Quantitative Fluorescence Analysis Research Articles

Rapid improvement of grain appearance in three-line hybrid rice via CRISPR/Cas9 editing of grain size genes

Key messageGenetic editing of grain size genes quickly improves three-line hybrid rice parents to increase the appearance quality and yield of hybrid rice.Grain size affects rice yield and quality. In this study, we used CRISPR/Cas9 to edit the grain size gene GW8 in the maintainer line WaitaiB (WTB) and restorer line Guanghui998 (GH998). The new slender sterile line WTEA (gw8) was obtained in the BC2F1 generation by transferring the grain mutation of the maintainer plant to the corresponding sterile line WantaiA (WTA, GW8) in the T1 generation. Two slender restorer lines, GH998E1 (gw8(II)) and GH998E2 (gw8(I)), were obtained in T1 generation. In the early stage, new sterile and restorer lines in grain mutations were created by targeted editing of GS3, TGW3, and GW8 genes. These parental lines were mated to detect the impact of grain-type mutations on hybrid rice yield and quality. Mutations in gs3, gw8, and tgw3 had a minimal impact on agronomic traits except the grain size and thousand-grain weight. The decrease in grain width in the combination mainly came from gw8/gw8, gs3/gs3 increased the grain length, gs3/gs3-gw8/gw8 had a more significant effect on the grain length, and gs3/gs3-gw8/gw8(I) contributed more to grain length than gs3/gs3-gw8/gw8(II). The heterozygous TGW3/tgw3 may not significantly increase grain length. Electron microscopy revealed that the low-chalky slender-grain variety had a cylindrical grain shape, a uniform distribution of endosperm cells, and tightly arranged starch grains. Quantitative fluorescence analysis of endospermdevelopment-related genes showed that the combination of slender grain hybrid rice caused by gs3 and gw8 mutations promoted endosperm development and improved appearance quality. An appropriate grain size mutation resulted in hybrid rice varieties with high yield and quality.

Genome-scale identification, expression and evolution analysis of B-box members in Dendrobium huoshanense

B-box (BBX) proteins have been recognized as vital determinants in plant development, morphogenesis, and adaptive responses to a myriad of environmental stresses. These zinc-finger proteins play a pivotal role in various biological processes. Their influence spans photomorphogenesis, the regulation of flowering, and imparting resilience to a wide array of challenges, encompassing both biotic and abiotic factors. Chromosome localization, gene structure and conserved motifs, phylogenetic analysis, collinearity analysis, expression profiling, fluorescence quantitative analysis, and tobacco transient transformation methods were used for functional localization and expression pattern analysis of the DhBBX gene. A total of 23 DhBBX members were identified from Dendrobium huoshanense. Subsequent phylogenetic evaluations effectively segregated these genes into five discrete evolutionary subsets. The predictions of subcellular localizations revealed that all these proteins were localized in the nucleus. The genetic composition and patterns showed that the majority of these genes consisted of several exons, with a few variations that could be attributed to transposon insertion. A comprehensive analysis using qRT-PCR was conducted to unravel the expression patterns of these genes in D. huoshanense, with a specific concentration on their responses to various hormone treatments and cold stress. Subcellular localization reveals that DhBBX21 and DhBBX9 are located in the nucleus. Our results provide a deep comprehension of the complex regulatory mechanisms of BBXs in response to various environmental and hormonal stimuli. These discoveries encourage further detailed and focused investigations into the operational dynamics of the BBX gene family in a wider range of plant species.

Abstract PO4-07-07: The construction and clinical research value of the prediction model of sentinel lymph node metastasis based on indocyanine green fluorescence quantification in breast cancer

