tool, has faced challenges in effectively targeting RNA with stable secondary structures. Here we report that CRISPR-Cas13d can be optimized to specifically target GGGGCC repeat RNA. Our results demonstrate that the CRISPR-Cas13d system can be harnessed to significantly diminish the translation of poly-dipeptides originating from the GGGGCC repeat RNA. This efficacy has been validated in various cell types, including induced pluripotent stem cells and differentiated motor neurons originating from C9orf72-ALS patients, as well as in C9orf72 repeat transgenic mice. These findings demonstrate the application of CRISPR-Cas13d in targeting RNA with intricate higher-order structures and suggest a potential therapeutic approach for ALS and FTD.

[Simultaneous determination of melamine and cyanuric acid residues in water and urine by solid phase extraction-hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry method].

OBJECTIVE: We aimed to establish a sensitive quantified method for the simultaneous determination of melamine and cyanuric acid residues in water and urine by hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry (HILIC-ESI-MS/MS) with the pretreatment of hydrophilic functional silica gel and cation exchange resin mixed solid phase extraction column(MCT), and to investigate the melamine and cyanuric acid residues in 501 water and 216 urine from several province and city. METHODS: About 100 ml water (or 10 ml urine) was adjusted to pH 3.0 with concentrated hydrochloric acid, and then mixed with the internal standard solution((15)N3-melamine and (15)N3-(13)C3 -cyanuric acid) and 100 ml acetonitrile (10 ml for urine). The solution was cleaned with MCT solid-phase extraction column, and eluted once by 3 ml methanol and twice by 2.5 ml methanol (containing 5% ammonia water). The effluent was collected and dried by N2 flow at 40 °C, and then diluted to 2 mmol/L ammonium acetate containing 90% volume fraction acetonitrile. The completely dissolved solution was then filtered with 0.22 µm organic membrane; and the filtrate was detected by high performance liquid chromatography-tandem mass spectrometry and quantified with internal standards. The repeatability and sensitivity of the assay were evaluated. Then we detected the melamine and cyanuric acid residues in 501 water and 216 urine samples collected from several province and city. RESULTS: By the quantification of internal standard (15)N3-melamine and (15)N3-(13)C3-cyanuric acid, the melamine and cyanuric acid were linear in the range of 2.0-1000.0 µg/L with correlation coefficient of 0.9998 and 0.9997. The detection limits of the method were separately 0.4 ng/L (melamine) and 0.3 ng/L (cyanuric acid) for water, and 4.0 ng/L (melamine) and 3.0 ng/L (cyanuric acid) for urine. The average recovery rate was around 95.3%-100.1% with the relative standard deviation (RSD) was <4.02%. Out of the 501 water samples, melamine was detected out in 19.9% (100/501) and cyanuric acid was detected out in 5.2% (26/501). The content was around 0.03-5.00 g/L. Melamine or cyanuric acid was detected out in 24.5% of the urine samples (53/216), with the content around 0.01-1.00 g/L. CONCLUSION: The established method of solid phase extraction-hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry can satisfy the requirement for detection of melamine and cyanuric acid residues in all sorts of water and urine. Meanwhile, the two substances widely existed in water and Chinese population.

[Simultaneous analysis the residues of 20 hormones in foods of animal origin by liquid chromatography tandem mass spectrometry].

OBJECTIVE: To develop a quick and accurate method for simultaneous determining the multi-residues of hormones in foods of animal origin by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to investigate these residues in swine, bovine, egg and milk collected from local markets in Shenzhen. METHOD: The sample was firstly extracted with acetonitrile, and then subjected to solid-phase extraction clean-up using HLB-NH2 cartridges after defatted with hexane, lastly detected by high performance liquid chromatography-tandem mass spectrometry. Identification was achieved by electrospray ionization (ESI) in both positive and negative mode using multiple reaction monitoring. Quantification was performed by internal standard calibration. RESULTS: The study showed that there was a certain amount of endogenous hormones in collected samples. The limits of quantification were 0.5 - 1.0 microg/kg for 20 hormones in swine, bovine, egg and milk. Average recoveries were 60.4% -118.2%, and the relative standard deviations were 2.5% - 16.2%. CONCLUSION: This method was quick and accurate which could be used for determination of hormones in foods of animal origin (such as swine, bovine, egg and milk).

Optimal search mapping among sensors in heterogeneous smart homes.

There are huge differences in the layouts and numbers of sensors in different smart home environments. Daily activities performed by residents trigger a variety of sensor event streams. Solving the problem of sensor mapping is an important prerequisite for the transfer of activity features in smart homes. However, it is common practice among most of the existing approaches that only sensor profile information or the ontological relationship between sensor location and furniture attachment are used for sensor mapping. The rough mapping seriously restricts the performance of daily activity recognition. This paper presents a mapping approach based on the optimal search for sensors. To begin with, a source smart home that is similar to the target one is selected. Thereafter, sensors in both source and target smart homes are grouped by sensor profile information. In addition, sensor mapping space is built. Furthermore, a small amount of data collected from the target smart home is used to evaluate each instance in sensor mapping space. In conclusion, Deep Adversarial Transfer Network is employed to perform daily activity recognition among heterogeneous smart homes. Testing is conducted using the public CASAC data set. The results have revealed that the proposed approach achieves a 7-10% improvement in accuracy, 5-11% improvement in precision, and 6-11% improvement in F1 score, compared with the existing methods.

DNA-initiated epigenetic cascades driven by C9orf72 hexanucleotide repeat.

The C9orf72 hexanucleotide repeat expansion (HRE) is the most frequent genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we describe the pathogenic cascades that are initiated by the C9orf72 HRE DNA. The HRE DNA binds to its protein partner DAXX and promotes its liquid-liquid phase separation, which is capable of reorganizing genomic structures. An HRE-dependent nuclear accumulation of DAXX drives chromatin remodeling and epigenetic changes such as histone hypermethylation and hypoacetylation in patient cells. While regulating global gene expression, DAXX plays a key role in the suppression of basal and stress-inducible expression of C9orf72 via chromatin remodeling and epigenetic modifications of the promoter of the major C9orf72 transcript. Downregulation of DAXX or rebalancing the epigenetic modifications mitigates the stress-induced sensitivity of C9orf72-patient-derived motor neurons. These studies reveal a C9orf72 HRE DNA-dependent regulatory mechanism for both local and genomic architectural changes in the relevant diseases.

[Determination of five microcystins in drinking water by HPLC/MS/MS].

OBJECTIVE: A high performance liquid chromatography-tandem mass spectrometric method was established for determination of five microcystins( MC-LR,MC-LW,MC-RR, MC-LF, MC-YR)in drinking water and source water. METHODS: The five microcystins in water was cleaned by 0.22 microm millipore filter, then detected by high performance liquid chromatography-tandem mass spectrometry. Identification was achieved by electrospray ionization (ESI) in positive mode using multiple reaction monitoring. RESULTS: The calibration curves of five microcystins showed good linearity in the range of 0.5-50 microg/L with correlation coefficient in the range of 0.9994 -1.0000. The detection limit of the method was from 0.06 microg/L to 0.08 microg/L, the recoveries of two spiking levels ranged from 91.2% to 102%, and RSDs of range from 2.11% to 3.26% were obtained. CONCLUSION: The method for determination of five microcystins in drinking water and source water by HPLC-MS/MS was of operation convenience, less interference from impurities and good accuracy, which could meet the requirements of national health standard method for the determination of microcystins in drinking water.