The enzyme encoded by Myrmecia incisa, a green microalga, phospholipase A2 gene preferentially hydrolyzes arachidonic acid at the sn-2 position of phosphatidylcholine.

The enzyme phospholipase A2 (PLA2) plays a crucial role in acyl remodeling of phospholipids via the Lands' cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In this study, a full-length cDNA sequence coding Myrmecia incisa phospholipase A2 (MiPLA2) was cloned using the technique of rapid amplification of cDNA ends. Comparison of the 1082-bp cDNA with its corresponding cloned DNA sequence revealed that MiPLA2 contained 3 introns. Mature MiPLA2 (mMiPLA2) had a conserved Ca2+-binding loop and a catalytic site motif that has been recognized in plant secretory PLA2 (sPLA2) proteins. Correspondingly, phylogenetic analysis illustrated that MiPLA2 was clustered within GroupXIA of plant sPLA2 proteins. To ascertain the function of MiPLA2, the cDNA coding for mMiPLA2 was subcloned into the vector pET-32a to facilitate the production of recombinant mMiPLA2 in Escherichia coli. Recombinant mMiPLA2 was purified and used for the in vitro enzyme reaction. Thin-layer chromatography profiles of the catalytic products generated by recombinant mMiPLA2 indicated a specificity for cleaving sn-2 acyl chains from phospholipids, thereby functionally characterizing MiPLA2. Although recombinant mMiPLA2 displayed a strong preference for phosphatidylethanolamine, it preferentially hydrolyzes arachidonic acid (ArA) at the sn-2 position of phosphatidylcholine. Results from the fused expression of p1300-sp-EGFP-mMiPLA2 illustrated that MiPLA2 was localized in the intercellular space of onion epidermis. Furthermore, the positive correlation between MiPLA2 transcription and free ArA levels were established. Consequently, the role of mMiPLA2 in the biosynthesis of ArA-rich TAG was elucidated. This study helps to understand how M. incisa preferentially uses ArA to synthesize TAG.

Lactobacillus rhamnosus GG triggers intestinal epithelium injury in zebrafish revealing host dependent beneficial effects.

Lactobacillus rhamnosus GG (LGG), the well-characterized human-derived probiotic strain, possesses excellent properties in the maintenance of intestinal homeostasis, immunoregulation and defense against gastrointestinal pathogens in mammals. Here, we demonstrate that the SpaC pilin of LGG causes intestinal epithelium injury by inducing cell pyroptosis and gut microbial dysbiosis in zebrafish. Dietary SpaC activates Caspase-3-GSDMEa pathways in the intestinal epithelium, promotes intestinal pyroptosis and increases lipopolysaccharide (LPS)-producing gut microbes in zebrafish. The increased LPS subsequently activates Gaspy2-GSDMEb pyroptosis pathway. Further analysis reveals the Caspase-3-GSDMEa pyroptosis is initiated by the species-specific recognition of SpaC by TLR4ba, which accounts for the species-specificity of the SpaC-inducing intestinal pyroptosis in zebrafish. The observed pyroptosis-driven gut injury and microbial dysbiosis by LGG in zebrafish suggest that host-specific beneficial/harmful mechanisms are critical safety issues when applying probiotics derived from other host species and need more attention.

[Rules of acupoint selection and pattern-acupoint relationship in treatment with acupuncture and moxibustion for endometriosis based on complex network analysis technology].

