Relationship Between the Hemoglobin, Albumin, Lymphocyte Count, Platelet Count (HALP) Score and Type 2 Diabetes Retinopathy.

Purpose: To explore the correlation between hemoglobin, albumin, lymphocyte count, platelet count (HALP) score and type 2 diabetic retinopathy (DR). Methods: The study was conducted on 674 patients with type 2 diabetes (T2DM). According to the results of the fundus examination, they were divided into non-diabetic retinopathy group (NDR, n=388) and diabetic retinopathy group (DR, n=286). Collected patients baseline data, calculated HALP score, analyzed the correlation between HALP score and DR. Results: In all patients, male patients and female patients, the HALP score of the DR group was lower than that of the NDR group (P<0.001), and the HALP score was negatively correlated with the incidence of DR (P<0.05). HALP score was independent risk factors for DR, regardless of gender. In male patients, HALP score and DR had a linear relationship, but in female patients, HALP score and DR showed a nonlinear relationship, and HALP score was more sensitive to the onset of DR in male patients. The combined diagnostic model of HALP score, course of disease, SBP and BUN was used to diagnose DR, and it was found that the diagnostic value was the highest among male patients, with AUC of 0.761, sensitivity of 58.3% and specificity of 80.3%. Conclusion: HALP score was an independent risk factor for DR, attention should be paid to monitoring HALP score, especially in male T2DM patients. The accuracy of HALP score, disease course, SBP and BUN combined model diagnosis of DR was high, which can become a biological indicator for early screening of DR.

DLM-GelMA/tumor slice sandwich structured tumor on a chip for drug efficacy testing.

The in vitro recapitulation of tumor microenvironment is of great interest to preclinical screening of drugs. Compared with culture of cell lines, tumor organ slices can better preserve the complex tumor architecture and phenotypic activity of native cells, but are limited by their exposure to fluid shear and gradual degradation under perfusion culture. Here, we established a decellularized liver matrix (DLM)-GelMA "sandwich" structure and a perfusion-based microfluidic platform to support long-term culture of tumor slices with excellent structural integrity and cell viability over 7 days. The DLM-GelMA was able to secrete cytokines and growth factors while providing shear protection to the tumor slice via the sandwich structure, leading to the preservation of the tumor microenvironment where immune cells (CD3, CD8, CD68), tumor-associated fibroblasts (α-SMA), and extracellular matrix components (collagen I, fibronectin) were well maintained. Furthermore, this chip presented anti-tumor efficacy at cisplatin (20 µM) on tumor patients, demonstrating our platform's efficacy to design patient-specific treatment regimens. Taken together, the successful development of this DLM-GelMA sandwich structure on the chip could faithfully reflect the tumor microenvironment and immune response, accelerating the screening process of drug molecules and providing insights for practical medicine.

Unconventional hcp/fcc Nickel Heteronanocrystal with Asymmetric Convex Sites Boosts Hydrogen Oxidation.

Developing non-platinum group metal catalysts for the sluggish hydrogen oxidation reaction (HOR) is critical for alkaline fuel cells. To date, Ni-based materials are the most promising candidates but still suffer from insufficient performance. Herein, we report an unconventional hcp/fcc Ni (u-hcp/fcc Ni) heteronanocrystal with multiple epitaxial hcp/fcc heterointerfaces and coherent twin boundaries, generating rugged surfaces with plenty of asymmetric convex sites. Systematic analyses discover that such convex sites enable the adsorption of *H in unusual bridge positions with weakened binding energy, circumventing the over-strong *H adsorption on traditional hollow positions, and simultaneously stabilizing interfacial *H2O. It thus synergistically optimizes the HOR thermodynamic process as well as reduces the kinetic barrier of the rate-determining Volmer step. Consequently, the developed u-hcp/fcc Ni exhibits the top-rank alkaline HOR activity with a mass activity of 40.6 mA mgNi-1 (6.3 times higher than fcc Ni control) together with superior stability and high CO-tolerance. These results provide a paradigm for designing high-performance catalysts by shifting the adsorption state of intermediates through configuring surface sites.

Oxidative stress induces ferroptosis in tendon stem cells by regulating mitophagy through cGAS-STING pathway.

