DNA barcoding, identification, and validation of the pufferfish (Order: Tetraodontiformes) in China coastal waters.

The order Tetraodontiformes are one of the most unique groups of teleostean fish, exhibiting highly derived and greatly diversified phenotypes. It is a difficult task for both professionals and nonprofessionals to accurately identify these species only according to morphological characteristics. DNA barcoding can identify species at the molecular level to overcome the limitations of morphological classification. In this study, we collected 616 specimens of pufferfish from the coastal waters of China. According to the morphological characteristics, they were preliminarily identified as 50 species. Further analysis using DNA barcodes identified these specimens as 46 species, belonging to 23 genera, 6 families. According to the species classification results of DNA barcoding, the three species identified by morphology as Takifugu pseudommus, Takifugu chinensis, and Takifugu rubripes should be the same species. Similarly, Lagocephalus wheeleri is the synonym of Lagocephalus spadiceus. Another important discovery of DNA barcoding analysis is that there are closer interspecific genetic distances within the genus Takifugu. If T. rubripes, T. pseudommus, and T. chinensis are taken as one species, the average interspecific to intraspecific genetic distance ratio of Takifugu is only 6.21 times, which does not reach the DNA barcoding threshold of more than 10 times proposed previously. Although the interspecific genetic distance in the genus Takifugu is relatively small, each species can be clustered into independent clades in the NJ tree. In conclusion, this study not only found that there are synonymous phenomena in the order Tetraodontiformes but also provided molecular evidence for the valid species names of Takifugu rubripes and Lagocephalus Spadiceus. The results can provide reliable DNA barcoding information for the identification of pufferfish species, help solve the problem of classification confusion in this order, and provide technical support for the identification of the original components of related commodities on the aquatic product market.

Humic Acid Extracts Leading to the Photochemical Bromination of Phenol in Aqueous Bromide Solutions: Influences of Aromatic Components, Polarity and Photochemical Activity.

Dissolved organic matter (DOM) is considered to play an important role in the abiotic transformation of organobromine compounds in marine environment, for it produces reactive intermediates photochemically and is recognized as a significant source of reactive halogen species in seawater. However, due to the complex composition of DOM, the relationship between the natural properties of DOM and its ability to produce organobromine compounds is less understood. Here, humic acid (HA) was extracted and fractionated based on the polarity and hydrophobicity using silica gel, and the influences of different fractions (FA, FB and FC) on the photochemical bromination of phenol was investigated. The structural properties of HA fractions were characterized by UV-vis absorption, Fourier transform infrared spectroscopy and fluorescence spectroscopy, and the photochemical reactivity of HA fractions was assessed by probing triplet dissolved organic matter (3DOM*), singlet oxygen (1O2) and hydroxyl radical (•OH). The influences of HA fractions on the photo-bromination of phenol were investigated in aqueous bromide solutions under simulated solar light irradiation. FA and FB with more aromatic and polar contents enhanced the photo-bromination of phenol more than the weaker polar and aromatic FC. This could be attributed to the different composition and chemical properties of the three HAs' fractions and their production ability of •OH and 3DOM*. Separating and investigating the components with different chemical properties in DOM is of great significance for the assessment of their environmental impacts on the geochemical cycle of organic halogen.

Application of DNA barcoding in fish identification of supermarkets in Henan province, China: More and longer COI gene sequences were obtained by designing new primers.

In recent years, DNA barcode technology has been widely used in food identification, especially in the identification of fish. In China, there are few studies on the authenticity of fish products in Henan province of China. In this study, 179 fish samples were collected from supermarkets in Zhengzhou city and Xinxiang city in Henan province, China. COI gene sequences were obtained with PCR technology by designing specific primers and universal primers. COI gene sequences of all samples were obtained to identify species, which is used to investigate species substitution and mislabeling of the fish sold in the two regional markets. The molecular identification results showed that 28.49% (51/179) fish samples were not consistent with the labels. Substitution of high-price fish by low-price fish was prevalent. For example, halibut (Pleuronectiformes) and cod (Gadus) are replaced by striped catfish (Pangasianodon hypophthalmus), and some merchants label bighead carp (Hypophthalmichthys nobilis) as cod (Gadus), there are also accidental labeling errors (such as labels for greenfin horse-faced filefish (Thamnaconus septentrionalis) have been identified as grass carp (Ctenopharyngodon idella) and Wuchang bream (Megalobrama amblycephala) etc. Most of the samples labeled correctly are the fish of low economic value and the fresh fish. This study shows that almost all the commercial fish can be identified by COI DNA barcoding by newly designed primers. Finally, this study also gives a reference of real species of fish fillet in Henan province in China.

[Determination of 2-methylisoborneol and geosmin in drinking water using headspace solid phase micro-extraction coupled with gas chromatography-mass-spectrometry].

