Codon usage bias of goose circovirus and its adaptation to host.

Goose circovirus (GoCV), a potential immunosuppressive virus possessing a circular single-stranded DNA genome, is widely distributed in both domesticated and wild geese. This virus infection causes significant economic losses in the waterfowl industry. The codon usage patterns of viruses reflect the evolutionary history and genetic architecture, allowing them to adapt quickly to changes in the external environment, particularly to their hosts. In this study, we retrieved the coding sequences (Rep and Cap) and the genome of GoCV from GenBank, conducting comprehensive research to explore the codon usage patterns in 144 GoCV strains. The overall codon usage of the GoCV strains was relatively similar and exhibited a slight bias. The effective number of codons (ENC) indicated a low overall extent of codon usage bias (CUB) in GoCV. Combined with the base composition and relative synonymous codon usage (RSCU) analysis, the results revealed a bias toward A- and G-ending codons in the overall codon usage. Analysis of the ENC-GC3s plot and neutrality plot suggested that natural selection plays an important role in shaping the codon usage pattern of GoCV, with mutation pressure having a minor influence. Furthermore, the correlations between ENC and relative indices, as well as correspondence analysis (COA), showed that hydrophobicity and geographical distribution also contribute to codon usage variation in GoCV, suggesting the possible involvement of natural selection. In conclusion, GoCV exhibits comparatively slight CUB, with natural selection being the major factor shaping the codon usage pattern of GoCV. Our research contributes to a deeper understanding of GoCV evolution and its host adaptation, providing valuable insights for future basic studies and vaccine design related to GoCV.

Simultaneous determination of 10 new psychoactive piperazine derivatives in urine using ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction combined with gas chromatography-tandem mass spectrometry.

With the rapid development of synthetic drugs, novel piperazine derivatives, as an increasingly important class of new psychoactive substances (NPS), have attracted global attention owing to their increasing demand in the illicit drug market. In this study, ten piperazine derivatives were analyzed in urine samples after pre-treatment with ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) combined with gas chromatography-tandem mass spectrometry (GC-MS/MS). This simple approach involved the use of urine samples (1 mL) adjusted to pH 12, which was added to 100 µL of n-hexane and subjected to ultrasonication for 3 min to completely disperse the sample in the n-hexane solution. The resulting turbid suspension was centrifuged at 10,000 rpm for 3 min, and the supernatant was extracted and analyzed using GC-MS/MS. Under the optimized conditions presented in this study, the linear relationship between the analytes was good within 10-1500 ng/mL, and the correlation coefficient (r) was between .9914 and .9983. The limit of detection (LOD) was 0.3-2 ng/mL (S/N = 3), and the lower limit of quantification (LLOQ) was 10 ng/mL (S/N = 10) with the recovery of the analytes of interest from the spiked samples being 76.3%-93.3%. This method has been used to analyze real-world samples; our study shows that the UA-LDS-DLLME approach can be used for rapid analysis while consuming minimal solvent for the simultaneous determination of a range of analytes. This method has the potential for use in clinical analyses and forensic toxicology.

Case studies on illegal production of ephedrine/pseudoephedrine within Fujian China.

Ephedrine/pseudoephedrine (EPH) is the most common precursor for the manufacture of methamphetamine and is controlled within China via criminal law and government regulations. Therefore, it is of great significance to systematically study the cases of illicit production of EPH in order to aid timely investigation into the production of precursor drugs. According to the literature, there are no comprehensive case studies on the illicit production of EPH. To address this, 50 cases involving the illicit production of EPH from Fujian Province in China were comprehensively explored and the quantitative data of the 762 collected samples were directly compared. In terms of the timeframe these cases occurred in, the results show that the number of such cases in Fujian Province increased significantly from 2012 to 2013 (10 cases -15 cases), but the number of cases decreased rapidly in 2016 (4 cases). Across the region of interest, the illegal production of EPH was mainly observed in Longyan, accounting for 32 cases (64.0 % of the total cases). Forty-two cases (84.0 % of the total cases) were located in remote mountains, abandoned pig farms, chicken farms and factories. In terms of the synthetic methodology used, initially (up until 2013) only the extraction of EPH from the ephedra plant and from commercially available tablets was observed. The manufacture of EPH via a direct chemical synthesis was only observed in this region after 2013 at which point a significant drop in the extraction methodologies was identified. The quality of the samples and the appearance of semi-finished products was shown to vary significantly across the cases with sample color ranging from light yellow, brown, tan for both seized solids and liquids. This data set gives some insights into the local issues specifically around EPH production in Chinese provinces and goes some way to help inform policing strategies.

Application of dispersive liquid-liquid microextraction and GC-MS/MS for the determination of GHB in beverages and hair.