Tendinopathy is one of the most prevalent sports injury diseases in orthopedics. However, there is no effective treatment or medicine. Recently, the discovery of tendon stem cells (TSCs) provides a new perspective to find new therapeutic methods for Tendinopathy. Studies have shown that oxidative stress will inevitably cause TSCs injury during tendinopathy, but the mechanism has not been fully elucidated. Here, we report the oxidative damage of TSCs induced by H2O2 via ferroptosis, as well, treatment with H2O2 raised the proportion of mitochondria engulfed by autophagosomes in TSCs. The suppression of mitophagy by Mdivi-1 significantly attenuates the H2O2-induced ferroptosis in TSCs. Mechanically, H2O2 actives the cGAS-STING pathway, which can regulate the level of mitophagy. Interfering with cGAS could impair mitophagy and the classical ferroptotic events. In the rat model of tendinopathy, interference of cGAS could relieve tendon injury by inhibiting ferroptosis. Overall, these results provided novel implications to reveal the molecular mechanism of tendinopathy, by which pointed to cGAS as a potential therapeutic target for the treatment of tendinopathy.

Structures of Native Doublet Microtubules from Trichomonas vaginalis Reveal Parasite-Specific Proteins as Potential Drug Targets.

Doublet microtubules (DMTs) are flagellar components required for the protist Trichomonas vaginalis ( Tv ) to swim through the human genitourinary tract to cause trichomoniasis, the most common non-viral sexually transmitted disease. Lack of DMT structures has prevented structure-guided drug design to manage Tv infection. Here, we determined the cryo-EM structure of native Tv- DMTs, identifying 29 unique proteins, including 18 microtubule inner proteins and 9 microtubule outer proteins. While the A-tubule is simplistic compared to DMTs of other organisms, the B-tubule features specialized, parasite-specific proteins, like Tv FAP40 and Tv FAP35 that form filaments near the inner and outer junctions, respectively, to stabilize DMTs and enable Tv locomotion. Notably, a small molecule, assigned as IP6, is coordinated within a pocket of Tv FAP40 and has characteristics of a drug molecule. This first atomic model of the Tv -DMT highlights the diversity of eukaryotic motility machinery and provides a structural framework to inform the rational design of therapeutics.

Stabilizing and Activating Active Sites: 1T-MoS2 Supported Pd Single Atoms for Efficient Hydrogen Evolution Reaction.

Metallic 1T-MoS2 with high intrinsic electronic conductivity performs Pt-like catalytic activity for hydrogen evolution reaction (HER). However, obtaining pure 1T-MoS2 is challenging due to its high formation energy and metastable properties. Herein, an in situ SO4 2--anchoring strategy is reported to synthesize a thin layer of 1T-MoS2 loaded on commercial carbon. Single Pd atoms, constituting a substantial loading of 7.2 wt%, are then immobilized on the 1T-phase MoS2 via Pd─S bonds to modulate the electronic structure and ensure a stable active phase. The resulting Pd1/1T-MoS2/C catalyst exhibits superior HER performance, featuring a low overpotential of 53 mV at the current density of 10 mA cm-2, a small Tafel slope of 37 mV dec-1, and minimal charge transfer resistance in alkaline electrolyte. Moreover, the catalyst also demonstrates efficacy in acid and neutral electrolytes. Atomic structural characterization and theoretical calculations reveal that the high activity of Pd1/1T-MoS2/C is attributed to the near-zero hydrogen adsorption energy of the activated sulfur sites on the two adjacent shells of atomic Pd.

High Apolipoprotein B/Apolipoprotein A1 is Associated with Vitamin D Deficiency Among Type 2 Diabetes Patients.