The odorous compounds of 2-methylisoborneol (2-MIB) and geosmin (GSM) heavily produced and released in water source are one of the most important factors leading to off-flavor emergencies and resident water consumption panic in drinking water. A headspace solid phase micro-extraction ( HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) method was established for the simultaneous determination of these two trace earthy and musty compounds in reservoir water, reservoir soil and tap water. The conditions of HS-SPME, such as salt amount, extraction time and extraction temperature, were optimized based on orthogonal analysis. The qualitative and quantitative analyses of 2-MIB and GSM were carried out in the electron impact (EI) -selective ion scanning mode. The results showed that the linear relationship between peak area and concentration of 2-MIB and GSM was good enough (r2 0.998) from 5 to 1 000 ng/L, the limits of detection were 0.72 ng/L for 2-MIB, 0.34 ng/L for GSM and the limits of quantification were 2.40 ng/L for 2-MIB, 1.13 ng/L for GSM. When the target samples spiked in the range of 10-600 ng/L, the average recoveries of the target compounds were 93.6% - 107.7% ( RSD ≤ 6.1%, n = 6). Based on the above method, the target compounds in reservoir water, reservoir soil and tap water in a certain region of Liaoning Province were analyzed. The results showed that the two target odors in reservoir water were 3.0 -3.6 ng/L. As for the extract of the soil around the reservoir, 2-MIB was 8.1 ng/L and GSM was 17.8 ng/L. The odorous substances were not detected in the tap water. This method is simple, accurate, reliable, highly sensitive and no need of organic solvents. And it is suitable for the detection of 2-MIB and GSM in drinking water.

Enantioseparations of amino acids by capillary array electrophoresis with 532 nm laser induced fluorescence detection.

Capillary array electrophoresis (CAE) is a promising technique for multiple enantiomeric separations. Carboxytetramethylrhodamine succinimidyl ester (TAMRA SE), a rhodamine-core fluorescent probe, has rarely been applied as an original precolumn derivatization reagent for chiral amino acid (AA) analysis so far. For these purposes, high-throughput enantiomeric separations of 12 TAMRA SE-AAs by a home-made 532 nm CAE-LIF scanner are presented. The effect of cyclodextrins (CDs) and a variety of organic modifiers was quickly investigated. Baseline separations were achieved in 100 mM Tris-borate buffer (pH 10.0) containing 2 mM ß-CD and 10 mM hexamethylenediamine (HDA). Multiple determination of the enantiomeric excess (ee) in non-racemic mixtures of alanine is successfully presented.

Isolation and identification of an iopromide-degrading strain and its application in an A2/O system.

An iopromide (IOPr)-degrading bacterium was isolated from activated sludge of a wastewater treatment plant in Shanghai. Based on its morphology, physiological-biochemical characteristics and a phylogenetic analysis of its 16S rRNA sequence, the bacterium was identified and named as Pseudomonas sp. I-24. The optimum condition for degrading IOPr was at 30°C and pH 7.0. After 5 days, strain I-24 could degrade 30 mg/L IOPr by 99% in a basal salts medium with a 5% (V/V) inoculum and 200 mg/L starch as the primary substrate. When applied to an Anaerobic-Anoxic/Aerobic (A2/O) process, with the coexistence of other bacteria, the strain I-24 got lower (61.3%) IOPr removal, but in two A2/O systems (with and without I-24 inoculation), the CODcr removal were both approximately 95%. The trial dosed with strain I-24 showed better IOPr removal than the un-dosed one. I-24 sustained its abundance in the A2/O system during the experiment.

Droplet-fused microreactors for room temperature synthesis of nanoscale needle-like hydroxyapatite.

A microfluidic device using droplet-fused microreactors is introduced for room temperature synthesis of nanoscale needle-shaped hydroxyapatite (HAp, Ca10(PO4)6(OH)2). The device is integrated with multifunctional units, e.g., T-junctions for droplet generation and fusion, winding channels for rapid mixing, and a delay line for simple visualization of the HAp formation process. The necessary conditions such as surfactant and fluid flow rate for an aqueous stream to merge with water-in-oil droplets are investigated. The nanoscale morphologies of the HAp produced by this method are also compared with HAp prepared by conventional bulk mixing. This paper shows that further reaction could be initiated by flowing additional reagent streams directly into the droplets of the initial reaction mixture, which is a novel approach for synthesizing a needle-like morphology of the HAp with a high aspect ratio under room temperature.

Influence of nebulizer type with different pediatric aerosol masks on drug deposition in a model of a spontaneously breathing small child.