Abstract Objective: To establish a prediction model of sentinel lymph node metastasis by quantitative indocyanine green fluorescence analysis in breast cancer. Methods: From May 2023 to June 2023, 20 patients with breast cancer underwent modified radical mastectomy in the General Surgery Department of Jiangsu Provincial People's Hospital were chosen for research and analysis. Sentinel lymph node were taken from all the patients. The sentinel lymph nodes were located by using the double staining method of subcutaneous injection of Methylene blue and intravenous injection of indocyanine green. The postoperative pathology is used as the standard for evaluating the presence or absence of metastatic cancer in lymph nodes. At the same time, the transcutaneous fluorescence of sentinel lymph node was recorded and the peak time of fluorescence intensity curve was plotted. To establish a logistic diagnostic prediction model and fluorescence threshold, the sentinel lymph node was incised during operation and the fluorescence of sentinel lymph node was recorded again. The criteria for ICG positive determination are determined by the Youden index, which was used to judge the lymph nodes above the threshold in the Logistic diagnostic prediction model. Lymph nodes below the threshold determined in the Logistic diagnostic prediction model are the criteria for ICG negative determination. Cases with less than 3 sentinel lymph node were excluded. Results: 19 cases were ultimately selected. Methylene blue staining data was taking as the judgment standard of sentinel lymph node. Fluorescence imaging was 100% in 14 patients, 66.7% (2/3) in 1 patient, and 125%-150% (5/4-6/4) in 4 patients. Fluorescence quantification values below 2.91 are negative lymph nodes, and values above 4.69 are positive lymph nodes. Moreover, caution is required between 2.91 and 4.69. Combining with the fluorescence dynamic attenuation curve, the positive group has a plateau curve, while the negative group mostly has a decline curve. Combining fluorescence quantification with fluorescence dynamic attenuation curve, the diagnostic rate of positive lymph nodes has a lower false negative rate compared to simple fluorescence quantification and fluorescence dynamic attenuation curve. Conclusion: The quantitative difference value of indocyanine green fluorescence angiography can be used to predict the positive metastasis of sentinel lymph node in breast cancer, making it an attempt to exempt sentinel lymph node biopsy. Citation Format: Yang Wu, Wei Zhang, Jinhai Tang. The construction and clinical research value of the prediction model of sentinel lymph node metastasis based on indocyanine green fluorescence quantification in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-07-07.

Seasonal and Spatial Fluctuations of Reactive Oxygen Species in Riparian Soils and Their Contributions on Organic Carbon Mineralization.

Reactive oxygen species (ROS) are ubiquitous in the natural environment and play a pivotal role in biogeochemical processes. However, the spatiotemporal distribution and production mechanisms of ROS in riparian soil remain unknown. Herein, we performed uninterrupted monitoring to investigate the variation of ROS at different soil sites of the Weihe River riparian zone throughout the year. Fluorescence imaging and quantitative analysis clearly showed the production and spatiotemporal variation of ROS in riparian soils. The concentration of superoxide (O2•-) was 300% higher in summer and autumn compared to that in other seasons, while the highest concentrations of 539.7 and 20.12 μmol kg-1 were observed in winter for hydrogen peroxide (H2O2) and hydroxyl radicals (•OH), respectively. Spatially, ROS production in riparian soils gradually decreased along with the stream. The results of the structural equation and random forest model indicated that meteorological conditions and soil physicochemical properties were primary drivers mediating the seasonal and spatial variations in ROS production, respectively. The generated •OH significantly induced the abiotic mineralization of organic carbon, contributing to 17.5-26.4% of CO2 efflux. The obtained information highlighted riparian zones as pervasive yet previously underestimated hotspots for ROS production, which may have non-negligible implications for carbon turnover and other elemental cycles in riparian soils.

Functional Differentiation of the Duplicated Gene BrrCIPK9 in Turnip (Brassica rapa var. rapa).

Gene duplication is a key biological process in the evolutionary history of plants and an important driving force for the diversification of genomic and genetic systems. Interactions between the calcium sensor calcineurin B-like protein (CBL) and its target, CBL-interacting protein kinase (CIPK), play important roles in the plant's response to various environmental stresses. As a food crop with important economic and research value, turnip (Brassica rapa var. rapa) has been well adapted to the environment of the Tibetan Plateau and become a traditional crop in the region. The BrrCIPK9 gene in turnip has not been characterized. In this study, two duplicated genes, BrrCIPK9.1 and BrrCIPK9.2, were screened from the turnip genome. Based on the phylogenetic analysis, BrrCIPK9.1 and BrrCIPK9.2 were found located in different sub-branches on the phylogenetic tree. Real-time fluorescence quantitative PCR analyses revealed their differential expression levels between the leaves and roots and in response to various stress treatments. The differences in their interactions with BrrCBLs were also revealed by yeast two-hybrid analyses. The results indicate that BrrCIPK9.1 and BrrCIPK9.2 have undergone Asparagine-alanine-phenylalanine (NAF) site divergence during turnip evolution, which has resulted in functional differences between them. Furthermore, BrrCIPK9.1 responded to high-pH (pH 8.5) stress, while BrrCIPK9.2 retained its ancestral function (low K+), thus providing further evidence of their functional divergence. These functional divergence genes facilitate turnip's good adaptation to the extreme environment of the Tibetan Plateau. In summary, the results of this study reveal the characteristics of the duplicated BrrCIPK9 genes and provide a basis for further functional studies of BrrCBLs-BrrCIPKs in turnip.

