Reference interval establishment and correlation research of urine thromboxane metabolites: Unveiling new possibilities in platelet activation.

BACKGROUND: Thromboxane metabolites could indirectly reflect platelet activation, among which 11-dehydro-thromboxane B2 (11dhTxB2) and 11-dehydro-2, 3-dinor thromboxane B2 (11dh23dinorTxB2) are two stable metabolites that are abundant in urine, and both are closely related to disease progression and drug use. However, most clinical application studies have focused on the single indicator of 11dhTxB2. We propose an LC-MS/MS method suitable for routine clinical screening with simultaneous determination of both metabolites and conduct preliminary studies in different populations. METHODS AND RESULTS: The thromboxane metabolites were extracted by liquid-liquid extraction and determined by LC-MS/MS. Reference intervals (RI) were established in 333 healthy adults and validated in 25 patients with coronary atherosclerosis (CA). This LC-MS/MS method was over a wide quantitative range (0.1-10 µmol/L), the imprecision and accuracy were 5.2 %-11 % and 89.3 %-106.5 %, and was suitable for clinical routine quantitative screening. The 95th percentile RI of unire 11dhTxB2 was 1220 (95 % CI: 1048, 1376) pg mg Cr -1, for 11dh23dinorTxB2, RI was 908 (95 % CI: 821, 1102) pg mg Cr -1. For the first time, we found a significant correlation between 11dhTxB2 and 11dh23dinorTxB2 in both healthy adults (r = 0.67, P < 0.001) and CA patients (r = 0.77, P < 0.001). CONCLUSION: The establishment of RI provides a reference for diseases related to platelet activation and the use of drugs, and the first discovery of the correlation between 11dhTxB2 and 11dh23dinorTxB2 in urine provides a new possibilitie for the diagnostic and prognostic of cardiovascular diseases.

Harnessing aptamers for the biosensing of cell surface glycans - A review.

Cell surface glycans (CSGs) are essential for cell recognition, adhesion, and invasion, and they also serve as disease biomarkers. Traditional CSG recognition using lectins has limitations such as limited specificity, low stability, high cytotoxicity, and multivalent binding. Aptamers, known for their specific binding capacity to target molecules, are increasingly being employed in the biosensing of CSGs. Aptamers offer the advantage of high flexibility, small size, straightforward modification, and monovalent recognition, enabling their integration into the profiling of CSGs on living cells. In this review, we summarize representative examples of aptamer-based CSG biosensing and identify two strategies for harnessing aptamers in CSG detection: direct recognition based on aptamer-CSG binding and indirect recognition through protein localization. These strategies enable the generation of diverse signals including fluorescence, electrochemical, photoacoustic, and electrochemiluminescence signals for CSG detection. The advantages, challenges, and future perspectives of using aptamers for CSG biosensing are also discussed.

Simultaneous determination of human plasma 5 amino acid neurotransmitters using liquid chromatography-tandem mass spectrometry: Establishment of reference intervals in Chinese adult population and application to patients with schizophrenia.

Schizophrenia is a serious mental disease with unknown etiology that affects approximately 1 % of the population around the world. Altered levels of amino acid neurotransmitters may underlie the physiopathology of schizophrenia (SZ). This study aimed to develop a rapid and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of glutamate acid (Glu), aspartic acid (Asp), γ-aminobutyric acid (GABA), glycine acid (Gly), and Taurine acid (Tau) in patients with schizophrenia plasma and establish reference intervals for Chinese adult populations, and applied to patients with schizophrenia for a preliminary exploration of changes in their plasma levels of five amino acid neurotransmitters. Sample treatment involved protein precipitation followed by dansyl chloride (DNS-Cl) derivatization and total run time is 5.8 min. The method was validated according to the latest national and international guidelines, which achieved acceptable precision (0.54-14.54 %) and accuracy (97.06-103.82 %). The reference interval for Glu, Asp, Gly, Tau, and GABA were 55.51-189.06, 27.51-92.38, 204.01-574.55, 107.50-227.65, and <1 µmol/L, respectively. Increased Tau levels and decreased Asp and Glu levels were shown in patients with schizophrenia. This method was suitable for clinical routine detection of plasma 5 amino acid neurotransmitters in Chinese adult populations.

An ultra performance liquid chromatography method for transthyretin variants screening and heart failure assisting diagnosis.

