Single-cell transcriptome analyses reveal critical regulators of spermatogonial stem cell fate transitions.

BACKGROUND: Spermatogonial stem cells (SSCs) are the foundation cells for continual spermatogenesis and germline regeneration in mammals. SSC activities reside in the undifferentiated spermatogonial population, and currently, the molecular identities of SSCs and their committed progenitors remain unclear. RESULTS: We performed single-cell transcriptome analysis on isolated undifferentiated spermatogonia from mice to decipher the molecular signatures of SSC fate transitions. Through comprehensive analysis, we delineated the developmental trajectory and identified candidate transcription factors (TFs) involved in the fate transitions of SSCs and their progenitors in distinct states. Specifically, we characterized the Asingle spermatogonial subtype marked by the expression of Eomes. Eomes+ cells contained enriched transplantable SSCs, and more than 90% of the cells remained in the quiescent state. Conditional deletion of Eomes in the germline did not impact steady-state spermatogenesis but enhanced SSC regeneration. Forced expression of Eomes in spermatogenic cells disrupted spermatogenesis mainly by affecting the cell cycle progression of undifferentiated spermatogonia. After injury, Eomes+ cells re-enter the cell cycle and divide to expand the SSC pool. Eomes+ cells consisted of 7 different subsets of cells at single-cell resolution, and genes enriched in glycolysis/gluconeogenesis and the PI3/Akt signaling pathway participated in the SSC regeneration process. CONCLUSIONS: In this study, we explored the molecular characteristics and critical regulators of subpopulations of undifferentiated spermatogonia. The findings of the present study described a quiescent SSC subpopulation, Eomes+ spermatogonia, and provided a dynamic transcriptional map of SSC fate determination.

Selectfluor Mediated Direct C-H Fluorination of 3-Heteroaryl-Oxindoles.

An efficient transition-metal-free fluorination synthesis of N-H-free 3-heteroaryl-oxindoles with Selectfluor was depicted. Under mild reaction conditions, a series of 3-heteroaryl-fluorooxindoles were produced in yield of 62-88% using Selectfluor as a fluorine source.

Single-cell analysis identifies critical regulators of spermatogonial development and differentiation in cattle-yak bulls.

Spermatogenesis is a continuous process in which functional sperm are produced through a series of mitotic and meiotic divisions and morphological changes in germ cells. The aberrant development and fate transitions of spermatogenic cells cause hybrid sterility in mammals. Cattle-yak, a hybrid animal between taurine cattle (Bos taurus) and yak (Bos grunniens), exhibits male-specific sterility due to spermatogenic failure. In the present study, we performed single-cell RNA sequencing analysis to identify differences in testicular cell composition and the developmental trajectory of spermatogenic cells between yak and cattle-yak. The composition and molecular signatures of spermatogonial subtypes were dramatically different between these 2 animals, and the expression of genes associated with stem cell maintenance, cell differentiation and meiotic entry was altered in cattle-yak, indicating the impairment of undifferentiated spermatogonial fate decisions. Cell communication analysis revealed that signaling within different spermatogenic cell subpopulations was weakened, and progenitor spermatogonia were unable or delayed receiving and sending signals for transformation to the next stage in cattle-yak. Simultaneously, the communication between niche cells and germ cells was also abnormal. Collectively, we obtained the expression profiles of transcriptome signatures of different germ cells and testicular somatic cell populations at the single-cell level and identified critical regulators of spermatogonial differentiation and meiosis in yak and sterile cattle-yak. The findings of this study shed light on the genetic mechanisms that lead to hybrid sterility and speciation in bovid species.

First Report of Cytospora ailanthicola Causing Canker on cherry blossom (Cerasus serrulata) in China.