Investigation of porcine circovirus type 2 and porcine circovirus type 3 infections based on dual TaqMan fluorescent quantitative PCR method and genetic evolutionary analysis of these two viruses.

Porcine circovirus type 2 (PCV2) is the pathogen of Porcine Circovirus Associated Diseases. Porcine circovirus type 3 (PCV3) is a novel porcine circovirus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2 is clearly pathogenic, while the pathogenicity of PCV3 remains controversial, so it is crucial to monitor the prevalence of PCV2 and PCV3 in healthy and diseased pigs to investigate the effects of PCV3 and PCV2 on the health status of pigs. Here, we developed a PCV2 and PCV3 dual TaqMan quantitative PCR (qPCR) method to test samples from healthy and diseased pigs, to clarify the differences in the positive rates and viral copy numbers of PCV2 and PCV3, and to analyze the genetic evolution and molecular characterization of the viral genomes obtained with sequence alignment and phylogenetic analysis, homology and structural analysis of Cap proteins, and selection pressure analysis. We successfully established a dual TaqMan qPCR method for PCV2 and PCV3 with good repeatability, specificity and sensitivity. In total, 1,385 samples from 15 Chinese provinces were tested with the established qPCR. The total positive rates were 37.47% for PCV3 and 57.95% for PCV2, and the coinfection rate for was 25.49%. The positive rates of PCV3 and PCV2 in 372 healthy pigs were 15.05 and 69.89%, respectively, and the coinfection rate was 12.90%. The positive rates of PCV3 and PCV2 in 246 diseased pigs were 55.69 and 83.33%, respectively, and the coinfection rate was 47.97%. Eighteen PCV3 genomes and 64 PCV2 genomes were identified, including nine each of the PCV3a-1 and PCV3b genotypes, eight of PCV2a, 16 of PCV2b, and 40 of PCV2d. The amino acid identity within the PCV3 Cap proteins was 94.00-100.0%, whereas the PCV2 Cap proteins showed an identity of 81.30-100.0%. PCV3 Cap was most variable at amino acid sites 24, 27, 77, 104 and 150, whereas PCV2 Cap had 10-13 unique sites of variation between genotypes. These results clarify the prevalence and variations of PCV2 and PCV3 in healthy and diseased pigs, which will provide a basis for the prevention and control of the two viral infections.

Establishing an efficient callus genetic transformation system for the tung tree (Vernicia fordii)

Tung tree (Vernicia fordii) as an important industrial raw material used to produce tung oil in China. Genetic transformation system has not been established for the tung tree, hindering research on the functions of tung tree genes and the cultivation of new stress-resistant varieties. In this study, we optimized the tissue culture regeneration system of tung tree hypocotyls, screened out a callus state suitable for Agrobacterium tumefaciens infection, and improved the efficiency of genetic transformation. RNA interference (RNAi) vector was constructed using FAD2 as the target gene. Adventitious shoots were obtained by infecting the hypocotyl calli cultured for 25 days through Agrobacterium-mediated infection. The positive adventitious shoot transformation rate of the pBI121 vector was 22.86%, and the adventitious shoot transformation rate of the RNAi vector was 7.84%. We established a complete genetic transformation system using two vectors, and the system was verified via GUS staining and real-time fluorescence quantitative analyses to ensure its stability. This system solves the technical problem of a genetic transformation system for the tung tree, and will provide technical support for studying the functions of tung tree genes and molecular breeding.

Comparative transcriptome analysis and identification of candidate R2R3-MYB genes involved in anthraquinone biosynthesis in Rheum palmatum L.