Purpose: To explore the relationship between vitamin D (VitD) deficiency and the apolipoprotein B/apolipoprotein A1 (apo B/A1) in type 2 diabetes mellitus (T2DM) patients. Methods: This was a retrospective study that lasted 2 years and 6 months, collecting information and laboratory data from 784 patients with T2DM. Patients were divided into VitD deficiency group (n = 433) and non-VitD deficiency group (n = 351) based on VitD levels. Calculated apo B/A1 ratio, and patients were further divided into high-apo B/A1 group (n = 392) and low-apo B/A1 group (n = 392) based on the median of the apo B/A1. All data were analyzed using Prism 8.0.1 and R version 4.3.1 software. Results: Apo B/A1 levels of T2DM patients combined with VitD deficiency was significantly higher than that of non-VitD deficiency patients, and the VitD levels of patients with high apo B/A1 was significantly lower than that patients with low apo B/A1 (all P<0.001). Spearman correlation analysis showed that VitD levels were negatively correlated with apo B/A1 (r=-0.238, P<0.001). Multiple linear regression analysis revealed after adjusting other factors, VitD levels were significantly negatively associated with apo B/A1 (ß=-0.123, P=0.001). Binary logistic regression analysis showed apoB/A1 was an independent risk factor for VitD deficiency in T2DM patients. Restrictive cubic spline indicated a significant linear relationship between apoB/A1 and VitD deficiency (P general trend <0.0001, P nonlinear = 0.0896), after stratification of gender, the results showed that apo B/A1 was more susceptible to VitD deficiency in female patients. The receiver operating characteristic (ROC) curve analysis showed that the area under the curve, sensitivity and specificity of the apo B/A1 for VitD deficiency were 0.654, 66.3% and 59.8%, respectively. Conclusion: The apo B/A1 was significantly negatively associated with VitD levels and an independent risk factor for VitD deficiency in patients with T2DM.

Interaction patterns and assembly mechanisms of dinoflagellates and diatoms in a coastal bay suffering from long-term eutrophication.

Dinoflagellates and diatoms are highly prevalent and ecologically important phytoplankton in coastal waters, greatly contributing to primary productivity in marine ecosystems. Although their composition and diversity have been extensively elucidated in the open ocean, their interaction patterns and community assembly in long-term eutrophic coastal waters remain poorly understood. This investigation aimed to elucidate the seasonal successional patterns of dinoflagellates and diatoms by 18S rRNA gene amplicon sequencing in a semi-enclosed bay. The results revealed that dinoflagellate and diatom communities have pronounced seasonal succession patterns, which are primarily associated with temperature. Furthermore, the most prevalent species throughout the year were Heterocapsa rotundata and Skeletonema costatum. Moreover, the assembly of dinoflagellate and diatom communities was mainly dominated by stochastic processes, with drift being the major factor. The co-occurrence of dinoflagellates and diatoms showed seasonal patterns, with the highest interactions observed in autumn. In addition, interactions of Syndiniales with dinoflagellates and diatoms highlighted the roles of parasites in eutrophic conditions. Flavobacteriaceae and Rhodobacteraceae are the bacterial taxa that most frequently interacted with dinoflagellates and diatoms, with interactions between dinoflagellates and bacteria being more complex than those between diatoms and bacteria. Overall, this study provides results that deepen our understanding of the phytoplankton dynamics in coastal eutrophic waters.IMPORTANCEDinoflagellates and diatoms are major phytoplankton groups in coastal waters. The composition and diversity of dinoflagellates and diatoms in the open ocean have been well documented; however, it remains uncertain to what extent their adaptation to long-term eutrophic conditions influences their response to environmental disturbances. Here, we investigated the interactions and assembly processes of dinoflagellates and diatoms in a eutrophic bay throughout the whole year. Our findings revealed that interactions between dinoflagellates and diatoms are primarily shaped by seasonal transitions, while prolonged eutrophic conditions tend to amplify stochastic processes in community assembly. These findings provide novel perspectives on the influence of long-term eutrophication on phytoplankton dynamics within eutrophic waters.

Elevated temperature alters bacterial community from mutualism to antagonism with Skeletonema costatum: insights into the role of a novel species, Tamlana sp. MS1.