BACKGROUND: Transthyretin (TTR) gene mutations are associated with hereditary amyloidosis (ATTR) caused by mutant TTR protein dissociation, misfolding, aggregation, and insoluble fibrils deposition. Herein, we reported a chromatographic approach for quantification and identification of TTR tetramer in human blood serum by ultra performance liquid chromatography (UPLC). METHODS: TTR proteins and serum were incubated with a fluorescent TTR tetramer sensor (A2). The A2 sensor specifically reacted with tetrameric TTR and released stoichiometric fluorescence that was detected by fluorescence detector coupled to UPLC. The external standard was used for quantification, the chromatographic peak parameters were used to identification certain mutation types. RESULTS: UPLC correctly distinguished 18 types of mutant TTR proteins from wild type. The results were consistent with follow-up analysis of two ATTR patients' blood serum samples. In addition, the tetrameric TTR of 30 heart failure (HF) patients showed strongly correlation (r = -0.63, p < 0.00) with NT-proBNP, a HF clinical biomarker. CONCLUSIONS: UPLC method has sufficient accuracy to eliminate the necessity of sequencing for certain types of TTR mutations and allows for facile initial screening of ATTR amyloidosis patients, carriers, and healthy individuals for time-saving and economical purposes. TTR tetramer may serve as a diagnostic biomarker to evaluate the risk of HF diseases.

A first-in-human phase 1 study of simnotrelvir, a 3CL-like protease inhibitor for treatment of COVID-19, in healthy adult subjects.

Safe and efficacious antiviral therapeutics are in urgent need for the treatment of coronavirus disease 2019. Simnotrelvir is a selective 3C-like protease inhibitor that can effectively inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the safety, tolerability, and pharmacokinetics of dose escalations of simnotrelvir alone or with ritonavir (simnotrelvir or simnotrelvir/ritonavir) in healthy subjects, as well as the food effect (ClinicalTrials.gov Identifier: NCT05339646). The overall incidence of adverse events (AEs) was 22.2% (17/72) and 6.3% (1/16) in intervention and placebo groups, respectively. The simnotrelvir apparent clearance was 135-369 L/h with simnotrelvir alone, and decreased significantly to 19.5-29.8 L/h with simnotrelvir/ritonavir. The simnotrelvir exposure increased in an approximately dose-proportional manner between 250 and 750 mg when co-administered with ritonavir. After consecutive twice daily dosing of simnotrelvir/ritonavir, simnotrelvir had a low accumulation index ranging from 1.39 to 1.51. The area under the curve of simnotrelvir increased 44.0 % and 47.3 % respectively, after high fat and normal diet compared with fasted status. In conclusion, simnotrelvir has adequate safety and tolerability. Its pharmacokinetics indicated a trough concentration above the level required for 90 % inhibition of SARS-CoV-2 in vitro at 750 mg/100 mg simnotrelvir/ritonavir twice daily under fasted condition, supporting further development using this dosage as the clinically recommended dose regimen.

A first-in-human phase I study of SHR-1906, a humanized monoclonal antibody against connective tissue growth factor, in healthy participants.

New therapeutic targets and drugs are urgently needed to halt the fibrosing process in idiopathic pulmonary fibrosis (IPF). SHR-1906 is a novel fully humanized monoclonal antibody against the connective tissue growth factor, which plays an essential role in the genesis of IPF. We assessed the safety, tolerability, pharmacokinetics (PKs), and immunogenicity of single dose SHR-1906 in healthy participants. This was a randomized, double-blind, placebo-controlled, dose-escalation, phase I study. Twelve healthy participants for each dose level were enrolled to receive single ascending doses of SHR-1906 intravenously (1.5, 6, 12, 20, 30, and 45 mg/kg) or placebo and followed for 71 days. The primary end points were safety and tolerability. Treatment-related treatment-emergent adverse events occurred in 25 participants (46.3%) in the SHR-1906 group and 11 (61.1%) in the placebo group. No serious adverse events occurred. Over the dose range investigated, the geometric mean clearance was 0.14-0.63 mL/h/kg, the geometric mean volume of distribution at steady-state was 47.4-75.5 mL/kg, and the terminal elimination half-life was 51.9-349 h. SHR-1906 showed nonlinear PKs. The peak concentration increased in a dose-proportional manner, whereas the area under the concentration-time curve showed a greater than dose-proportional increase. Anti-drug antibodies of SHR-1906 were detected in nine of 54 participants (16.7%). A single dose of SHR-1906 up to 45 mg/kg demonstrated a favorable tolerability profile in healthy participants. The PKs and immunogenicity of SHR-1906 were evaluated, supporting further clinical development.