Cherry blossom (Cerasus serrulata) is a plant with important garden applications. It is a newly introduced exotic plants in the Arar region of Xinjiang, China (40°41'18.19″N,81°43'50.55″E). In October 2022, it was discovered that about 30% of cherry blossoms had a canker disease. The leaves of the sick branches were dired, the branches themselves were damaged, with dark brown color inside. Orange-yellow conidia horns were produced in humid condition. Samples were collected from fifteen trees exhibiting notable symptoms. The diseased junctions of the infected shoots were chopped into small pieces and subjected to surface sterilization by using 70% ethanol for 30s, 1% NaClO solution for one minute, and sterile distilled water three times (Chen et al. 2016). The representative strain YINGHUA-1 was chosen for identification by molecular biology and morphology. After five days of incubation at 26℃ on PDA media, colonies of white fluffy mycelium were produced from the YINGHUA-1 strain. After 25 days of PDA culture, the production of pycnidia was first observed, circular, black. The pycnidia began to produce conidia at 30 days. The conidia was translucent without septum, with a slightly curved single cell and smooth surface. Pycnidia was spherical and flat, with a single black aperture at the top that resembles a nearly round hole, the chamber was made up of several tiny chambers separated by a shared wall, and its diameter ranges from 900-1900 µm. The size of the conidium was 3.7-6.6×1.1-1.9 µm (n=20). The intrinsic transcriptional spacer (ITS), transcriptional elongation factor (tef-1α), and ß-tubulin (tub2) gene moieties of rDNA were sequenced using ITS1/ITS4, EF1-728F/EF1-986R, and Bt2a/Bt2b primers, respectively(Zhang et al. 2014). The amplified sequences of ITS region (Accession No. OR855907), tub2 (Accession No. OR865863) and tef-1α (Accession No. OR865864) were deposited in the GenBank. BLAST searches of the sequences revealed 99.59% identity (474/476 bp) of the ITS sequence, 98.63% identity (216/219 bp) of the tef-1α sequence, and 98.55% identity of the tub2 sequence (339/344 bp) with C. ailanthicola CFCC59446 (accessions OR826163, OR832040, and OR832062, respectively.) Phylogenetic analyses were performed with Iqtree v.1.6.12 for maximum likelihood (ML). Confidence levels for the nodes were determined using 1000 replicates of bootstrapping methods. Based on phylogenetic analysis and morphological characteristics, the pathogen was identified as C. ailanthicola. The pathogenicity of C. ailanthicola was confirmed by inoculation of 1-year-old shoots (5 replicates of this experiment). After 7 days, symptoms of inner bark discoloration were visible on xylem of branches and the same fungus was re-isolated from the inoculated shoots, with no lesions on the control shoots. C. ailanthicola is only known from a single host plant, Ailanthus altissima,in China (Fan et al.2020). As far as we know, this is the first report of C. ailanthicola harmings C. serrulata in China.

Progress on the role of LIN28A/B in tumor development and progression.

LIN28A and its homolog LIN28B are highly conserved RNA-binding proteins that play important roles in early embryonic development, somatic cell reprogramming, metabolism and tumorigenesis. LIN28A/B are highly expressed in a variety of malignant tumors such as breast cancer. They play important roles in the initiation, maintenance, and metastasis of tumors and are associated with poor prognosis. Previous studies have shown that the main regulatory mechanisms of LIN28A/B include let-7s dependent ways and let-7s independent ways, such as directly targeting mRNA. In this review, we summarize the function and molecular regulatory mechanisms of LIN28A/B in malignant tumors such as liver cancer, breast cancer and colorectal cancer, in order to provide references for further exploring the function and mechanism of LIN28A/B and their possible roles in clinical applications.

Comparative proteomic analysis identifies differentially expressed proteins associated with meiotic arrest in cattle-yak hybrids.

Cattle-yak, the interspecific hybrid between yak and taurine cattle, exhibits male-specific sterility. Massive loss of spermatogenic cells, especially spermatocytes, results in azoospermia in these animals. Currently, the mechanisms underlying meiosis block and defects in spermatocyte development remain elusive. The present study was designed to investigate the differences in the protein composition of spermatocytes isolated from 12-month-old yak and cattle-yak testes. Histological analysis confirmed that spermatocytes were the most advanced germ cells in the testes of yak and cattle-yak at this developmental stage. Comparative proteomic analysis identified a total of 452 differentially abundant proteins (DAPs) in the fluorescence-activated cell sorting (FACS) isolated spermatocytes from cattle-yak and yak. A total of 291 proteins were only present in yak spermatocytes. Gene Ontology analysis revealed that the downregulated DAPs were mostly enriched in the cellular response to DNA damage stimulus and double-strand breaks (DSBs) repair via break-induced replication, while the proteins specific for yak were related to cell division and cycle, spermatogenesis, and negative regulation of the extrinsic apoptotic signaling pathway. Ultimately, these DAPs were related to the critical process for spermatocyte meiotic events, including DSBs, homologous recombination, synapsis, crossover formation, and germ cell apoptosis. The database composed of proteins associated with spermatogenesis, including KPNA2, HTATSF1, TRIP12, STIP1, LZTFL1, LARP7, MTCH2, STK31, ROMO1, CDK5AP2, DNMT1, RBM44, and CHRAC1, is the focus of further research on male hybrid sterility. In total, these results provide insight into the molecular mechanisms underlying failed meiotic processes and male infertility in cattle-yak.