OBJECTIVE: To explore the rules of acupoint selection and pattern-acupoint relationship in treatment with acupuncture and moxibustion for endometriosis (EMs) based on complex network analysis technology. METHODS: The articles for clinical trial of EMs treated with acupuncture and moxibustion were searched from CNKI, Wanfang, VIP, SinoMed, PubMed, EMbase and Cochrane Library from the inception of the databases to December 14, 2022. Using Microsoft Excel 2019 software, the database was established to collect the use frequency of acupoint, meridian tropism, location and pattern-acupoint relationship. SPSS Modeler 18.0 Apriori algorithm was adopted to conduct the association rule analysis, Cytoscape3.7.2 software was used to plot the complex co-occurrence network map; and SPSS Statistics 26.0 was adopted to perform hierarchical cluster analysis on high-frequency acupoints and a tree diagram was drawn. RESULTS: A total of 163 articles were included, and 167 core acupoint prescriptions and 74 pattern-associated acupoint prescriptions were extracted, involving 92 acupoints, with a cumulative frequency of 1 223 times. The top five acupoints with the highest use frequency were Guanyuan (CV 4), Sanyinjiao (SP 6), Zhongji (CV 3), Zigong (EX-CA 1) and Qihai (CV 6). The selected acupoints were mostly distributed in the chest, abdomen and lower limbs; and the involved meridians included the conception vessel, the spleen meridian of foot-taiyin and the stomach meridian of foot-yangming. The acupoint compatibility of high frequency referred to Guanyuan (CV 4) - Sanyinjiao (SP 6), Guanyuan (CV 4) - Zhongji (CV 3), and Guanyuan (CV 4) - Zigong (EX-CA 1). The close association was presented among Guanyuan (CV 4), Sanyinjiao (SP 6), Qihai (CV 6) and Zhongji (CV 3), which had the strongest connection with the other acupoints; among the top 25 acupoints with the highest use frequency, 5 acupoint prescriptions with high frequency were obtained by the cluster analysis. Guanyuan (CV 4), Qihai (CV 6), Sanyinjiao (SP 6), Zigong (EX-CA 1) and Zhongji (CV 3) were selected for cold and blood stagnation; Guanyuan (CV 4), Sanyinjiao (SP 6), Zhongji (CV 3), Dahe (KI 12) and Taixi (KI 3) for kidney deficiency and blood stagnation; Zhongji (CV 3), Guanyuan (CV 4), Sanyinjiao (SP 6), Xuehai (SP 10) and Diji (SP 8) for qi and blood stagnation; Qihai (CV 6), Guanyuan (CV 4), Zusanli (ST 36), Xuehai (SP 10), and Zigong (EX-CA 1) for qi deficiency and blood stagnation; Sanyinjiao (SP 6), Fenglong (ST 40), Zhongliao (BL 33), Ciliao (BL 32) and Xialiao (BL 34) for interaction of phlegm and stasis; and Daheng (SP 15), Guanyuan (CV 4), Zhongji (CV 3), Qihai (CV 6) and Zhongwan (CV 12) for retention of damp and heat. CONCLUSION: The core acupoints are Guanyuan (CV 4), Sanyinjiao (SP 6), Zhongji (CV 3), Qihai (CV 6) and Zigong (EX-CA 1) in treatment of endometriosis with acupuncture and moxibustion. Six patterns/syndromes are involved in clinical practice. In terms of the properties, functions and indications, the supplementary acupoints are selected on the basis of the core acupoints for different patterns/sydnromes of the disease.

Serum metabolomics analyses reveal biomarkers of osteoporosis and the mechanism of Quanduzhong capsules.

With the aging of the population, the prevalence of osteoporosis (OP) is rising rapidly, making it an important public health concern. Early screening and effective treatment of OP are the primary challenges facing the management of OP today. Quanduzhong capsule (QDZ) is a single preparation composed of Eucommia ulmoides Oliv., which is included in the Pharmacopoeia of the People's Republic of China. It is used to treat OP in clinical practice, but its mechanisms are unclear. This study involved 30 patients with OP, 30 healthy controls (HC), and 28 OP patients treated with QDZ to identify potential biomarkers for the early diagnosis of OP and to investigate the potential mechanism of QDZ in treating OP. The serum samples were analyzed using targeted amino acid metabolomics. Significant differences in amino acid metabolism were identified between the OP cohort and the HC group, as well as between OP patients before and after QDZ treatment. Compared with HC, the serum levels of 14 amino acids in OP patients changed significantly. Kynurenine, arginine, citrulline, methionine, and their combinations are expected to be potential biomarkers for OP diagnosis. Notably, QDZ reversed the changes in levels of 10 amino acids in the serum of OP patients and significantly impacted numerous metabolic pathways during the treatment of OP. This study focuses on screening potential biomarkers for the early detection of OP, which offers a new insight into the mechanism study of QDZ in treating OP.

LncRNA 4930581F22Rik promotes myogenic differentiation by regulating the ERK/MAPK signaling pathway.