Recently, gamma-hydroxybutyrate (GHB) was determined in "Kawa Trendy Drink" which was labeled as a functional beverage containing gamma-aminobutyric acid and quickly became popular in various club in China. GHB has a strong sedative effect and is often used as a date rape drug and euphoriant in drug-facilitated sexual assaults. However, it is believed that same beverages themselves contain natural GHB. Whether GHB contained in seized beverages was added or exists in themselves resulted in complicated interpretation of the nature of cases. The detection window of GHB in blood (5 h) and urine (<12 h) was narrow, make documentation of GHB exposure difficult, while hair can extend the detection time of GHB from several hours to several days/months. Thus, a sensitive detection method based on dispersive liquid-liquid microextraction (DLLME) and GC-MS/MS for GHB in beverages and hair had been established. Under the optimum extraction conditions, acceptable linear relationship was achieved in the range of 1.5-500 ng/mL with the correlation coefficient (r) of 0.9986 for spiked water samples and in the range of 0.03-10 ng/mg with the correlation coefficient (r) of 0.9979 for spiked melanin samples. The LOD (S/N = 3) was estimated to be 0.5 ng/mL and 0.01 ng/mg, respectively. A recovery of 70.6-88.5% were obtained for spiked samples. The mean relative error (MRE) was within ±6.5% and the relative standard error (RSD) was less than 4.9%. The combination of DLLME with GC-MS/MS offers an alternative analytical approach for the sensitive detection of GHB in real beverages sold in Chinese markets and in hair of Chinese population for forensic purposes. The proposed methods had the advantages of shorter extraction time and were suitable for simultaneous pretreatment of samples in batches. To our knowledge, this is the first report to present GHB in beverages and human hair using DLLME.

Recent Progress in NIR-II Contrast Agent for Biological Imaging.

Fluorescence imaging technology has gradually become a new and promising tool for in vivo visualization detection. Because it can provide real-time sub-cellular resolution imaging results, it can be widely used in the field of biological detection and medical detection and treatment. However, due to the limited imaging depth (1-2 mm) and self-fluorescence background of tissue emitted in the visible region (400-700 nm), it fails to reveal biological complexity in deep tissues. The traditional near infrared wavelength (NIR-I, 650-950 nm) is considered as the first biological window, because it reduces the NIR absorption and scattering from blood and water in organisms. NIR fluorescence bioimaging's penetration is larger than that of visible light. In fact, NIR-I fluorescence bioimaging is still interfered by tissue autofluorescence (background noise), and the existence of photon scattering, which limits the depth of tissue penetration. Recent experimental and simulation results show that the signal-to-noise ratio (SNR) of bioimaging can be significantly improved at the second region near infrared (NIR-II, 1,000-1,700 nm), also known as the second biological window. NIR-II bioimaging is able to explore deep-tissues information in the range of centimeter, and to obtain micron-level resolution at the millimeter depth, which surpass the performance of NIR-I fluorescence imaging. The key of fluorescence bioimaging is to achieve highly selective imaging thanks to the functional/targeting contrast agent (probe). However, the progress of NIR-II probes is very limited. To date, there are a few reports about NIR-II fluorescence probes, such as carbon nanotubes, Ag2S quantum dots, and organic small molecular dyes. In this paper, we surveyed the development of NIR-II imaging contrast agents and their application in cancer imaging, medical detection, vascular bioimaging, and cancer diagnosis. In addition, the hotspots and challenges of NIR-II bioimaging are discussed. It is expected that our findings will lay a foundation for further theoretical research and practical application of NIR-II bioimaging, as well as the inspiration of new ideas in this field.

Ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction for the determination of 4 designer benzodiazepines in urine samples by gas chromatography-triple quadrupole mass spectrometry.

A novel microextraction technique based on ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) had been applied for the determination of 4 designer benzodiazepines (phenazepam, diclazepam, flubromazepam and etizolam) in urine samples by gas chromatography- triple quadrupole mass spectrometry (GC-QQQ-MS). Ethyl acetate (168µL) was added into the urine samples after adjusting pH to 11.3. The samples were sonicated in an ultrasonic bath for 5.5min to form a cloudy suspension. After centrifugation at 10000rpm for 3min, the supernatant extractant was withdrawn and injected into the GC-QQQ-MS for analysis. Parameters affecting the extraction efficiency have been investigated and optimized by means of single factor experiment and response surface methodology (Box-Behnken design). Under the optimum extraction conditions, a recovery of 73.8-85.5% were obtained for all analytes. The analytical method was linear for all analytes in the range from 0.003 to 10µg/mL with the correlation coefficient ranging from 0.9978 to 0.9990. The LODs were estimated to be 1-3ng/mL. The accuracy (expressed as mean relative error MRE) was within ±5.8% and the precision (expressed as relative standard error RSD) was less than 5.9%. UA-LDS-DLLME technique has the advantages of shorter extraction time and is suitable for simultaneous pretreatment of samples in batches. The combination of UA-LDS-DLLME with GC-QQQ-MS offers an alternative analytical approach for the sensitive detection of these designer benzodiazepines in urine matrix for clinical and medico-legal purposes.

[Ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction for the determination of eight drugs in biological samples by gas chromatography-triple quadrupole mass spectrometry].

A novel microextraction technique based on ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) has been developed for the determination of multiple drugs of abuse in biological samples by gas chromatography-triple quadrupole mass spectrometry (GC-QQQ-MS). A total of 100 µL of toluene as extraction solvent was dropped into the sample solution. Then the mixture was sonicated drastically in an ultrasonic bath for 3 min with occasional manual shaking to form a cloudy suspension. After centrifugation at 10,000 r/min for 3 min, the upper layer of low-density extractant was withdrawn and injected into the GC-QQQ-MS for analysis. The parameters affecting extraction efficiency have been investigated and optimized. Under the optimum conditions, good linearities were observed for all analytes with the correlation coefficients ranging from 0. 998 4 to 0. 999 4. The recoveries of 79.3%-100.3% with RSDs < 5.7% were obtained. The LODs (S/N = 3) were in the range from 0.05 to 0.40 µg/L. UA-LDS-DLLME technique has the advantages of less extraction time, suitable for batches of sample pretreatment simultaneously, and higher extraction efficiency. It was successfully applied to the analysis of amphetamines in real human urine samples.

Comparison of hollow fiber liquid-phase microextraction and ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction for the determination of drugs of abuse in biological samples by gas chromatography-mass spectrometry.

Two microextraction techniques based on hollow fiber liquid-phase microextraction (HF-LPME) and ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) had been applied for the determination of drugs of abuse (methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, methcathinone, ketamine, meperidine, and methadone) in urine and blood samples by gas chromatography-mass spectrometry. Parameters affecting extraction efficiency have been investigated and optimized for both methods. Under the optimum conditions, linearities were observed for all analytes in the range 0.0030-10 µg/ml with the correlation coefficient (R) ranging from 0.9985 to 0.9995 for HF-LPME and in the range 0.0030-10 µg/ml with the R ranging from 0.9985 to 0.9994 for DLLME. The recovery of 79.3-98.6% with RSDs of 1.2-4.5% was obtained for HF-LPME, and the recovery of 79.3-103.4% with RSDs of 2.4-5.7% was obtained for DLLME. The LODs (S/N=3) were estimated to be in the range from 0.5 to 5 ng/ml and 0.5 to 4 ng/ml, respectively. Compared with HF-LPME, the UA-LDS-DLLME technique had the advantages of less extraction time, suitability for batches of sample pretreatment simultaneously, and higher extraction efficiency, while HF-LPME has excellent sample clean-up effect, and is a robust and suitable technique for various sample matrices with better repeatability. Both methods were successfully applied to the analysis of drugs of abuse in real human blood sample.

Inspection and analysis of mixed drugs recently seized in China.

Recently a new type of mixed drug oral solution has become prevalent in China, the identification and quantitative inspection of which are demanded in conviction and sentencing as well as drug intelligence. In 2013, inspection was carried out on 86 bottles of substances seized in 13 cases with the adoption of GC-MS methodology for qualitative identification and GC-FID for quantitative identification. 94.1% of the samples were light yellow turbid liquid while 95.3% were detected with more than two drugs. Detection rate of methamphetamine, MDMA and ketamine was up to 89.5%, 37.2% and 83.7% respectively. Quantitative inspection results indicated that concentrations of methamphetamine, MDMA, ketamine were 0.04-13 mg/mL, 0.04-29 mg/mL and 0.1-34 mg/mL, respectively. Appearance, composition and drug concentration of samples vary from case to case.

[Determination of tramadol hydrochloride in serum samples by disk solid phase extraction and gas chromatography-mass spectrometry in selected ion monitoring].

OBJECTIVE: Disk solid phase extraction (SPE) was assessed for tramadol hydrochloride from serum. METHODS: The SPE was performed with SPEC C18AR/MP3 Disk SPE cartridge, offering hydrophobic C18 and strong cation ionic exchange interactions for the analytes, before being added into extraction column, 1 mL serum was diluted by 2 mL 0.1 mol/L phosphate buffer solution (pH 6) and the eluent was ethyl acetate containing 2% ammonia. Then SKF525 was added as internal standard into samples, which would be extracted simultaneously with analyte,quantitatively, determined by GC/MS/SIM. RESULTS: The extraction recovery of tramadol hydrochloride was 98.9%, 92.5% and 84.8% for serum samples with corresponding amounts of standard addition of 0.1 microg/mL, 0.2 microg/mL and 0.5 microg/mL. And RSD measured 5 times was 3.2%, 8.7% and 10.9% respectively. The linear range varied from 0.1 microg/mL to 4 microg/mL. The multinomial regression correlation coefficient (r2) equaled 0.9939, and the detection limit was 21 ng/mL. After the same extraction column was continuously used for 5 times, there was no jam, pollution and decline of recovery and RSD. CONCLUSION: This method is suitable for forensic toxicological analysis.