First report of Diplodia mutila causing canker and branch dieback of walnut trees in Xinjiang, China.

Walnut (Juglans regia L.) has become an important economic fruit tree in China. In April 2022, branch dieback was observed in 15-year-old walnut trees (cv Wen 185) in a commercial orchard in Aksu, Xinjiang, China (40°21'55''N, 80°1'48''E), with an incidence of 2% (4 out of 200 trees) of affected trees. The symptoms observed, included depressed and shrunken cankers, twigs and branches dieback. Cross-sections of diseased branches revealed dark-brown wedge-shaped lesions. To isolate the potential causal pathogen, four specimens were isolated from diseased branches, and small pieces taken from the edge of canker samples (0.5 × 0.5 cm), were disinfected by immersion in 75% ethanol for 30 s and 2% NaClO solution for 3 min, and rinsed three times with sterile water. The disinfected wood samples were then placed on potato dextrose agar (PDA) and incubated in the dark at 25°C for 2-3 days. Then, we applied the mycelial tip purification method and repeated this purification process until a single colony was formed. Four pure isolates (3-1-51A, W-2-54, K-1-43A, K-1-43B) developed white to white-gray fast-growing colonies with abundant aerial mycelium after seven days at 25°C on PDA and gradually became dark olive green over subsequent growth stages. Conidia production was then induced on 2% w/v water agar containing sterilized pine needles under near-U/V light (Alves et al., 2004). The conidia were initially hyaline, thick-walled, oblong to ovoid with one septum and a size range of 19.47-24.16 × 9.78-13.51 µm (n = 40). Based on morphological characteristics these isolates were tentatively identified as Diplodia mutila (Fr.) Mont. (Alves et al., 2004).To confirm the pathogen identified, the representative isolate 3-1-51A was amplified and sequenced using specific primer pairs (ITS1/ITS4, EF1-986/EF1-728E, BT2a/BT2b) to the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and beta-tubulin (TUB) (White et al. 1990; Carbone and Kohn 1999; O' Donnell and Cigelnik 1997), respectively. The sequences showed 100% similarity to two D. mutila strains CBS230.30 and CBS112553. Maximum likelihood analysis was performed based on a concatenated dataset (ITS + TEF1-α + TUB) gene using MEGA 11. 0 and isolate 3-1-51A formed a single clade with the reference ex-type of D. mutila. The isolate 3-1-51A was deposited into GenBank as OP006733, OP373140, and OP373139 for ITS, TEF1-α, and TUB, respectively. To fulfill Koch's postulates, pathogenicity tests were performed using isolate 3-1-51A on one-year-old healthy walnut branches cv. Wen 185 of walnut trees (n=5). Five twigs of healthy walnut branches were cleaned, submerged in 1% NaClO for 15 minutes and then dried. Then, a sterile hole punch (5mm in diameter) was used to create a wound in the middle of each walnut branch, and placing mycelial plugs(3 days old; 5 mm in diameter) and sealed with parafilm. An equal number of twigs inoculated with sterile agar plugs served as controls. On the 7th day after inoculation, dark brown coloration was developed on the branches with symptoms of shrinkage dryness, and dieback. D. mutila isolate was re-isolated only from the inoculated branches. In negative control twigs, lesions and re-isolated were absent, thus fulfilling Koch's postulates. D. mutila has been previously reported causing canker and branch dieback in walnut trees in Chile (Díaz et al. 2018) and California (Chen et al. 2014). Previously, D. seriata (Zhang et al. 2017), Botryosphaeria dothidea (Guo et al. 2016), Lasiodiplodia pseudotheobromae (Guo et al. 2016), Dothiorella gregaria (Liu et al. 1986) and Neofusicoccum parvum (Yu et al. 2015) have been identified on walnut trees in China. To our knowledge, this is the first report of canker and branch dieback caused by D. mutila in walnut trees in Xinjiang, China. Further studies are now required to better understand the etiology of canker and branch dieback on walnut trees from different areas in China.