The skeletal muscle is the largest organ in mammals and is the primary motor function organ of the body. Our previous research has shown that long non-coding RNAs (lncRNAs) are significant in the epigenetic control of skeletal muscle development. Here, we observed progressive upregulation of lncRNA 4930581F22Rik expression during skeletal muscle differentiation. Knockdown of lncRNA 4930581F22Rik hindered skeletal muscle differentiation and resulted in the inhibition of the myogenic markers MyHC and MEF2C. Furthermore, we found that lncRNA 4930581F22Rik regulates myogenesis via the ERK/MAPK signaling pathway, and this effect could be attenuated by the ERK-specific inhibitor PD0325901. Additionally, in vivo mice injury model results revealed that lncRNA 4930581F22Rik is involved in skeletal muscle regeneration. These results establish a theoretical basis for understanding the contribution of lncRNAs in skeletal muscle development and regeneration.

Dietary Bacillus velezensis T23 fermented products supplementation improves growth, hepatopancreas and intestine health of Litopenaeus vannamei.

This study aimed to elucidate the effects of dietary fermented products of Bacillus velezensis T23 on the growth, immune response and gut microbiota in Pacific white shrimp (Litopenaeus vannamei). Shrimp were fed with diets containing fermentation products of B. velezensis T23 at levels of (0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 g/kg) for 4 weeks, to assess the influence on shrimp growth. The results showed that 0.3 and 0.4 g/kg T23 supplementation improved shrimp growth and feed utilization. Based on these results we selected these three diets (Control, 0.3T23 and 0.4T23) to assess the effect on immune response and gut microbiota of shrimp. Compared with the control, the 0.3T23 and 0.4T23 groups enhanced lipase and α-amylase activities in the gut significantly. Moreover, the 0.4T23 group decreased TAG and MDA levels in hepatopancreas, ALT and AST levels of serum significantly (P < 0.05). In hepatopancreas, CAT and SOD activities were improved observably and the MDA content was reduced markedly in both T23 groups. The expressions of antimicrobial related genes, Cru and peroxinectin in the 0.3T23 group, and proPO and peroxinectin in the 0.4T23 group were up-regulated remarkably (P < 0.05). Moreover, hepatopancreas of shrimp fed with a diet amended with T23 showed a significant down-regulated expression of nf-kb and tnf-α genes, while expressions of tgf-ß was considerably up-regulated. Furthermore, serum LPS and LBP contents were reduced markedly in T23 groups. Intestinal SOD and CAT were noteworthy higher in T23 groups (P < 0.05). In the intestine of shrimp fed on the diet enriched with T23 the expression of nf-κb and tnf-α exhibited markedly down-regulated, whereas hif1α was up-regulated (P < 0.05). Besides, in the intestine of shrimp grouped under T23, Cru and peroxinectin genes were markedly up-regulated (P < 0.05). Dietary 0.3 g/kg T23 also upregulated the ratio of Rhodobacteraceae to Vibrionaceae in the gut of the shrimp. Taken together, the inclusion of B. velezensis T23 in the diet of shrimp enhanced the growth and feed utilization, enhanced hepatopancreas and intestine health.

Repeat biopsy versus initial biopsy in terms of complication risk factors and clinical outcomes for patients with non-small cell lung cancer: a comparative study of 113 CT-guided needle biopsy of lung lesions.

Objectives: The safety and feasibility of repeat biopsy after systemic treatment for non-small cell lung cancer have received extensive attention in recent years. The purpose of this research was to compare complication rates between initial biopsy and rebiopsy in non-small cell lung cancer patients with progressive disease and to assess complication risk factors and clinical results after rebiopsy. Methods: The study included 113 patients initially diagnosed with non-small cell lung cancer who underwent lung biopsy at initial biopsy and rebiopsy after progression while on epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and/or chemotherapy from January 2018 to December 2021. We compared the incidence of complications between the initial biopsy and rebiopsy and analyzed the predictors factors that influenced complications in patients who underwent rebiopsy. Results: The successful rate of rebiopsy was 88.5% (100/113). With the exception of two cases where lung adenocarcinoma changed into small cell lung cancer with gefitinib treatment, 98 individuals retained their initial pathological type. The secondary EGFR T790M mutation accounts for 55.6% of acquired resistance. The total number of patients with complications in initial biopsy was 25 (22.1%) and 37 (32.7%) in the rebiopsy. The incidence of pulmonary hemorrhage increased from 7.1% at the initial biopsy to 10.6% at rebiopsy, while the incidence of pneumothorax increased from 14.2% to 20.4%. Compared with the initial biopsy, the incidence of overall complications, parenchymal hemorrhage, and pneumothorax increased by 10.6%, 3.5%, and 6.2%, respectively. In all four evaluations (pneumorrhagia, pneumothorax, pleural reaction, and overall complication), there were no significant differences between the rebiopsy and initial biopsy (all p > 0.05). The multivariate logistic regression analysis suggested that male sex (odds ratio [OR] = 5.064, p = 0.001), tumor size ≤ 2 cm (OR = 3.367, p = 0.013), EGFR-TKIs with chemotherapy (OR = 3.633, p =0.023), and transfissural approach (OR = 7.583, p = 0.026) were independent risk factors for overall complication after rebiopsy. Conclusion: Compared with the initial biopsy, the complication rates displayed a slight, but not significant, elevation in rebiopsy. Male sex, tumor size ≤ 2 cm, transfissural approach, and EGFR-TKIs combined with chemotherapy were independent risk factors for rebiopsy complications.