BackgroundRheum palmatum L. has important medicinal value because it contains biologically active anthraquinones. However, the key genes and TFs involved in anthraquinone biosynthesis and regulation in R. palmatum remain unclear.MethodsBased on full length transcriptome data, in this study, we screened the differentially expressed genes in the anthraquinone biosynthesis pathway. The R2R3-MYB family genes of R. palmatum were systematically identified based on full-length transcriptome sequencing followed by bioinformatics analyses. The correlation analysis was carried out by using co-expression analysis, protein interaction analysis, and real-time fluorescence quantitative analysis after MeJA treatment. The RpMYB81 and RpMYB98 genes were amplified by RT-PCR, and their subcellular localization and self-activation characteristics were analyzed.ResultsComparative transcriptome analysis results revealed a total of 3525 upregulated and 6043 downregulated DEGs in the CK versus MeJA group; 28 DEGs were involved in the anthraquinone pathway. Eleven CHS genes that belonged to the PKS family were differentially expressed and involved in anthraquinone biosynthesis. Twelve differentially expressed MYBs genes were found to be co-expressed and interact with CHS genes. Furthermore, 52 MYB genes were identified as positive regulators of anthraquinone biosynthesis and were further characterized. Three MYB genes including RpMYB81, RpMYB98, and RpMYB100 responded to MeJA treatment in R. palmatum, and the levels of these genes were verified by qRT-PCR. RpMYB81 was related to anthraquinone biosynthesis. RpMYB98 had an interaction with genes in the anthraquinone biosynthesis pathway. RpMYB81 and RpMYB98 were mainly localized in the nucleus. RpMYB81 had self-activation activity, while RpMYB98 had no self-activation activity.ConclusionRpMYB81, RpMYB98, and RpMYB100 were significantly induced by MeJA treatment. RpMYB81 and RpMYB98 are located in the nucleus, and RpMYB81 has transcriptional activity, suggesting that it might be involved in the transcriptional regulation of anthraquinone biosynthesis in R. palmatum.

Identification and expression of genome of uridine diphosphate glycosyltransferase (UGT) gene family from Chrysanthemum indicum

Uridine diphosphate glycosyltransferase(UGT) is involved in the glycosylation of a variety of secondary metabolites in plants and plays an important role in plant growth and development and regulation of secondary metabolism. Based on the genome of a diploid Chrysanthemum indicum, the UGT gene family from Ch. indicum was identified by bioinformatics methods, and the physical and chemical properties, subcellular localization prediction, conserved motif, phylogeny, chromosome location, gene structure, and gene replication events of UGT protein were analyzed. Transcriptome and real-time fluorescence quantitative polymerase chain reaction(PCR) were used to analyze the expression pattern of the UGT gene in flowers and leaves of Ch. indicum. Quasi-targeted metabolomics was used to analyze the differential metabolites in flowers and leaves. The results showed that a total of 279 UGT genes were identified in the Ch. indicum genome. Phylogenetic analysis showed that these UGT genes were divided into 8 subfamilies. Members of the same subfamily were distributed in clusters on the chromosomes. Tandem duplications were the main driver of the expansion of the UGT gene family from Ch. indicum. Structural domain analysis showed that 262 UGT genes had complete plant secondary metabolism signal sequences(PSPG box). The analysis of cis-acting elements indicated that light-responsive elements were the most ubiquitous elements in the promoter regions of UGT gene family members. Quasi-targeted metabolome analysis of floral and leaf tissue revealed that most of the flavonoid metabolites, including luteolin-7-O-glucoside and kaempferol-7-O-glucoside, had higher accumulation in flowers. Comparative transcriptome analysis of flower and leaf tissue showed that there were 72 differentially expressed UGT genes, of which 29 genes were up-regulated in flowers, and 43 genes were up-regulated in leaves. Correlation network and phylogenetic analysis showed that CindChr9G00614970.1, CindChr2G00092510.1, and CindChr2G00092490.1 may be involved in the synthesis of 7-O-flavonoid glycosides in Ch. indicum, and real-time fluorescence quantitative PCR analysis further confirmed the reliability of transcriptome data. The results of this study are helpful to understand the function of the UGT gene family from Ch. indicum and provide data reference and theoretical basis for further study on the molecular regulation mechanism of flavonoid glycosides synthesis in Ch. indicum.

A novel Foley catheter made of high-intensity near-infrared fluorescent silicone rubber for image-guided surgery of lower rectal cancer