Promotion Effect of Coexposure to a High-Fat Diet and Nano-Diethylnitrosamine on the Progression of Fatty Liver Malignant Transformation into Liver Cancer.

Overconsumption of high-fat foods increases the risk of fatty liver disease (FLD) and liver cancer with long pathogenic cycles. It is also known that the intake of the chemical poison nitrosamine and its nanopreparations can promote the development of liver injuries, such as FLD, and hepatic fibrosis, and significantly shorten the formation time of the liver cancer cycle. The present work confirmed that the coexposure of a high-fat diet (HFD) and nano-diethylnitrosamine (nano-DEN) altered the tumor microenvironment and studied the effect of this coexposure on the progression of fatty liver malignant transformation into liver cancer. Gene transcriptomics and immunostaining were used to evaluate the tumor promotion effect of the coexposure in mice. After coexposure treatment, tumor nodules were obviously increased, and inflammation levels were elevated. The liver transcriptomics analysis showed that the expression levels of inflammatory, fatty, and fibrosis-related factors in the coexposed group were increased in comparison with the nano-DEN- and high-fat-alone groups. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that coexposure aggravated the high expression of genes related to the carcinomatous pathway and accelerated the formation of the tumor microenvironment. The immunohistochemical staining results showed that the coexposure significantly increased the abnormal changes in proteins related to inflammation, proliferation, aging, and hypoxia in mouse liver tissues. The coexposure of high fat and nano-DEN aggravated the process of steatosis and carcinogenesis. In conclusion, the habitual consumption of pickled foods containing nitrosamines in a daily HFD significantly increases the risk of liver pathology lesions progressing from FLD to liver cancer.

A comparative study on the efficacy and safety of propofol combined with different doses of alfentanil in gastroscopy: a randomized controlled trial.

PURPOSE: Propofol can be used alone or in combination with opioids during gastroscopy. This study aimed to assess the efficacy and safety of intravenous propofol and different doses of alfentanil in patients undergoing gastroscopy. METHODS: A total of 300 patients undergoing sedative gastroscopy were randomly divided into four groups, and 0.9% saline (group A), 2 µg/kg alfentanil (group B), 3 µg/kg alfentanil (group C) or 4 µg/kg alfentanil (group D) were injected intravenously 1 min before the intravenous injection of 1.5 mg/kg propofol. If body movement and coughing occurred during the procedure, 0.5 mg/kg propofol would be administered intravenously. The primary outcome (awakening time) and secondary outcomes were recorded and analyzed, including hemodynamic changes, the incidences of body movement, coughing, hypoxemia, hypotension, hypertension, bradycardia, tachycardia, nausea and vomiting, drowsiness and dizziness. RESULTS: Patients in group C (7.0 [5.0 to 8.0] min) and group D (6.0 [5.0 to 7.0] min) woke up significantly earlier than those in group A (8.0 [6.0 to 10.0] min) (P < 0.001). Patients in group A experienced more body movement (P = 0.001) and coughing (P < 0.001) than the other groups. With the increasing dose of alfentanil, the morbidity of hypotension and bradycardia increased significantly (P = 0.001), while the incidence of dizziness decreased significantly (P = 0.037). The incidences of hypoxemia, tachycardia, drowsiness, nausea and vomiting were similar among the four groups (P > 0.05). CONCLUSIONS: Intravenous 1.5 mg/kg propofol combined with 3 µg/kg alfentanil is more suitable for patients undergoing gastroscopy, and the dose of alfentanil can be reduced according to the patient's actual physical condition.