In Vitro Culture and Biological Characterization of Meniscal Fibrochondrocytes.

Background: Currently, there is a lot of discussion in clinical and scientific research over the best ways to delay meniscal degeneration and speed up its healing. Meniscal injury and degeneration are significant contributors to the development of knee osteoarthritis in a pathological state. Objective: To isolate, culture and characterize rat meniscal fibrochondrocytes in vitro, and to provide a simple and feasible cell culture method for the study of damage repair of rat meniscal fibrochondrocytes. Methods: The rat medial and lateral meniscus of both knees was surgically isolated. Trypsin and type II collagenase were used to remove the cells, and toluidine blue staining and type II collagen immunofluorescence were used to identify the cells. The cells were then routinely cultured in low-sugar DMEM complete culture medium. Results: At different time points, cells showed different physiological shapes, from polygonal or short spindle to spindle shape, and finally to triangle or ellipse, and cell proliferation ability gradually increased with time. The OD values of cells cultured at 48h and 72h were higher than those at 24h. Comparing OD values of cells cultured for 48h and 72h, although OD value of 72h increased. Toluidine blue staining and type I collagen immunofluorescence staining were positive. Conclusion: A more dependable technique for fibrochondrocyte isolation and culture is offered for the study of meniscus in molecular biology and tissue engineering. The cells cultured using this method are morphologically stable, have a strong proliferation ability, and possess the fundamental biological properties of fibrochondrocytes in vivo.

Corrigendum: The effect and underlying mechanism of yeast ß-glucan on antiviral resistance of zebrafish against spring viremia of carp virus infection.

[This corrects the article DOI: 10.3389/fimmu.2022.1031962.].

Pricing strategy research in the dual-channel pharmaceutical supply chain considering service.

In the context of developing a new era, the pharmaceutical supply chain market has gradually transformed from a seller's market to a buyer's market. The closer the consumers are, the greater the market pricing power, so the pharmaceutical market power of manufacturers and retailers has also changed. This study considers the effect of service on the pricing strategy of the pharmaceutical platform supply chain. The study aimed to coordinate optimization, and the coordination strategy of the pharmaceutical platform supply chain of complementary products is discussed mainly by researching the price and service factors. Various situations are studied by hypothesis and model solving. This study uses Stackelberg game decision-making. Manufacturers are at the forefront of platform supply chain decisions. The research found that the price was lower under centralized decision-making than under decentralized decision-making. Coordination between price and service levels needs attention in the pharmaceutical platform supply chain of complementary products, and the service level should be controlled within a certain range. Only by improving the service level can enterprises maximize profits, providing a theoretical basis for pharmaceutical supply chain pricing strategy research. Supply chain members must strive to improve service levels to improve medical supply consumers' (patients) psychological satisfaction level. Service levels do not fully mitigate channel conflict. Therefore, pharmaceutical complements have become a way to alleviate the conflicts in the pharmaceutical platform supply chain.

Case report: Retroperitoneal solid pseudopapillary neoplasm associated with multiple hepatic metastases.