BackgroundUrethral injury occurs in 1–6 % of male cases during minimally invasive surgery of lower rectal cancer. A Foley catheter emitting near-infrared (NIR) fluorescence of sufficient intensity has been expected to locate the urethra during image-guided surgery. Although it has been difficult to impart NIR fluorescent properties to biocompatible thermosetting polymers, we have recently succeeded in developing a NIR fluorescent compound for silicone rubber and a NIR fluorescent Foley catheter (HICARL). Here, we evaluated its NIR fluorescence properties and visibility performance using porcine anorectal isolation specimens. MethodsThe HICARL catheter was made of a mixture of solid silicone rubber and a NIR fluorescent compound that emits fluorescence with a wavelength of 820–880 nm, while a conventional transparent Foley catheter was made of solid silicone rubber only. As a standard for comparison of the intensity of NIR fluorescence, a transparent Foley catheter the lumen of which was filled with a mixture of indocyanine green (ICG) and human plasma was used. As a comparison to assess the visibility performance of the HICARL catheter, a transparent Foley catheter into which a commercially available NIR fluorescent polyurethane ureteral catheter (NIRC) was placed was used. ResultsA NIR fluorescence quantitative imaging analysis revealed that the Foley-NIRC catheter and the HICARL catheter emitted 3.42 ± 0.42 and 6.43 ± 0.07 times more fluorescence than the Foley-ICG catheter, respectively. The location of the HICARL catheter placed in the anorectum with a wall thickness of 3.8 ± 0.1 mm was clearly delineated in its entirety by NIR fluorescence, while that of the Foley-NIRC catheter was faintly or only partially visible. ConclusionsThe HICARL catheter emitting NIR fluorescence of sufficient intensity is a promising and easy-to-use tool for urethral visualization during image-guided surgery of lower rectal cancer.

Erythrocyte membrane-coated nanocarriers modified by TGN for Alzheimer's disease

Alzheimer's disease (AD) is an aging-related neurodegenerative disease, and the main pathological feature was β-amyloid protein (Aβ) deposition. Recently, bioactive materials-based drug delivery system has been widely investigated for the treatment of AD. In this study, we developed a red blood cells (RBC) membrane-coated polycaprolactone (PCL) nanoparticles (NPs) loading with a therapeutic agent for AD, curcumin (Cur). A functional peptide TGNYKALHPHN (TGN) was conjugated to the surface of membrane for blood-brain barrier (BBB) transport (TGN-RBC-NPs-Cur). TGN peptide can be recognized by receptors on the BBB and has great potential for brain transport. To confirm the targeted delivery of Cur to the brain, a cell co-culturing immortalized human cerebral microvascular endothelial cells and human brain astrocytes glioblastoma (hCMEC/D3 and U-118MG) in vitro model was established. As a result, the BBB transporting ratio of TGN-RBC-NPs-FITC was 29.64% at 12 h which was approximately eight-fold than RBC-NPs-FITC. The improvement of drug accumulation in the AD lesion was confirmed by the NPs modified with the BBB-penetrating peptide in the fluorescence imaging and quantitative analysis with UPLC-MS/MS in vivo. The neuroprotective effects were evaluated with new object recognition behavioral test, in vitro AD cell model, dendritic spine stain, GFAP and IBA1 immunofluorescence stain. The spatial learning and memory abilities of the AD model mice treated with TGN-RBC-NPs-Cur were obviously enhanced compared with the AD control mice and were also better than Cur at the same dosage. These results were consistent with the values of protection index of rat adrenal pheochromocytoma cells (PC12 cells) treated by Aβ25–35. TGN-RBC-NPs-Cur increased the dendritic segments densities and restrained activation of microglia and astrocytes of AD mice, as well as reversed cognitive function of AD mice. All of the results demonstrated TGN-RBC-NPs-Cur a promising therapeutic strategy for delaying the progression of AD by designing biomimetic nanosystems to deliver drugs into the brain.

Malvidin promotes PGC-1α/Nrf2 signaling to attenuate the inflammatory response and restore mitochondrial activity in septic acute kidney injury