The effect of music intervention on sedation in elderly patients undergoing transurethral resection of prostate under spinal anesthesia: a prospective randomized controlled clinical trial.

PURPOSE: Music intervention is commonly used as a non-pharmacologic therapeutic modality to alleviate anxiety in perioperative patients. This study aimed to assess the sedative and anxiolytic effects of music on elderly patients receiving transurethral resection of prostate (TURP) under spinal anesthesia. METHODS: This was a prospective randomized controlled trial on patients who aged over 60 and received TURP under spinal anesthesia. Participants were randomized to the music group or the control group (no music). The primary outcome was perioperative BIS values, and the secondary outcomes were patient's perioperative anxiety levels, heart rate (HR), blood pressure, and patient satisfaction score. RESULTS: A total of 82 patients were analyzed. The perioperative BIS values in the music group were significantly lower than those of the control group at almost all time points (P < 0.001), as well as showed a significant reduction compared with baseline (P < 0.001), whereas the control group did not. In comparison with the control group, systolic blood pressure (SBP) significantly decreased in the music group at the beginning (mean difference, - 8.0 mmHg; 95% CI - 15.70 to 0.35; P = 0.041) and the 60th minute (mean difference, - 7.9 mmHg; 95% CI - 15.30 to 0.51; P = 0.037) of TURP. Furthermore, compared with baseline within the music group, diastolic blood pressure (DBP) and HR significant reduced at whole time points (P < 0.05), yet the control group not. CONCLUSION: Music intervention effectively provided slight sedation for elderly patients when undergoing TURP under spinal anesthesia without sedatives.

An Iron(III)-Based Metal-Organic Gel-Catalyzed Dual Electrochemiluminescence System for Cytosensing and In Situ Evaluation of the VEGF165 Subtype.

The recent surge of interest in metal-organic gels (MOGs) has emerged for their soft porous structure, large surface area, and abundant active metal sites, making them a promising candidate for building catalyst matrices. In this work, facilely synthesized Fe(III)-organic gel was directly used as a robust electrode matrix. Detailed studies illustrated that their Fe(III) centers can speed up the electro-oxidation/reduction of the H2O2 coreactant to produce reactive oxygen species for enhancing a potential-resolved dual electrochemiluminescence (ECL) emission. Among them, the anodic signal of luminol varied with the cell concentration based on the impedance ECL mechanism, while the cathodic signal of CdS quantum dots traced the VEGF165 subtype at cell surface by specific aptamer recognition. Based on this, a ratiometric strategy was proposed for accurate cytosensing by eliminating environmental interference. Moreover, by cooperating these two signals, a novel strategy was developed for direct evaluation of the VEGF165 subtype, further realizing rapid drug screening and subtype assessment on different cell lines. This work not only opens up the promising application of MOGs as an effective catalyst matrix but also develops reliable cell assays and protein subtype identification for clinical diagnosis and research.

Chiral-at-Cage Carboranes for Circularly Polarized Luminescence and Aggregation-Induced Electrochemiluminescence.

Herein, we report the structures of chiral-at-cage carborane derivatives bearing carbazole chromophores that emit circularly polarized luminescence (CPL) and aggregation-induced electrochemiluminescence (AIECL). By adjusting the substituent positions on the carborane derivatives, two chiral luminescent molecules, Cb1 and Cb2, with different properties were obtained. The photoluminescence dissymmetry factors |gPL | of both (R/S)-Cb1 and (R/S)-Cb2 enantiomers in neat films were as high as 6.24×10-3 and 7.38×10-3 , respectively. Cb1 showed a deep blue emission peak at 434 nm in n-pentane. Interestingly, distinct fluorescence and CPL spectra were observed in solvents of different polarities due to the twisted intramolecular charge transfer effect, suggesting its potential use in solvent recognition. Meanwhile, Cb2 exhibited good AIECL property, excellent ECL stability and could be used for determining dopamine concentrations, suggesting its potential applications in biology and diagnosis.

The circular RNA circSlc7a11 promotes bone cancer pain pathogenesis in rats by modulating LLC-WRC 256 cell proliferation and apoptosis.