Skeletonema costatum, a cosmopolitan diatom primarily inhabiting coastal ecosystems, exhibits a typically close yet variable relationship with heterotrophic bacteria. The increasing temperature of surface seawater is expected to substantially affect the viability and ecological dynamics of S. costatum, potentially altering its relationship with bacteria. However, it remains unclear to what extent the elevated temperature could change these relationships. Here, the relationship between axenic S. costatum and natural seawater bacteria underwent a dramatic shift from mutualism to antagonism as the co-culture temperature increased from 20°C to 25°C. The co-occurrence network indicated significantly increased complexity of interaction between S. costatum and bacteria community after temperature elevation, especially with Flavobacteriaceae, implying their potential role in eliminating S. costatum under higher temperatures. Additionally, a Flavobacteriaceae isolate, namely MS1 identified as Tamlana genus, was isolated from the co-culture system at 25°C. MS1 had a remarkable ability to eliminate S. costatum, with the mortality rate at 25°C steadily rising from 30.2% at 48 h to 92.4% at 120 h. However, it promoted algal growth to some extent at 20°C. These results demonstrated that increased temperature promotes MS1 shifts from mutualism to antagonism with S. costatum. According to the comparative genomics analysis, changes in the lifestyle of MS1 were attributed to the increased gliding motility and attachment of MS1 under elevated temperature, enabling it to exert an algicidal effect through direct contact with alga. This investigation provided an advanced understanding of interactions between phytoplankton and bacteria in future warming oceanic ecosystems. IMPORTANCE: Ocean warming profoundly influences the growth and metabolism of phytoplankton and bacteria, thereby significantly reshaping their interactions. Previous studies have shown that warming can change bacterial lifestyle from mutualism to antagonism with phytoplankton, but the underlying mechanism remains unclear. In this study, we found that high temperature promotes Tamlana sp. MS1 adhesion to Skeletonema costatum, leading to algal lysis through direct contact, demonstrating a transition in lifestyle from mutualism to antagonism with increasing temperature. Furthermore, the gliding motility of MS1 appears to be pivotal in mediating the transition of its lifestyle. These findings not only advance our understanding of the phytoplankton-bacteria relationship under ocean warming but also offer valuable insights for predicting the impact of warming on phytoplankton carbon sequestration.

Metabolic differences in MSTN and FGF5 dual-gene edited sheep muscle cells during myogenesis.

Dynamic metabolic reprogramming occurs at different stages of myogenesis and contributes to the fate determination of skeletal muscle satellite cells (MuSCs). Accumulating evidence suggests that mutations in myostatin (MSTN) have a vital role in regulating muscle energy metabolism. Here, we explored the metabolic reprogramming in MuSCs and myotube cells in MSTN and FGF5 dual-gene edited sheep models prepared previously, and also focused on the metabolic alterations during myogenic differentiation of MuSCs. Our study revealed that the pathways of nucleotide metabolism, pantothenate and CoA biosynthesis were weakened, while the unsaturated fatty acids biosynthesis were strengthened during myogenic differentiation of sheep MuSCs. The MSTN and FGF5 dual-gene editing mainly inhibited nucleotide metabolism and biosynthesis of unsaturated fatty acids in sheep MuSCs, reduced the number of lipid droplets in per satellite cell, and promoted the pentose phosphate pathway, and the interconversion of pentose and glucuronate. The MSTN and FGF5 dual-gene editing also resulted in the inhibition of nucleotide metabolism and TCA cycle pathway in differentiated myotube cells. The differential metabolites we identified can be characterized as biomarkers of different cellular states, and providing a new reference for MSTN and FGF5 dual-gene editing in regulation of muscle development. It may also provide a reference for the development of muscle regeneration drugs targeting biomarkers.

Characterization and correlations of dominant microorganisms and volatile compounds in fermentation process of Yangjiang douchi.

BACKGROUND: Yangjiang douchi (YD) is a traditional fermented soybean product, which is popular in Chinese cuisine for its unique flavor. However, due to its high salt content and unstable flavor, its competitiveness in the international market is gradually weakening. Microorganisms have a key role in the production process of YD because it is a fermented food but the effect of microorganisms on the volatile compounds of YD is also not currently clear. RESULTS: In this paper, aroma compounds and microbial diversity in different fermentation stages of YD were analyzed using gas chromatography-mass spectrometry/olfactometry (GC-MS/O) and IlluminaMiseq system sequencing. A total of 78 aroma-active compounds were detected throughout the fermentation process and they influenced the formation of flavor in YD. Fungi flora were relatively single in YD, and bacteria were rich and varied. A total of 418 species of bacteria were present during fermentation, with unclassified_Staphylococcus, Staphylococcus_kloosii, and Bacillus_velezensis_Bacillus predominating. There were 25 species of fungi at the species level, and Aspergillus minisclerotigenes (OTU 4) played a dominant role in the whole fermentation process. CONCLUSION: Staphylococcus and Bacillus in the bacterial genus were strongly correlated with most flavor compounds detected, and A. minisclerotigenes in the fungi were more relevant to flavor compounds. This research provides a theoretical basis for the enhancement of the flavor of traditional fermented douchi in China. © 2024 Society of Chemical Industry.