Solid pseudopapillary neoplasm (SPN) is a rare tumor mostly occurring in the pancreas. They are low-grade malignant tumors of the exocrine pancreas that occasionally metastasize, usually to the liver or peritoneum. Additionally, multiple metastases of extrapancreatic SPN to the liver are extremely rare and have been reported before. This study presents a case of a 13-year-old male patient with retroperitoneal SPN and multiple hepatic metastases. The patient presented with abdominal trauma and underwent enhanced CT, which revealed upper pancreatic occupancy and three hypodense foci in the right lobe of the liver. Moreover, increased spleen size was noted. The patient's serum tumor marker CA125 was increased to 39.00 U/mL (N < 35.0 U/mL), and circulating tumor cells were elevated to 10.2 FU/3 mL (N < 8.7 FU/3 mL). The patient underwent retroperitoneal occupancy resection and splenectomy, followed by resection of liver metastases 7 months after the surgery. Furthermore, multiple liver metastases from retroperitoneal SPN were confirmed postoperatively. The patient recovered for 1 year without tumor recurrence. This case emphasizes the importance of evaluating serum tumor markers and medical imaging in young patients as well as the fact that surgery appears to be the preferred treatment option for multiple metastases in SPN.

Chestnut-Inspired Hollow Hydroxyapatite 3D Printing Scaffolds Accelerate Bone Regeneration by Recruiting Calcium Ions and Regulating Inflammation.

This study aims to overcome the drawbacks associated with hydroxyapatite (HAP) dense structures after sintering, which often result in undesirable features such as large grain size, reduced porosity, high crystallinity, and low specific surface area. These characteristics hinder osseointegration and limit the clinical applicability of the material. To address these issues, a new method involving the preparation of hollow hydroxyapatite (hHAP) microspheres has been proposed. These microspheres exhibit distinctive traits including weak crystallization, high specific surface area, and increased porosity. The weak crystallization aligns more closely with early mineralization products found in the human body and animals. Moreover, the microspheres' high specific surface area and porosity offer advantages for protein loading and facilitating osteoblast attachment. This innovative approach not only mitigates the limitations of conventional HAP structures but also holds the potential for improving the effectiveness of hydroxyapatite in biomedical applications, particularly in enhancing osseointegration. Three-dimensional printed hHAP/chitosan (CS) scaffolds with different hHAP concentration gradients were manufactured, and the physical and biological properties of each group were systematically evaluated. In vitro and in vivo experiments show that the hHAP/CS scaffold has excellent performance in bone remodeling. Furthermore, in-scaffold components, hHAP and CS were cocultured with bone marrow mesenchymal stem cells to explore the regulatory role of hHAP and CS in the process of bone healing and to reveal the cell-level specific regulatory network activated by hHAP. Enrichment analysis showed that hHAP can promote bone regeneration and reconstruction by recruiting calcium ions and regulating inflammatory reactions.

Characterization of a novel γ-type carbonic anhydrase, Sjγ-CA2, in Saccharina japonica: Insights into carbon concentration mechanism in macroalgae.

Carbonic anhydrase (CA) is a crucial component of CO2-concentrating mechanism (CCM) in macroalgae. In Saccharina japonica, an important brown seaweed, 11 CAs, including 5 α-, 3 ß-, and 3 γ-CAs, have been documented. Among them, one α-CA and one ß-CA were localized in the periplasmic space, one α-CA was found in the chloroplast, and one γ-CA was situated in mitochondria. Notably, the known γ-CAs have predominantly been identified in mitochondria. In this study, we identified a chloroplastic γ-type CA, Sjγ-CA2, in S. japonica. Based on the reported amino acid sequence of Sjγ-CA2, the epitope peptide for monoclonal antibody production was selected as 165 Pro-305. After purification and specificity identification, anti-SjγCA2 monoclonal antibody was employed in immunogold electron microscopy. The results illustrated that Sjγ-CA2 was localized in the chloroplasts of both gametophytes and sporophytes of S. japonica. Subsequently, immunoprecipitation coupled with LC-MS/MS analysis revealed that Sjγ-CA2 mainly interacted with photosynthesis-related proteins. Moreover, the first 65 amino acids at N-terminal of Sjγ-CA2 was identified as the chloroplast transit peptide by the transient expression of GFP-SjγCA2 fused protein in tabacco. Real-time PCR results demonstrated an up-regulation of the transcription of Sjγ-CA2 gene in response to high CO2 concentration. These findings implied that Sjγ-CA2 might contribute to minimizing the leakage of CO2 from chloroplasts and help maintaining a high concentration of CO2 around Rubisco.