Treatment of bone cancer pain (BCP) caused by bone metastasis in advanced cancers remains a challenge in clinical oncology, and the underlying mechanisms of BCP are poorly understood. This study aimed to investigate the pathogenic roles of circular RNAs (circRNAs) in regulating cancer cell proliferation and BCP development. Eight differentially expressed circRNAs in the rat spinal cord were validated by agarose gel electrophoresis and Sanger sequencing. Expression of circRNAs and mRNAs was detected by quantitative RT-PCR. MTS assay and flow cytometry were performed to analyze cell proliferation and apoptosis, respectively. Differentially expressed mRNA profiles were characterized by deep RNA sequencing, hierarchical clustering, and functional categorization. The interactions among circRNAs, microRNAs (miRNAs), and mRNAs were predicted using TargetScan. Additionally, western blot was performed to determine the protein levels of Pax8, Isg15, and Cxcl10. Multiple circRNAs were differentially expressed in the spinal cords of BCP model rats; of these, circSlc7a11 showed the greatest increase in expression. The overexpression of circSlc7a11 significantly promoted cell proliferation and repressed apoptosis of LLC-WRC 256 and UMR-106 cells, whereas circSlc7a11 silencing produced the opposite effects. Altered expression of circSlc7a11 also induced substantial changes in the mRNA expression profiles of LLC-WRC 256 cells; these changes were linked to multiple apoptotic processes and signaling pathways, such as the chemokine signaling pathway, and formed a complex circRNA/miRNA/mRNA network. Additionally, Pax8, Isg15, and Cxc110 protein level in LLC-WRC 256 cells was consistent with the mRNA results. The circRNA circSlc7a11 regulates rat BCP development by modulating LLC-WRC 256 cell proliferation and apoptosis through multiple-signaling mechanisms.

[Dysregulation of MAD2L1/CAMK2A/PTTG1 Gene Cluster Maintains the Stemness Characteristics of Uterine Corpus Endometrial Carcinoma].

Objective To study the stemness characteristics of uterine corpus endometrial carcinoma(UCEC)and its potential regulatory mechanism.Methods Transcriptome sequencing data of UCEC was obtained from The Cancer Genome Atlas.Gene expression profile was normalized by edgeR package in R3.5.1.A one-class logistic regression machine learning algorithm was employed to calculated the mRNA stemness index(mRNAsi)of each UCEC sample.Then,the prognostic significance of mRNAsi and candidate genes was evaluated by survminer and survival packages.The high-frequency sub-pathways mining approach(HiFreSP)was used to identify the prognosis-related sub-pathways enriched with differentially expressed genes(DEGs).Subsequently,a gene co-expression network was constructed using WGCNA package,and the key gene modules were analyzed.The clusterProfiler package was adopted to the function annotation of the modules highly correlated with mRNAsi.Finally,the Human Protein Atlas(HPA)was retrieved for immunohistochemical validation.Results The mRNAsi of UCEC samples was significantly higher than that of normal tissues(t=25.095,P<0.001),and the lower degree of differentiation corresponded to higher mRNAsi in tumor tissues.The mRNAsi of UCEC increased gradually with tumor staging.The prognostic analysis showed that high mRNAsi was correlated with short overall survival in patients with UCEC(χ2=6.864,P=0.0088).There were 570 DEGs between the high-and low-mRNAsi groups.By using the HiFreSP algorithm,we identified that the oocyte meiosis sub-pathway(Oocyte meiosis_1)and cell cycle sub-pathway(Cell cycle_3)had significant prognostic significance.These pathways contained 11 DEGs(MAD2L1,CAMK2A,PTTG1,PLK1,CCNE1,CCNE2,ESPL1,CDC20,CCNB1,CCNB2,and SMC1B),which were significantly associated with the prognosis of UCEC patients.Gene co-expression network showed that mRNAsi,as well as MAD2L1,CAMK2A,and PTTG1,was associated with three gene modules.The immunohistochemical analysis demonstrated that MAD2L1 and PTTG1 showed up-regulated expression while CAMK2A showed down-regulated expression in UCEC,which was consistent with the results of transcriptome sequencing.Conclusions On the basis of machine learning,this study characterizes the stemness characteristics of UCEC.We identify the key sub-pathways related to prognosis and demonstrate that MAD2L1,CAMK2A,PTTG1 are closely related to the stemness of UCEC,which provides insight into the regulatory mechanism of cancer stemness and reveals the potential therapeutic targets of UCEC.