Correction: A differential-targeting core-shell microneedle patch with coordinated and prolonged release of mangiferin and MSC-derived exosomes for scarless skin regeneration.

Correction for 'A differential-targeting core-shell microneedle patch with coordinated and prolonged release of mangiferin and MSC-derived exosomes for scarless skin regeneration' by Shang Lyu et al., Mater. Horiz., 2024, https://doi.org/10.1039/D3MH01910A.

Controllable radical polymerization of TEMPO redox for stable and sensitive enzyme electrode interface.

Assembling functional molecules on the surface of an enzyme electrode is the most basic technique for constructing a biosensor. However, precise control of electron transfer interface or electron mediator on the electrode surface remains a challenge, which is a key step that affects the stability and sensitivity of enzyme-based biosensors. In this study, we propose the use of controllable free radical polymerization to grow stable 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) polymer as electron mediator on enzyme surface for the first time. Through scanning electron microscopy (SEM), Raman spectroscopy, electrode surface coverage measurement, static contact angle (SCA), and a series of electrochemical methods, it has been demonstrated that the TEMPO-based enzyme electrode exhibits a uniform hydrophilic morphology and stable electrochemical performance. Furthermore, the results show that the sensor demonstrates high sensitivity for detecting glucose biomolecules in artificial sweat and serum. Attributing to the quantitative and controllable radical polymerization of TEMPO redox assembled enzyme electrode surface, the as-proposed biosensor providing a use, storage, and inter-batch sensing stability, providing a vital platform for wearable/implantable biochemical sensors.

Epidemiological Characterisation of Chronic Diseases Among Civil Servants in Hebei Province, China: A Cross-Sectional Real-World Study of Approximately 50,000 Adults.

Background: The objective of this investigation was to explore the health status and epidemiological features of civil servants working in Hebei Province, China. Methods: Data was collected through a cross-sectional survey that involved 50,039 adult civil servants in Hebei Province. The research was conducted at the Hebei Provincial Medical Examination Centre and included inquiries about demographics, health behaviours, chronic illnesses, and abnormal check-up indicators. Statistical data were used to determine the prevalence of chronic diseases and abnormal health examination indicators. Subgroup analyses by sex were then conducted to calculate the similarities and differences in health status between men and women. Results: The findings of the survey indicate that 14.2% of individuals who work in civil service suffer from obesity. Additionally, a considerable proportion of this group display poor health behaviours, including smoking and alcohol consumption. Hypertension (21.03%), dyslipidaemia (10.88%), and diabetes mellitus (8.56%) were identified as the top three chronic diseases, while cardiovascular diseases were prevalent at 6.27% among civil servants. Ultrasound showed a high prevalence of fatty liver, non-smooth gallbladder and liver cysts (44.61%, 33.24% and 23.26% respectively). The occurrence of pulmonary nodules was alarmingly high at 88.48%, while thyroid nodules were found in as much as 62.65% of patients. Abnormal E/A values≤1, prostate hyperplasia, breast nodules, and carotid plaques were also prevalent, with percentages ranging from 46.76% to 63.04%. In subgroup analyses by gender, the prevalence of chronic diseases and abnormal screening indicators was consistently higher in men than in women, with the exception of thyroid nodules and thyroid surgery. Conclusion: The survey revealed a large proportion of civil servants in Hebei Province, China, suffering from chronic diseases, underscoring the necessity for enhanced attention to chronic disease prevention and management in this group and emphasising the requirement for focused interventions aimed at improving health outcomes.

Rapid and highly sensitive colorimetric LAMP assay and integrated device for visual detection of monkeypox virus.