Metabolomics analysis of amino acid and fatty acids in colorectal cancer patients based on tandem mass spectrometry.

Metabolic differences between colorectal cancer (CRC) and NI (NI) play an important role in early diagnoses and in-time treatments. We investigated the metabolic alterations between CRC patients and NI, and identified some potential biomarkers, and these biomarkers might be used as indicators for diagnosis of CRC. In this study, there were 79 NI, 50 CRC I patients, 52 CRC II patients, 56 CRC III patients, and 52 CRC IV patients. MS-MS was used to measure the metabolic alterations. Univariate and multivariate data analysis and metabolic pathway analysis were applied to analyze metabolic data and determine differential metabolites. These indicators revealed that amino acid and fatty acids could separate these groups. Several metabolites indicated an excellent variables capability in the separation of CRC patients and NI. Ornithine, arginine, octadecanoyl carnitine, palmitoyl carnitine, adipoyl carnitine, and butyryl carnitine/propanoyl carnitine were selected to distinguish the CRC patients and NI. And methionine and propanoyl carnitine, were directly linked to different stages of CRC. Receiver operating characteristics curves and variables importance in projection both represented an excellent performance of these metabolites. In conclusion, we assessed the difference between CRC patients and NI, which supports guidelines for an early diagnosis and effective treatment.

Feeding Behavior, Growth Performance and Meat Quality Profile in Broiler Chickens Fed Multiple Levels of Xylooligosaccharides.

A total of 240 1-day-old Arbor Acres broiler chickens were randomly distributed to 4 treatment groups with 6 replicates and 10 birds per replicate. Chickens were fed with corn-soybean meal diet supplementation with additions of 0, 150, 300, and 450 mg/kg XOS for 42 days. At 4 weeks of age, the average feeding time was reduced in the 450 mg/kg XOS group (p < 0.05), and the percentage of feeding time was increased in the 300 mg/kg XOS group (p < 0.05). At 5 weeks of age, broilers fed with 300 mg/kg XOS had increased the percentage of feeding time (p < 0.05), and 450 mg/kg XOS had increased the feeding frequency and percentage of feeding time (p < 0.05). At 6 weeks of age, the feeding frequency was highest in the 450 mg/kg XOS group (p < 0.05). During 4 to 6 weeks of age, the average feeding time was increased in 300 mg/kg XOS group (p < 0.05), the frequency was improved in the 450 mg/kg XOS group (p < 0.05), and the percentage of feeding time was longer in the XOS group than that in the control group (p < 0.05). The average daily gain was improved during days 22-42 and days 1-42 in the 150 mg/kg XOS group (p < 0.05). Broilers fed with 300 mg/kg XOS had an increased eviscerated rate (p < 0.05). The pH45min of breast muscle was highest in the 450 mg/kg XOS group (p < 0.05), as well as the pH45min and pH24h of thigh muscle, which improved in the 300 mg/kg and 450 mg/kg XOS groups (p < 0.05). In addition, the cooking loss of thigh muscle was reduced in the 300 mg/kg XOS group (p < 0.05). In conclusion, dietary supplementation with XOS had positive effects on the feeding behavior, growth performance, and meat quality of broiler chickens.

An efficient LC-MS/MS method for simultaneous determination of four substances in the Hcy metabolism pathway.

Aim: This study aimed to develop and validate an efficient LC-MS/MS method for the simultaneous determination of Hcy, Cys, Met and 5-methyltetrahydrofolate in human serum and to apply this method to patients with coronary artery disease. Methodology and results: Serum samples were prepared by reduction with dithiothreitol followed by protein precipitation. The analytical run time was 2.2 min. The linearity was good in the range of 2-100 µmol/l-1 for Hcy and Met, 10-500 µmol/l-1 for Cys, and 1-50 ng/ml-1 for 5-methyltetrahydrofolate. Conclusion: An accurate and precise method that was rapid, robust and with high-throughput for the routine clinical monitoring of patients with coronary artery disease was developed.

CRISPR/Cas12a-mediated ultrasensitive and on-site monkeypox viral testing.