A Novel Putative Role of TNK1 in Atherosclerotic Inflammation Implicating the Tyk2/STAT1 Pathway.

OBJECTIVE: Atherosclerosis is a chronic inflammatory disease which is responsible for many clinical manifestations. The present study was to investigate the anti-inflammatory functions and mechanisms of TNK1 in atherosclerosis. METHODS: The ApoE(-/-) mice and human carotid endarterectomy (CEA) atherosclerotic plaques were used to investigate the differential expression of TNK1. The ApoE(-/-) mice were fed with high-fat diet (HFD) or normal-fat diet (NFD) for 8 weeks; the aorta was separated and stained with oil red O to evaluate the formation of atherosclerosis. TNK1 in mice aorta was measured by qPCR. The human CEA were obtained and identified as ruptured and stable plaques. The level of TNK1 was measured by qPCR and Western-blot staining. Further studies were conducted in THP-1 cells to explore the anti-inflammatory effects of TNK1. We induced the formation of macrophages by incubating THP-1 cells with PMA (phorbol 12-myristate 13-acetate). Afterwards, oxidized low-density lipoprotein (oxLDL) was used to stimulate the inflammation, and the secretion of inflammatory factors was measured by ELISA and qPCR. The levels of TNK1, total STAT1 and Tyk2, and the phosphorylation of STAT1 and Tyk2 were measured by western blot to uncover the mechanisms of TNK1. RESULTS: The oil red O staining indicated obvious deposition of lipid on the aorta of ApoE(-/-) mice after 8-week HFD treatment. The TNK1 level was much higher in both the HFD-fed ApoE(-/-) mice aorta arch and the ruptured human CEA plaques. We found that TNK1 was highly expressed in THP-1 cells, compared to other atherosclerotic related cells (HUVEC, HBMEC, and HA-VSMC), indicating TNK1 might be involved in the inflammation. Suppressing the expression of TNK1 by shTNK1 inhibited the oxLDL-induced secretion of inflammatory factors, such as IL-12, IL-6, and TNF-α. ShTNK1 also inhibited the uptake of lipid and decreased the cellular cholesterol content in THP-1 cells. Furthermore, the shTNK1 suppressed the oxLDL-induced phosphorylation of Tyk2 and STAT1. CONCLUSION: TNK1 participated in the inflammation in atherosclerosis. shTNK1 suppressed the oxLDL-induced inflammation and lipid deposition in THP-1 cells. The mechanism might be related to the Tyk2/STAT signal pathway.

An electrochemiluminescence biosensor for p53 antibody based on Zn-MOF/GO nanocomposite and Ag+-DNA amplification.

An ultrasensitive electrochemiluminescence biosensor was established based on the Zn-MOF/GO nanocomposite. Ag(I)-embedded DNA complexes were used as a signal amplification reagent. In this work, 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (TCPP) and Zn2+ were integrated into a porphyrin paddlewheel framework (Zn-MOF) by a hydrothermal method. The synthesized Zn-MOF material has electrochemiluminescence property, and the luminescence intensity is improved after being composited with graphene oxide (GO). Based on the composite material, we constructed an ultrasensitive ECL biosensor for the p53 antibody detection. The composite material acted as an admirable substrate and then loaded plenty of p53 antigens to recognize the target (p53 antibody) accurately. Because of the bridging effect of streptavidin and biotin-conjugated goat anti-rabbit IgG (bio-ab2), the rich-C DNA with positive correlation with the target was modified on the electrode and then captured the co-reactant accelerator Ag+ to amplify the signal. Therefore, the ECL biosensor response increases with increasing p53 antibody concentration. In the range 0.1 fg/mL-0.01 ng/mL, the response signal of the biosensor has a good linear relationship with the p53 antibody concentration. The detection limit is 0.03 fg/mL (S/N = 3). Impressively, the biosensor not only featured high sensitivity, good stability, and excellent specificity for the detection of p53 antibody, but also provides a new way for early detection of cancer. Graphical abstract Schematic representation of the electrochemiluminescence sensor based on a Zn-MOF/GO nanocomposite, which can be applied to the determination of p53 antibody.