Utility of spectral CT with orthopedic metal artifact reduction algorithms for 125I seeds implantation in mediastinal and hepatic tumors.

Background: Virtual monochromatic image (VMI) combined with orthopedic metal artifact reduction algorithms (VMI + O-MAR) can effectively reduce artifacts caused by metal implants of different types. Nevertheless, so far, no study has systematically evaluated the efficacy of VMI + O-MAR in reducing various types of metal artifacts induced by 125I seeds. The aim of this study was to assess the effectiveness of combining spectral computed tomography (CT) images with O-MAR in reducing metal artifacts and improving the image quality affected by artifacts in patients after 125I radioactive seeds implantation (RSI). Methods: A total of 45 patients who underwent dual-layer detector spectral CT (DLCT; IQon, Philips Healthcare) scanning of mediastinal and hepatic tumors after 125I RSI were retrospectively included. Spectral data were reconstructed into conventional image (CI), VMI, CI combined with O-MAR (CI + O-MAR), and VMI + O-MAR to evaluate the de-artifact effect and image quality improvement. Objective indicators included signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and artifact index (AI) of lesions affected by artifacts. Subjective indicators included assessment of overcorrected artifacts and new artifacts, different morphology of artifacts, and overall image quality. Results: In artifact-affected lesion areas, SNR and CNR in the CI/VMI + O-MAR groups were better than those in CI groups (all P values <0.05). The AI showed a downward trend as VMI keV increased (all P values <0.001). The AI values of the CI/VMI (50-150 keV) group were all higher than the groups of CI/VMI + O-MAR (50-150 keV) (P<0.001). Overcorrection artifacts and new artifacts were concentrated in the VMI50/70 keV groups. In the evaluation of artifact morphology, as the VMI keV increased, the number of near-field banding artifacts in hyperdense artifacts gradually decreased, whereas the number of minimal or no artifacts increased, and the total number of hyperdense artifacts were decreased. The diagnostic and image quality scores of hyperdense artifacts were higher than those of hypodense artifacts as VMI keV increased. Conclusions: High VMI level combined with O-MAR substantially improve objective and subjective image quality, lesion display ability, and diagnostic confidence of CT follow-up after 125I RSI, especially at the VMI + O-MAR 150 keV level.

UCP2 promotes NSCLC proliferation and glycolysis via the mTOR/HIF-1α signaling.

BACKGROUND: Metabolic disturbance is a hallmark of cancers. Targeting key metabolic pathways and metabolism-related molecular could be a potential therapeutic approach. Uncoupling protein 2 (UCP2) plays a pivotal part in the malignancy of cancer and its capacity to develop resistance to pharmaceutical interventions. However, it is unclear about the mechanism of how UCP2 acts in the tumor growth and metabolic reprogramming process in non-small cell lung cancer (NSCLC). METHODS: Here, we conducted qRT-PCR to investigate the expression of UCP2 in both NSCLC tissues and cell lines. Subsequent functional studies including colony formation assay, CCK-8 assay, and glycolysis assay were conducted to investigate the functions of UCP2 in NSCLC. The regulatory mechanism of UCP2 toward the mammalian target of rapamycin (mTOR) and hypoxia-inducible factor-1 alpha (HIF-1α) signaling in NSCLC was confirmed through western blotting. RESULTS: We observed a significant upregulation of UCP2 in both NSCLC tissues and cell lines. The increased expression of UCP2 has a strong association with a worse outlook. Silencing UCP2 remarkably dampened NSCLC cell proliferation and glycolysis capacities. Mechanically, UCP2 promoted NSCLC tumorigenesis partially via regulating the mTOR/HIF-1α axis. CONCLUSION: Taken together, we explored the functions as well as the mechanisms of the UCP2/mTOR/HIF-1α axis in NSCLC progression, uncovering potential biological signatures and targets for NSCLC treatment.

Deciphering the gut microbiome of grass carp through multi-omics approach.