The spread of the monkeypox virus (MPXV) from Central and West Africa to previously non-endemic regions has caused a global panic. In this context, the rapid, specific, and ultrasensitive detection of MPXV is crucial to contain its spread, though such technology has seldom been reported. Herein, we proposed an MPXV assay combining recombinase-aided amplification (RAA) and CRISPR/Cas12a. This assay targeted the highly conserved MPXV F3L gene and demonstrates a low detection limit (LOD) of 101 copies per µL. By leveraging the high specificity nature of RAA and CRISPR/Cas12a, we rationally optimized probes and conditions to achieve high selectivity that differentiates MPXV from other orthopox viruses and current high-profile viruses. To facilitate on-site screening of potential MPXV carriers, a kit integrating lateral flow strips was developed, enabling naked-eye MPXV detection with a LOD of 104 copies per µL. Our RAA-Cas12a-MPXV assay was able to detect MPXV without the need for sophisticated operation and expensive equipment. We believe that this assay can be rapidly deployed in emerging viral outbreaks for on-site surveillance of MPXV.

A simple and rapid liquid chromatography-tandem mass spectrometry method for the simultaneous determination of 15 bile acids in human serum and its application to patients with decompensated cirrhosis.

Liver cirrhosis is currently the twelfth leading cause of death globally and the sixth leading cause of death in China. Its treatment is expensive. Changes in the composition of the serum bile acid pool are sensitive indicators of the severity of liver cirrhosis. In this study, a novel LC-tandem mass spectrometry (LC-MS/MS) method was developed and used to simultaneously determine 15 bile acids in human serum in patients with decompensated cirrhosis. Sample preparation involved spiking with isotope internal standards followed by protein precipitation. The analytical run time was 5 min. The LC-MS/MS method was fully validated according to Clinical and Laboratory Standards Institute (CLSI) C62A and the consensus of method development and validation of liquid chromatography-tandem mass spectrometry in clinical laboratories. The method achieved an acceptable coefficient of variation for precision (0.83%-14.80%) and accuracy (89.39%-107.62%). Finally, as proof of applicability, the method was applied to patients with decompensated cirrhosis in routine clinical sample analysis. The degree of variation of different bile acids was clearly shown. These results indicated that abnormal metabolic pathways might play important roles in decompensated cirrhosis.

Integrating CRISPR/Cas12a with strand displacement amplification for the ultrasensitive aptasensing of cadmium(II).

Cadmium ion (Cd(II)) is a pernicious environmental pollutant that has been shown to contaminate agricultural lands, accumulate through the food chain, and seriously threaten human health. At present, Cd(II) monitoring is dependent on centralized instruments, necessitating the development of rapid and on-site detection platforms. Against this backdrop, the present study reports on the development of a fluorometric aptasensor designed to target Cd(II), which is achieved through the integration of strand displacement amplification (SDA) and CRISPR/Cas12a. In the absence of Cd(II), the aptamer initiates SDA, resulting in the generation of a profusion of ssDNA that activates Cas12a, leading to a substantial increase in fluorescence output. Conversely, the presence of Cd(II) curtails the SDA efficiency, culminating in a significant reduction in fluorescence output. The proposed approach has been demonstrated to enable the selective detection of Cd(II) at concentrations of 60 pM, with the performance of the aptasensor validated in real water and rice samples. The proposed platform based on aptamer-target interaction holds immense promise as a signal-amplified and precise method for the detection of Cd(II) and has the potential to transform current hazard detection practices in food samples.

Evaluation of analytical performance of homocysteine LC-MS/MS assay and design of internal quality control strategy.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become a common technique in clinical laboratories in recent years. Because most methods are laboratory-developed tests (LDTs), their reproducibility and quality control (QC) have been controversial. In this study, Westgard Sigma Rules were used to evaluate the analytical performance and establish an individualised internal QC (IQC) strategy for these LDTs. METHODS: Taking the LC-MS/MS LDT method for homocysteine (Hcy) as an example, the 'desirable specifications' from the Biological Variation Database were used as quality goals. Based on the external quality assessment (EQA) samples, bias was calculated and the coefficient of variation (CV) was also calculated by IQC measurements for six consecutive months. The analytical performance was evaluated by calculated sigma metrics and an IQC strategy was designed using the Westgard Sigma Rules with run size. RESULTS: Over 116 days within 6 months, a total of 850 data points were collected for each of IQC 1 and IQC 2. The monthly coefficient of variation CV% was 2.57-4.01%, which was non-significant (p-value: 0.75). The absolute bias% for IQC1 and IQC2 was 1.23 and 1.87%, respectively. The allowable total error (TEa) was selected as 15.5%, Sigma metrics were 4.02 and 4.30, and the analytical performance was 'Good'. The 13s/22s/R4s/41s multi rules (n=4, r=1) with a run size of 200 samples were suggested for the Hcy IQC scheme. The quality goal index (QGI) values were over 1.2, indicating that trueness needed to be improved. CONCLUSIONS: The analytical performance of the Hcy LC-MS/MS LDT conformed to the Six Sigma rating level, achieving 'good' (four Sigma). Clinical practice indicated that calibration bias was the primary factor affecting trueness.