Acute kidney injury (AKI) in sepsis is a vital and dangerous organ failure caused by an infection-induced dysregulation of the host reaction. Malvidin possesses significant anti-inflammatory and antioxidant bioactivities. This study explored the critical roles of malvidin in sepsis AKI and the crosstalk among mitochondrial function, nucleotide-binding oligomerization-like receptor 3 (NLRP3) inflammasome and nuclear factor erythroid 2 (Nrf2) signaling pathway. First, C57BL/6 mice were administered lipopolysaccharide intraperitoneally for 6 h to create an AKI model of sepsis. Hematoxylin-eosin staining and serum biomarker assays showed that malvidin protected from AKI in sepsis. Real-time fluorescence quantitative polymerase chain reaction analysis revealed that malvidin was able to inhibit inflammatory cytokines and mediators. Western blot assays indicated that malvidin suppressed NLRP3 inflammasome activation and enhanced antioxidant properties. Additionally, human renal tubular epithelial cells were stimulated by lipopolysaccharide/adenosine triphosphate to establish an NLRP3 inflammasome activation model in vitro, and in line with findings in vivo, malvidin significantly inhibited NLRP3 inflammasome activation. Furthermore, our data indicate that malvidin restored mitochondrial quality and function, reduced reactive oxygen species production, increased mitochondrial membrane potential, enhanced mitochondrial DNA copy number, and promoted peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) nuclear translocation. Moreover, inhibitor blockade assays indicated that both PGC-1α and Nrf2 affected the inhibition of the NLRP3 inflammasome by malvidin. Finally, immunoprecipitation assays showed that malvidin promoted PGC-1α and Nrf2 interactions. Overall, malvidin alleviated lipopolysaccharide-induced sepsis AKI, improved mitochondrial function and mitochondrial biogenesis, and inhibited the NLRP3 inflammasome through the PGC-1α/Nrf2 signaling pathway, suggesting that malvidin might translate into clinical applications for sepsis AKI therapy.

Arsenic-Induced Ferroptosis in Chicken Hepatocytes via the Mitochondrial ROS Pathway.

Arsenic has been shown to be highly toxic and can cause liver damage. Previous studies have shown that arsenic causes severe liver damage and induces accumulation of reactive oxygen species (ROS). This study aimed to investigate the effects of ferroptosis on the liver in arsenic trioxide (ATO) and to explore the underlying mechanisms. We confirmed the hepatotoxic effects of arsenic by in vivo and in vitro experiments. After 28days of administration of arsenic trioxide (4-mg/kg, 8-mg/kg) by gavage, chickens exhibited body weight loss and liver damage in a dose-dependent manner. In addition, in vivo and in vitro western blot and real-time fluorescence quantitative PCR analyses simultaneously indicated that ferroptosis might be the main pathway of arsenic-induced liver injury. Finally, Mito-TEMPO effectively eliminated the ROS accumulation in mitochondria, significantly attenuating the process of cellular ferroptosis. In summary, the hepatotoxic effects of arsenic are related to ferroptosis, and the hepatic ferroptosis process of arsenic is regulated by mitochondrial ROS (MtROS). Our study reveals new mechanisms of arsenic toxicity to the liver, which may deepen our understanding of arsenic toxicology.

Establishment of a Spontaneous Liver Fibrosis Model in NOD/SCID Mice Induced by Natural Aging.

Liver fibrosis, a critical pathological feature of chronic liver diseases, arises from a multitude of pathogenic factors. Consequently, establishing an appropriate animal model to simulate liver fibrosis holds immense significance for comprehending its underlying pathogenesis. Despite the numerous methodologies available for generating liver fibrosis models, they often deviate substantially from the spontaneous age-related liver fibrosis process. In this study, compared with young (12 weeks) and middle-aged NOD/SCID mice (32 weeks), there were a large number of fibrous septum and collagen in the liver tissue of old NOD/SCID mice (43 weeks, 43 W). Immunohistochemical analysis unequivocally indicated heightened α-SMA content within the liver tissue of the 43 W mice, thereby underscoring aging's role in triggering the epithelial-to-mesenchymal transition. In addition, SA-β-gal staining as well as P21 expression were increased, and SIRT1 and SIRT3 expression were decreased in 43 W mice. A comprehensive evaluation encompassing transmission electron microscopy and fluorescence quantitative analysis elucidated compromised mitochondrial function and reduced antioxidant capacity in hepatocytes of the 43 W mice. Furthermore, the aging process activated the pro-fibrotic TGF-β-SMAD pathway, concurrently inducing hepatocellular inflammation. The results of the present study not only validate the successful construction of a spontaneous liver fibrosis mouse model through natural aging induction but also provide initial insights into the mechanisms underpinning age-induced liver fibrosis.

Growth Axis Somatostatin, Growth Hormone Receptor, and Insulin-like Growth Factor-1 Genes Express and Are Affected by the Injection of Exogenous Growth Hormone in Chinemys reevesii.