BACKGROUND: Aquaculture plays an important role in global protein supplies and food security. The ban on antibiotics as feed additive proposes urgent need to develop alternatives. Gut microbiota plays important roles in the metabolism and immunity of fish and has the potential to give rise to novel solutions for challenges confronted by fish culture. However, our understanding of fish gut microbiome is still lacking. RESULTS: We identified 575,856 non-redundant genes by metagenomic sequencing of the intestinal content samples of grass carp. Taxonomic and functional annotation of the gene catalogue revealed specificity of the gut microbiome of grass carp compared with mammals. Co-occurrence analysis indicated exclusive relations between the genera belonging to Proteobacteria and Fusobacteria/Firmicutes/Bacteroidetes, suggesting two independent ecological groups of the microbiota. The association pattern of Proteobacteria with the gene expression modules of fish gut and the liver was consistently opposite to that of Fusobacteria, Firmicutes, and Bacteroidetes, implying differential functionality of Proteobacteria and Fusobacteria/Firmicutes/Bacteroidetes. Therefore, the two ecological groups were considered as two functional groups, i.e., Functional Group 1: Proteobacteria and Functional Group 2: Fusobacteria/Firmicutes/Bacteroidetes. Further analysis revealed that the two functional groups differ in genetic capacity for carbohydrate utilization, virulence factors, and antibiotic resistance. Finally, we proposed that the ratio of "Functional Group 2/Functional Group 1" can be used as a biomarker that efficiently reflects the structural and functional characteristics of the microbiota of grass carp. CONCLUSIONS: The gene catalogue is an important resource for investigating the gut microbiome of grass carp. Multi-omics analysis provides insights into functional implications of the main phyla that comprise the fish microbiota and shed lights on targets for microbiota regulation. Video Abstract.

Simultaneous determination of seven toxic components in ShenFuTuoDu capsules by HPLC-MS/MS.

An accurate and reliable HPLC-MS/MS method has been established for the simultaneous determination of seven toxic components in the Chinese medicine ShenFuTuoDu capsules. The seven toxic components were separated on a Shimadzu Shim-pack GIST C18 column (3.0 mm×50 mm, 3.0 µm) with methanol and water (containing 0.1% formic acid) as the mobile phase by gradient elution. The flow rate was 0.5 mL•min-1. The column temperature was 25°C and the injection volume was 5µL. An ESI+ scan combined with MRM was adopted and the instrument parameters were as follows: ion source voltage, 5.5 kV; ion source temperature, 600oC; curtain gas, 68.95 kPa; atomized gas, 344.75 kPa; auxiliary gas, 344.75 kPa. The linear relationships of the seven components were good (R2>0.9937). The average recoveries were 95.2%-106.7% with RSD of 0.79%-5.27% (n=6). The seven toxic components of scopolamine, atropine, rhynchophylline, isorhynchophylline, benzoylaconine, benzoylmesaconine and benzoylhypaconine in six batches of ShenFuTuoDu capsules were 5.99-18.48µg•g-1, 6.36-14.79µg•g-1, 3.71-15.45µg•g-1, 7.90-15.08µg•g-1, 19.05-44.58µg•g-1, 117.38-248.26µg•g-1 and 19.74-79.49µg•g-1, respectively. Precision, stability and repeatability test RSDs were less than 7.17% (n=6). The method is suitable for the simultaneous determination of scopolamine, atropine, rhynchophylline, isorhynchophylline, benzoylaconine, benzoylmesaconine and benzoylhypaconine. It can be used for the quality control of ShenFuTuoDu capsules.

Developing the Limosilactobacillus reuteri Chassis through an Endogenous Programmable Endonuclease-Based Genome Editing Tool.

Using genetically tractable probiotics to engineer live biotherapeutic products (LBPs) for disease treatment is urgently needed. Limosilactobacillus reuteri is an important vertebrate gut symbiont, which has great potential for developing LBPs. However, in L. reuteri, synthetic biology work is largely limited by the long editing cycle. In this study, we identified a subtype II-A CRISPR-Cas9 system in L. reuteri 03 and found the endogenous Cas9 (LrCas9) recognizing a broad protospacer-adjacent motif (PAM) sequence (3'-NDR; N = A, G, T, C; D = A, G, T; R = A, G). We reprogrammed the LrCas9 for efficient gene deletion (95.46%), point mutation (86.36%), large fragment deletion (40 kb), and gene integration (1743 bp, 73.9%), which uncovered the function of the repeated conserved domains in mucus-binding protein. Moreover, we analyzed the distribution of endogenous endonucleases in 304 strains of L. reuteri and found the existence of programmable endonucleases in 98.36% of L. reuteri strains suggesting the potential to reprogram endogenous endonucleases for genetic manipulation in the majority of L. reuteri strains. In conclusion, this study highlights the development of a new probiotic chassis based on endogenous endonucleases in L. reuteri 03, which paves the way for the development of genome editing tools for functional genetic studies in other L. reuteri. We believe that the development of an endogenous endonuclease-based genetic tool will greatly facilitate the construction of LBPs.