Population pharmacokinetics and pharmacogenetics of apatinib in adult cancer patients.

AIMS: Apatinib is widely used in Chinese cancer patients. As the in vivo drug disposition of apatinib has large individual differences, adverse events are prone to occur. Cytochrome P450 (CYP)3A5 and cancer types maybe the main factors affecting this individual differences. The objective of our study was to establish a population pharmacokinetics (PK) model of apatinib in adult cancer patients, and to explore optimal dosage regimens for individualized treatment. METHODS: Adult patients with various types of cancer treated with apatinib were enrolled. The concentration of apatinib in plasma was determined by high-performance liquid chromatography-tandem mass spectrometry. CYP3A5 genotype was determined using TaqMan allelic discrimination technique. The population PK model was developed by NONMEM V7.4. The dosing regimen was optimized based on Monte Carlo simulations. RESULTS: A population PK model of apatinib in adult cancer patient was established. CYP3A5 genotype and systemic cancer type (digestive system cancers, nondigestive system cancers) were the most significant covariates for PK parameters. Patients with CYP3A5*1 expressers (CYP3A5*1/*1 and CYP3A5*1/*3) had lower apparent clearance and apparent volume of distribution than patients who do not express CYP3A5*1 (CYP3A5*3/*3). Patients with nondigestive system cancer had higher apparent volume of distribution and absorption rate constant than digestive system cancer. The results of dose simulation suggest that the apatinib dose in patients who do not express CYP3A5*1 should be 33.33-50.00% higher than that in CYP3A5*1 expressers. CONCLUSIONS: A population PK model of apatinib in adult cancer patients was established. CYP3A5 genotype and systemic cancer type had concurrent effects on PK parameters. CYP3A5 patients who do not express CYP3A5*1 required higher doses.

Comparative Study of Two Common In Vitro Models for the Pancreatic Islet with MIN6.

BACKGROUND: Islet transplantation is currently considered the most promising method for treating insulin-dependent diabetes. The two most-studied artificial islets are alginate-encapsulated ß cells or ß cell spheroids. As three-dimensional (3D) models, both artificial islets have better insulin secretory functions and transplantation efficiencies than cells in two-dimensional (2D) monolayer culture. However, the effects of these two methods have not been compared yet. Therefore, in this study, cells from the mouse islet ß cell line Min6 were constructed as scaffold-free spheroids or alginate-encapsulated dispersed cells. METHODS: MIN6 cell spheroids were prepared by using Agarose-base microwell arrays. The insulin secretion level was determined by mouse insulin ELISA kit, and the gene and protein expression status of the MIN6 were performed by Quantitative polymerase chain reaction and immunoblot, respectively. RESULTS: Both 3D cultures effectively promoted the proliferation and glucose-stimulated insulin release (GSIS) of MIN6 cells compared to 2D adherent cells. Furthermore, 1% alginate-encapsulated MIN6 cells demonstrated more significant effects than the spheroids. In general, three pancreatic genes were expressed at higher levels in response to the 3D culture than to the 2D culture, and pancreatic/duodenal homeobox-1 (PDX1) expression was higher in the cells encapsulated in 1% alginate than that in the spheroids. A western blot analysis showed that 1% alginate-encapsulated MIN6 cells activated the phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (AKT)/forkhead transcription factor FKHR (FoxO1) pathway more than the spheroids, 0.5% alginate-, or 2% alginate-encapsulated cells did. The 3D MIN6 culture, therefore, showed improved effects compared to the 2D culture, and the 1% alginate-encapsulated MIN6 cells exhibited better effects than the spheroids. The upregulation of PDX1 expression through the activation of the PI3K/AKT/FoxO1 pathway may mediate the improved cell proliferation and GSIS in 1% alginate-encapsulated MIN6 cells. CONCLUSION: This study may contribute to the construction of in vitro culture systems for pancreatic islets to meet clinical requirements.