In this study, to explore the effect of growth hormone changes on the related genes and regulatory roles of the turtle, PCR amplification, real-time fluorescence quantitative analysis, and enzyme cutting technology were used to clone and sequence the somatostatin (SS) gene, growth hormone receptor (GHR), and insulin-like growth factor-1 (IGF-I) sequence of Chinemys reevesii. The effects of human growth hormone on the mRNA expression of growth-axis-related genes SS, GHR, and IGF-1 in different sexes were observed. The study of the SS gene in turtles using real-time fluorescence quantitative PCR showed that the SS gene was mainly expressed in the nervous system and the digestive system, with the highest expression found in the brain, while the GHR gene and the IGF-I gene were expressed in all tissues of Chinemys reevesii. The SS gene was expressed in the brain, pituitary, liver, stomach, and intestine, with the highest expression in the brain and the lowest expression in the liver. Within 4 weeks of the injection of exogenous growth hormone, the expression level of the SS gene in the brain of both sexes first increased and then decreased, showing a parabolic trend, and the expression level of the experimental group was lower than that of the control group. After the injection of growth hormone (GH), the expression of the GHR gene in the liver of both sexes showed a significant increase in the first week, decreasing to the control group level in the second week, and then gradually increasing. Finally, a significant level of difference in the expression of the GHR gene was reached at 3 and 4 weeks. In terms of the IGF-I gene, the changing trend of the expression level in the liver was the same as that of the GHR gene. After the injection of exogenous growth hormone, although the expression of the SS gene increased the inhibition of the secretion of the GHR gene by the Reeves' turtle, exogenous growth hormone could replace the synthesis of GH and GHR, accelerating the growth of the turtle. The experiments showed that the injection of recombinant human growth hormone affects the expression of SS, GHR, and IGF-1 genes, and promotes the growth of the Reeves' turtle.

Biofilm formation and development during the pyrite bioleaching of moderately thermophilic microorganisms

Bioleaching is a process that uses microorganisms to recover metals from intractable metal sulfide ores. Metal sulfide bioleaching is an interfacial process, and biofilm growth is crucial during the early phases. To study the interactions between microbes and minerals during the bioleaching of moderately thermophilic microorganisms, the formation and development of biofilms on pyrite coupons incubated in shake flasks for 40 days were evaluated. The biofilm biomass progressively increased as bioleaching proceeded. The most extensive biofilm was formed on day 30, when bacteria were attached to the mineral surface or submerged in extracellular polymeric substances. Confocal laser scanning microscopy indicated that the biofilm biomass of pyrite on day 40 was 6.55, 5.28, 3.17, and 1.70 times higher than that on days 5, 10, 20, and 30, respectively. Extracellular protein content exhibited an increasing trend throughout the process, whereas extracellular polysaccharides played a significant role during the early stages. Live/dead staining showed the highest mortality rate of 94% when cells were in contact with pyrite in the early stage, and the percentage of live bacteria increased in the later period. In the bioleaching environment, mature biofilms appeared to form relatively quickly after day 20. Real-time quantitative fluorescent quantitative analysis indicated that biofilm formation played an important role in defense against hostile external environments. In this study, biofilm formation and the associated RNA transcript numbers of moderately thermophilic microorganisms during pyrite bioleaching were investigated, contributing to the understanding of cell-mineral interactions.

Actin-Depolymerizing Factor Gene Family Analysis Revealed That CsADF4 Increased the Sensitivity of Sweet Orange to Bacterial Pathogens.

The actin-depolymerizing factor (ADF) gene family regulates changes in actin. However, the entire ADF family in the sweet orange Citrus sinensis has not been systematically identified, and their expressions in different organs and biotic stress have not been determined. In this study, through phylogenetic analysis of the sweet orange ADF gene family, seven CsADFs were found to be highly conserved and sparsely distributed across the four chromosomes. Analysis of the cis-regulatory elements in the promoter region showed that the CsADF gene had the potential to impact the development of sweet oranges under biotic or abiotic stress. Quantitative fluorescence analysis was then performed. Seven CsADFs were differentially expressed against the invasion of Xcc and CLas pathogens. It is worth noting that the expression of CsADF4 was significantly up-regulated at 4 days post-infection. Subcellular localization results showed that CsADF4 was localized in both the nucleus and the cytoplasm. Overexpression of CsADF4 enhanced the sensitivity of sweet orange leaves to Xcc. These results suggest that CsADFs may regulate the interaction of C. sinensis and bacterial pathogens, providing a way to further explore the function and mechanisms of ADF in the sweet orange.

Overexpression of Bruton Tyrosine Kinase Inhibits the Proliferation, Migration, and Invasion of Non-Small Cell Lung Cancer Cells.