BACKGROUND: The performance of nebulizers varies with the design type as well as the breathing patterns of various age groups. The present study quantified aerosol delivery using spontaneously breathing parameters of a small child (2-4 years) by a lung simulator to determine the influence of nebulizer type, actuation mechanisms, and pediatric aerosol masks. METHODS: Three types of nebulizer (constant-output, breath-enhanced, and breath-actuated nebulizer) and 3 masks (standard pediatric mask, the Fish mask, and a valved mask) were chosen for the testing. The actuation mechanism of the breath-actuated nebulizer was tested by manual synchronization with inspiration, breath actuation, and continuous nebulization. The nebulizer performance was determined by determining mass median aerodynamic diameter and analyzing drug deposition distal to the trachea (inhaled mass), on the face, on the mask, residual drug in the nebulizer, and the time of nebulization. The quantity of salbutamol deposited was determined by spectrophotometry (276 nm). RESULTS: Mass median aerodynamic diameter was similar across nebulizers. Breath-actuated nebulization generated a lower inhaled dose and higher nebulization time than continuous nebulization (P = .001). Breath synchronized aerosol generation, whether breath-actuated or manually actuated, yielded 10-20 times lower inhaled mass than continuous nebulization (0.1-0.6% vs 5-11%, respectively). The AeroEclipse, operated continuously, delivered greater inhaled dose than the LC Plus (P = .025). Higher inhaled dose was achieved with the Fish mask than standard or valved mask, with all nebulizers tested (P = .001). CONCLUSIONS: In this model using ventilatory parameters associated with a 2-4-year-old child, breath-actuated nebulization was not as effective as continuous nebulization. Aerosol mask design can impact inhaled drug dose across the range of nebulizers tested.

A microfluidic device integrated with multichamber polymerase chain reaction and multichannel separation for genetic analysis.

This work describes a microfluidic device integrated with multichamber polymerase chain reaction (PCR) and multichannel separation for parallel genetic analysis. The microdevice consists of three functional units: temperature control, multiple PCR (four chambers PCR), and multiple channel separation (four separation channels, each channel connected to a PCR chamber). Platinum (Pt)/titanium (Ti) microheater was used to ensure homogeneous temperature field, and Pt-chip sensor was used for temperature monitoring. The interface between chip-PCR and chip separation was simplified by connecting the PCR chamber with separation channel directly. After chip-PCR, PCR products were introduced into parallel separation channels for subsequent separation/detection by applying an electric field automatically. This microdevice was successfully applied for detection of pathogens including hepatitis B virus (HBV) and Mycobacterium tuberculosis (MTB), and genotyping of human leucocyte antigen (HLA)-B27 as well, demonstrating the feasibility of the integrated microdevice for parallel genetic analysis.

[Optimization of L-glutamic acid racemization reaction with capillary array electrophoresis].

L-Glutamic acid (Glu) racemization research is of great importance for the preparation of optical pure D-Glu. In the present work, we studied L-Glu racemization reaction through Schiff base intermediate by using capillary array electrophoresis (CAE) with 532 nm laser-induced confocal fluorescence detection. The racemization products were labeled by carboxytetramethylrhodamine succinimidyl ester (TAMRA) and analyzed in a 100 mmol/L Tris-borate buffer (pH 10) system containing 2 mmol/L beta-CD by CAE. Under this condition, the TAMRA-Glu enantiomer can be completely separated. The influences of the type and dosage of the aldehyde, organic acids, as well as the water content in the reaction system on the racemization were examined in detail, and L-Glu was racemized quickly in the presence of 0. 2 molar ratio of L-Glu to salicylaldehyde (as the catalyzer) in acetic acid containing 20% (v/v) water.

Home-made capillary array electrophoresis for high-throughput amino acid analysis.

A capillary array electrophoresis (CAE) with confocal rotary scanner for high-throughput carboxytetramethylrhodamine succinimidyl ester (TAMRA)-labeled amino acid (AA) analysis is presented. Performance of the CAE setup was evaluated with AA samples. Up to 128 capillaries could be detected in parallel. For the first time, the device was applied to separate the enantiomers of isoleucine, cysteine, and glutamic acid with cyclodextrin-modified electrolytes by capillary zone electrophoresis. Baseline separation of seven AAs is also demonstrated using micellar electrokinetic chromatography method.

High-throughput screening for the asymmetric transformation reaction of L-histidine to D-histidine by capillary array electrophoresis.

Asymmetric transformation reaction of L-histidine to D-histidine was studied by homemade capillary array electrophoresis for the first time. The enantiomeric excess value of asymmetric histidine products can be directly determined from the electrophoretogram of capillary array electrophoresis. The experiment results showed that the optimized asymmetric transformation reaction condition was in the presence of salicylaldehyde as catalyst and acetic acid as solvent.

Capillary array electrophoresis for the research of asymmetric transformation reaction of L-proline to D-proline.

One asymmetric transformation reaction of L-proline (L-Pro) to D-proline was studied by a home-made capillary array electrophoresis (CAE) for the first time. The aldehyde catalysts and the organic acid solvents for the asymmetric transformation reaction were rapidly screened and the enantiomeric excess values of the asymmetric product of L-Pro were directly obtained from the electrophoretogram of CAE.