Assessment of the inhibition risk of chlorophenol substances on cytochrome P450 via cocktail inhibition assays.

Chlorophenols (CPs) are widespread pollutants in nature. CPs have raised significant concern due to their potential hepatotoxic effects on humans. This research aimed to ascertain the inhibitory potential of eleven CPs (2-CP, 3-CP, 4-CP, 2,4-DCP, 2,3,4-TCP, 2,4,5-TCP, 2,4,6-TCP, 2,3,4,5-TeCP, 2,3,4,6-TeCP, 2,3,5,6-TeCP, and PCP) on nine human CYP isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4). The CPs that inhibit the activity of CYP isoforms were detected with human liver microsomes (HLM) using a cocktail approach in vitro. The results demonstrated that trichlorophenols, tetrachlorophenols, and PCP strongly inhibited CYP2C8 and CYP2C9. The half inhibition concentration (IC50) value of 2,3,4,6-TeCP and PCP for CYP2C8 inhibition was 27.3 µM and 12.3 µM, respectively. The IC50 for the inhibition of 2,4,6-TCP, 2,3,4,6-TeCP and PCP towards CYP2C9 were calculated to be 30.3 µM, 5.8 µM and 2.2 µM, respectively. 2,3,4,6-TeCP, and PCP exhibited non-competitive inhibition towards CYP2C8. 2,4,6-TCP, 2,3,4,6-TeCP, and PCP exhibited competitive inhibition towards CYP2C9. The inhibition kinetics parameters (Ki) were 51.51 µM, 22.28 µM, 37.86 µM, 7.27 µM, 0.68 µM for 2,3,4,6-TeCP-CYP2C8, PCP-CYP2C8, 2,4,6-TCP-CYP2C9, 2,3,4,6-TeCP-CYP2C9, PCP-CYP2C9, respectively. This study also defined clear structure-activity relationships (SAR) of CPs on CYP2C8, supported by molecular docking studies. Overall, CPs were found to cause inhibitory effects on CYP isoforms in vitro, and this finding may provide a basis for CPs focused on CYP isoforms inhibition endpoints.

Nutrient dynamics and microbial community response in macrophyte-dominated lakes: Implications for improved restoration strategies.

Although lakes dominated by macrophytes are conducive to ecological balance, this balance is easily disrupted by excessive nutrients flowing into the lake. However, knowledge of whether excessive nutrients lead to different microbial environmental vulnerabilities in the lake sediment between macrophyte-dominated areas and macrophyte-free areas is a prerequisite for the implementation of targeted protection measures. In this study, we investigated bacterial communities in sediments using high-throughput sequencing of 16S rRNA genes. Our results showed that the sources of total nitrogen (TN) and organic matter (OM) were related to the macrophytes. The structure, drivers, and interspecific associations of bacterial community, which were more susceptible to increased changes in TN and OM, differed significantly between macrophyte-dominated areas and macrophyte-free areas. More precisely, the lake edge, where was occupied by macrophytes, had a higher proportion of deterministic phylogenetic turnover (88.89%) than other sites, as well as a wider ecological niche and a tighter network structure. Further, as the difference in TN increased, the main assembly processes in surface sediments changed from stochastic to deterministic. However, the majority of phyla from the lake edge showed a greater correlation with excessive nutrients, and the selection of the community by excessive nutrients was more obvious at the edge of the lake. In addition, our results demonstrated that the stability of the bacterial community in macrophyte-free areas is greater than in macrophyte-dominated areas, while an excessively high deterministic process ratio and nutrient (TN and OM) concentration significantly reduced bacterial community stability at macrophyte-dominated areas. Taken together, these results provide a better understanding of the effects of excessive nutrients derived from macrophytes on bacterial community patterns, and highlight the importance of avoiding the accumulation of TN and OM in macrophyte-dominated areas to enhance the sustainability of the ecosystem after restoration of lakes with macrophytes.

Identification and characterization of an envelope protein 168L in Cherax quadricarinatus iridovirus (CQIV).