BACKGROUND: The monkeypox virus (MPXV) is a linear double-stranded DNA virus with a large genome that causes tens of thousands of infections and hundreds of deaths in at least 40 countries and regions worldwide. Therefore, timely and accurate diagnostic testing could be an important measure to prevent the ongoing spread of MPXV and widespread epidemics. RESULTS: Here, we designed multiple sets of primers for the target region of MPXV for loop-mediated isothermal amplification (LAMP) detection and identified the optimal primer set. Then, the specificity in fluorescent LAMP detection was verified using the plasmids containing the target gene, pseudovirus and other DNA/RNA viruses. We also evaluated the sensitivity of the colorimetric LAMP detection system using the plasmid and pseudovirus samples, respectively. Besides, we used monkeypox pseudovirus to simulate real samples for detection. Subsequent to the establishment and introduction of a magnetic beads (MBs)-based nucleic acid extraction technique, an integrated device was developed, characterized by rapidity, high sensitivity, and remarkable specificity. This portable system demonstrated a visual detection limit of 137 copies/mL, achieving sample-to-answer detection within 1 h. SIGNIFICANCE: The device has the advantages of integration, simplicity, miniaturization, and visualization, which help promote the realization of accurate, rapid, portable, and low-cost testing. Meanwhile, this platform could facilitate efficient, cost-effective and easy-operable point-of-care testing (POCT) in diverse resource-limited settings in addition to the laboratory.

A UDP-glycosyltransferase gene PcUGT202A9 was associated with abamectin resistance in Panonychus citri (McGregor).

Panonychus citri (McGregor) strains have developed a high level of resistance to abamectin, but the underlying molecular mechanism is unknown. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are critical for the removal of a variety of exogenous and endogenous substances. In this study, an enzyme activity assay revealed that UGTs potentially contribute to P. citri abamectin resistance. Spatiotemporal expression profiles showed that only PcUGT202A9 was significantly overexpressed in the abamectin-resistant strain (AbR) at all developmental stages. Moreover, UGT activity decreased significantly, whereas abamectin susceptibility increased significantly, in AbR after PcUGT202A9 was silenced. Three-dimensional modeling and molecular docking analyses revealed that PcUGT202A9 can bind stably to abamectin. Recombinant PcUGT202A9 activity was detected when α-naphthol was used, but the enzymatic activity was inhibited by abamectin (50 % inhibitory concentration: 803.3 ±â€¯14.20 µmol/L). High-performance liquid chromatography and mass spectrometry analyses indicated that recombinant PcUGT202A9 can effectively degrade abamectin and catalyze the conjugation of UDP-glucose to abamectin. These results imply PcUGT202A9 contributes to P. citri abamectin resistance.

Adaptively resettable microfluidic patch for sweat rate and electrolytes detection.

Skin-interfaced microfluidic patch has become a reliable device for sweat collection and analysis. However, the intractable problems of emptying the microchannel for reuse, and the channel's volumetric capacity limited by the size of the patch, directly hinder the practical application of sweat sensors. Herein, we report an adaptively resettable microfluidic sweat patch (Art-Sweat patch) capable of continuously monitoring both sweat rate (0.2-4.0 µL min-1) and total ionic charge concentration (10-200 mmol L-1). We develop a platform with a vertical and horizontal microchannel combined strategy, enabling repeatedly filling sweat and emptying the microchannel for autonomously resetting and detecting. The variation in the emptied volume is designed to be adaptively identified by the sensor, resulting in enhanced stability and an enlarged volumetric capacity of over 300 µL. By integrating with self-designed wireless transmission modules, the proposed Art-Sweat patch shows product-level wearability and high performance in monitoring variations in regional sweat rate and concentration for hydration status assessment.

Unravelling and reconstructing the biosynthetic pathway of bergenin.

Bergenin, a rare C-glycoside of 4-O-methyl gallic acid with pharmacological properties of antitussive and expectorant, is widely used in clinics to treat chronic tracheitis in China. However, its low abundance in nature and structural specificity hampers the accessibility through traditional crop-based manufacturing or chemical synthesis. In the present work, we elucidate the biosynthetic pathway of bergenin in Ardisia japonica by identifying the highly regio- and/or stereoselective 2-C-glycosyltransferases and 4-O-methyltransferases. Then, in Escherichia coli, we reconstruct the de novo biosynthetic pathway of 4-O-methyl gallic acid 2-C-ß-D-glycoside, which is the direct precursor of bergenin and is conveniently esterified into bergenin by in situ acid treatment. Moreover, further metabolic engineering improves the production of bergenin to 1.41 g L-1 in a 3-L bioreactor. Our work provides a foundation for sustainable supply of bergenin and alleviates its resource shortage via a synthetic biology approach.

An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293 T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.