The Initial Efficacy of Comprehensive Treatment of External Treatment of Traditional Chinese Medicine on Acute Mastitis During Lactation and its Influence on Patients' Symptoms.

Objective: To evaluate the initial efficacy of comprehensive treatment of external treatment of traditional Chinese medicine in the therapy of acute mastitis during lactation and its influence on sufferers' symptoms. Methods: From January 2021 to January 2023, a retrospective analysis was performed on 100 sufferers with acute mastitis during lactation who were received in our hospital as the study objects. Divided them into a control group (n = 50) and an observation group (n = 50). Among them, the control one was received in 50% magnesium sulfate for moist heat compress, and the observation one was treated with comprehensive external therapy of traditional Chinese medicine. After different treatment measures were carried out in the 2 groups, the total effective ratio of clinical therapy, excellent and good rates of lactation, quality of life measurements, pain scores before and after treatment, and TCM symptom points at pre-therapy and post-therapy, negative emotion scores at pre-therapy and post-therapy related to patients were analyzed. Results: (1) In the data comparison of the total effective ratio of clinical therapy between the 2 groups of sufferers after therapy, the data in the observation one were greater than the control one, and the distinction was obvious, with P < .05. (2) In the data comparison of the excellent and good lactation rate between the 2 groups of sufferers at post-therapy, the data in observation one were greater than the control one, and the distinction was obvious, with P < .05. (3) In the data comparison of quality of life measurements between the 2 groups of sufferers at post-therapy, the data in the observation one were greater than the control one, and the distinction was obvious, with P < .05. (4) Before treatment, it had no obvious distinction in the pain scores between the 2 groups, with P > .05; in the data comparison of the pain points between the 2 groups after therapy, the data in observation one were less than the control one, and the distinction was obvious, with P < .05. (5) Before therapy, it had no obvious distinction in TCM symptom points between the 2 groups, with P > .05; in the data comparison of TCM symptom points between the 2 groups after therapy, the data in the observation one were less than the control one, and the distinction was obvious, with P < .05. (6) Before therapy, it had no obvious distinction in negative emotion points between the 2 groups, with P > .05; in the data comparison of negative emotion scores between the 2 groups of sufferers at post-therapy, the data in the observation one were less than those in the control one, and the distinction was obvious, with P < .05. Conclusion: The comprehensive therapy of TCM external treatment for acute mastitis during lactation has a significant therapeutic effect, which is conducive to improving the clinical symptoms of sufferers as soon as possible and at the same time, greatly improving the life quality of sufferers and improving their negative emotions, which can relieve the pain of sufferers and promote the recovery of the sufferers.

Synthesis of lipoic acid ferulate and evaluation of its ability to preserve fish oil from oxidation during accelerated storage.

Lipoic acid ferulate (LAF) was synthesized and its anti-free radical ability in vitro was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) assays. Protective effects of LAF in stabilizing fish oil were tested, compared to antioxidants such as lipoic acid, ferulic acid and tert-butylhydroxyquinone (TBHQ) by measuring peroxide values, thiobarbituric acid reactants, p-anisidine values, nuclear magnetic resonance (NMR) spectra and gas chromatography-mass spectrometry (GC-MS) spectra of fish oil during accelerated storage (12 days, 80 °C). The inhibitory effect of these antioxidants on fish oil oxidation followed the order TBHQ ≧ LAF > ferulic acid > lipoic acid. In addition, the omega-3 polyunsaturated fatty acids were the first to be oxidized. The formation of oxidation products followed a first-order kinetic model, and the addition of LAF effectively reduced the reaction rate constants. Therefore, LAF can effectively slow down the formation of oxidative products and prolong the shelf life of fish oil.