Lung cancer is one of the most lethal malignant tumors in the world. Non-small cell lung cancer (NSCLC) is the most common pathological subtype. However, the molecular mechanism of NSCLC progress is still unclear. We extracted the expression data of the Bruton's tyrosine kinase (BTK) gene in NSCLC tissues from the TCGA database. The results of paired t-test showed that the BTK gene was significantly underexpressed in NSCLC tissues. To further verify the above results, we detected the expression of the BTK gene in NSCLC cell lines A549, H1299, and H1650 at the RNA and protein levels by real-time fluorescent quantitative polymerase chain reaction and Western Blot analysis, respectively. The results showed that BTK was low expressed in NSCLC tissues and cells. More importantly, the expression of the BTK gene is also significantly related to the patient's age, gender, tumor range (T), lymph node invasion (N), tumor stage, and prognosis, and its expression level gradually decreases with the progress of the disease. It is speculated that BTK may be an independent prognostic factor of NSCLC. Our experimental results are consistent with the above clinical correlation analysis results. Overexpression of BTK can significantly inhibit the proliferation, migration, and invasion of NSCLC cells and can block the G0/G1 tumor cell cycle, indicating that overexpression of BTK can inhibit the growth, migration, and invasion of NSCLC cells.

Identification of CDK gene family and functional analysis of CqCDK15 under drought and salt stress in quinoa

as one of the oldest cultivated crops in the world, quinoa has been widely valued for its rich nutritional value and green health. In this study, 22 CDK genes (CqCDK01-CqCDK22) were identified from quinoa genome using bioinformatics method. The number of amino acids was 173–811, the molecular weight was 19,554.89 Da-91,375.70 Da, and the isoelectric point was 4.57–9.77. The phylogenetic tree divided 21 CqCDK genes into six subfamilies, the gene structure showed that 12 (54.5%) CqCDK genes (CqCDK03, CqCDK04, CqCDK05, CqCDK06, CqCDK07, CqCDK11, CqCDK14, CqCDK16, CqCDK18, CqCDK19, CqCDK20 and CqCDK21) had UTR regions at 5’ and 3’ ends. Each CDK protein had different motifs (3–9 motifs), but the genes with the same motifs were located in the same branch. Promoter analysis revealed 41 cis-regulatory elements related to plant hormones, abiotic stresses, tissue-specific expression and photoresponse. The results of real-time fluorescence quantitative analysis showed that the expression level of some CDK genes was higher under drought and salt stress, which indicated that CDK genes could help plants to resist adverse environmental effects. Subcellular localization showed that CqCDK15 gene was localized to the nucleus and cytoplasm, and transgenic plants overexpressing CqCDK15 gene showed higher drought and salt tolerance compared to the controls. Therefore, CDK genes are closely related to quinoa stress resistance. In this study, the main functions of quinoa CDK gene family and its expression level in different tissues and organs were analyzed in detail, which provided some theoretical support for quinoa stress-resistant breeding. Meanwhile, this study has important implications for further understanding the function of the CDK gene family in quinoa and our understanding of the CDK family in vascular plant.

Fluorescent/Phosphorescent Dual-Emissive Probe for Simultaneous and Independent Sensing of Intracellular Hypochlorite and pH via Time-Resolved Luminescence Imaging

In this work, we present a dual-emissive polymeric probe capable of simultaneous and independent sensing of intracellular hypochlorite and pH. The probe integrates an anthracene–carboxamide-based fluorescent compound with a phosphorescent iridium(III) complex. The fluorescence and phosphorescence spectra are significantly overlapped at 500 to 650 nm, but their lifetimes differ substantially at 14.8 and 1113.6 ns, respectively, thereby allowing for monitoring in different time intervals through time-resolved luminescence analysis. The fluorescence is quenched upon oxidation by hypochlorite, whereas an acidic environment favors protonation of pyridyl groups on the iridium(III) complex, which causes phosphorescence quenching. During hypochlorite sensing, a linear plot of the fluorescence/phosphorescence ratio over hypochlorite concentration was obtained. During pH sensing, the pKa value was calculated to be about 4.6. The utilization of this probe for imaging cellular endogenous hypochlorite and intracellular pH values was demonstrated. The short-lived and long-lived signals were successfully extracted via time-resolved luminescence imaging microscopy, thereby allowing independent, simultaneous, and quantitative analysis of fluorescence and phosphorescence signals and their response toward hypochlorite and pH values. This study offers potential implications for the development of biosensing and bioimaging applications and paves the way for the simultaneous multiplexed detection of physiological and pathological activities within living cells.