Cherax quadricarinatus iridovirus (CQIV), a new member of family Iridoviridae, mainly infects the shrimps and crayfish with a high mortality rate. Previous gel-based LC-MS/MS study on CQIV has identified 30 structural proteins. In this study, one of the structural proteins, CQIV-168L, was selected for further analysis. RT-PCR and Western-blotting (WB) detection revealed that the transcript and the protein appeared late during infection of C. quadricarinatus cells and that the transcript was blocked by viral DNA replication inhibitor, indicating that CQIV-168L is a late expression gene. The specific antiserum against CQIV-168L was raised and immunofluorescence analysis showed that CQIV-168L was localized in the cytoplasm and associated with virus factories. Western-blotting (WB) assay suggested that CQIV-168L antiserum bound specifically to a 57-kDa protein in both the intact virions and the envelope fraction. As revealed by immunogold labeling, CQIV-168L was a component of the viral envelope. Findings in this work help to further understand the structure and entry mechanism of CQIV.

Pharmacokinetics and bioavailability of a new long-acting insulin analog in healthy Chinese volunteers: an open, randomized, single-dose, two-period and two-sequence cross-over study.

Objectives: INS068 is a novel, soluble, and long-acting insulin analog. In this study, we evaluated the pharmacokinetics and relative bioavailability of two formulations of INS068 in healthy Chinese subjects: a reference formulation packaged in vials and administered via syringe (R), and a test formulation packaged and administered via pen injector (T). Methods: A randomized, open-label, two-period, two-sequence crossover study was conducted with 24 healthy Chinese subjects. Subjects were randomized and administered subcutaneously in the abdomen at 0.4 U/kg of test or reference INS068 injection according to an open crossover design. INS068 concentrations in the serum were measured using LC-MS/MS, and the pharmacokinetic parameters of maximum concentration (Cmax) and area under the concentration-time curve (AUC0-t and AUC0-∞) were used to evaluate relative bioavailability. Results: After a single dose at 0.4 U/kg, the median Tmax of INS068 was 12 h for both formulations, and the mean t1/2 for T and R was 13.0 h and 12.6 h, respectively. The geometric means of Cmax and AUC0-∞ were 3.99 nmol/L and 120 h·nmol/L for the T, and 4.05 nmol/L and 117 h·nmol/L for the R, respectively. The geometric mean ratios of Cmax, AUC0-t and AUC0-∞ of T over R were 98.7% (90% CI: 92.7%-105.2%), 102.6% (90% CI: 100.0%-105.3%) and 102.8% (90% CI: 100.1%-105.5%). Conclusion: The overall PK profile of the two formulations of INS068 injection was comparable in healthy subjects, and the pen injector of INS068 had adequate safety and tolerability, supporting it as a new formulation in a phase III study and bridging PK data from early phase clinical trials. Clinical Trial Registration: clinicaltrials.gov, identifier: NCT05336071.

Molecular Detection and Multilocus Genotyping of Giardia duodenalis in Pigs in Fujian Province, Southeastern China.

Giardia duodenalis, an intestinal parasite, is widely distributed in humans and various animals, such as pigs, cattle and cats. The clinical symptoms of giardiasis are characterized as including abdominal pain, acute or chronic diarrhea, and bloating and weight loss in humans and animals, leading to public and veterinary health problems worldwide. However, the prevalence and genotypes of G. duodenalis in pigs in Fujian Province, southeastern China, have not been reported. In the present study, 725 fecal samples were collected from six cities (Fuqing, Putian, Nanping, Longyan, Sanming, Zhangzhou) in Fujian Province and analyzed for G. duodenalis prevalence and genotypes using nested PCR targeting the beta-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes. The results shown that total occurrence rate of G. duodenalis was 26.9% (195/725) in pigs, with significant differences in the prevalence among different regions (χ2 = 86.508, p < 0.05) and groups (χ2 = 12.748, p < 0.05). 195, 11 and 6 samples were detected at the bg, tpi and gdh loci, respectively. Each one belonged to a subtype of assemblage E and was analyzed using sequences obtained in this study. Based on phylogenetic analyses of sequences from the three genetic loci, only one MLG E1 was found. The results indicated that pigs may present a potential zoonotic risk of spreading G. duodenalis infection from animals to humans in this area. The findings of the present study also provide basic data for the prevention and control of G. duodenalis infection in pigs and humans in China.