The early diagnosis of Parkinson's disease through combined biomarkers.

OBJECTIVE: This study primarily aims to explore the value of combining the measurement of plasma α-synuclein oligomer levels with enhanced T2 star-weighted angiography (ESWAN) in the early diagnosis of Parkinson's disease. METHODS: Sixty patients with early Parkinson's disease and 30 normal adults, with similar ages and genders, were enrolled in the study. Their levels of plasma α-synuclein oligomers were measured, and ESWAN was performed. The amplitudes, phases and R2* values of the head, body and tail of the ipsilateral and contralateral substantia nigra pars compacta (SNc) were measured, at the side of the limb with severe symptoms or early symptoms. The receiver operating characteristic (ROC) curve was used to explore the value of these indexes in the early diagnosis of Parkinson's disease. RESULTS: The plasma level of α-synuclein oligomer was significantly higher in the experimental group than in the control group (P < 0.05). The amplitude values of the head and tail of contralateral SNcs were significantly lower in the experimental group than in the control group (P < 0.05). In the single-index assessment, the serum α-synuclein oligomer had the highest specificity (70%), while the sensitivity of the amplitude of the head and tail of the contralateral SNc was 75% and 80%, respectively. The area under the curve, for the combination of these three indicators, was 0.827, diagnostic efficiency was particularly high, and sensitivity and specificity both reached 80%. CONCLUSION: The combined detection of plasma α-synuclein oligomer and amplitude of the head and tail of the SNc has high diagnostic specificity and sensitivity.

Asiatic acid enhances intratumor delivery and the antitumor effect of pegylated liposomal doxorubicin by reducing tumor-stroma collagen.

Tumor-targeted drug delivery systems (Tt-DDSs) are proposed as a promising strategy for cancer care. However, the dense collagen network in tumors stroma significantly reduces the penetration and efficacy of Tt-DDS. In order to investigate the effect of asiatic acid (AA) on antitumor effect of pegylated liposomal doxorubicin (PLD) by attenuating stroma-collagen, colon cancer xenograft mice (SW620 cell line) were treated by PLD, AA, or combined regimes, respectively; the collagen levels were estimated by Sirius red/fast green dual staining and immunohistochemistry (IHC) staining; the intratumor exposure of doxorubicin was visualized by ex vivo fluorescence imaging and quantified by HPLC/MS analysis. In addition, the impact of AA on collagen synthesis of fibroblast cell (HFL-1) and cytotoxic effect of PLD and doxorubicin to cancer cell (SW620) were studied in vitro. In the presence of AA (4 mg/kg), the intratumor collagen level was restricted in vivo (reduced by 22%, from 4.14% ± 0.30% to 3.24% ± 0.25%, P = 0.051) and in vitro. Subsequently, doxorubicin level was increased by ~30%. The antitumor activity of PLD was significantly improved (57.3% inhibition of tumor growth and 44% reduction in tumor weight) by AA combination. Additionally, no significant improvement in cytotoxic effect of PLD or doxorubicin induced by AA was observed. In conclusion, AA is a promising sensitizer for tumor treatment by enhancing intratumor drug exposure via stromal remodeling.

Metabolism of SKLB-TB1001, a Potent Antituberculosis Agent, in Animals.

Tuberculosis is a major global health problem, and the emergence of multidrug-resistant and extensively drug-resistant strains has increased the difficulty of treating this disease. Among the novel antituberculosis drugs in the pipeline, decaprenylphosphoryl-beta-d-ribose-2-epimerase (DprE1) inhibitors such as BTZ043 and pBTZ169 exhibited extraordinary antituberculosis potency. Here, the metabolites of the new DprE1 inhibitor SKLB-TB1001 in vivo and its inhibition of cytochrome P450 isoforms and plasma protein binding (PPB) in vitro were studied. The results showed that rapid transformation and high PPB resulted in inadequate exposure in vivo and thus led to the moderate potency of SKLB-TB1001 in vivo This study provided explanations for the discrepant potency of this scaffold in vivo and in vitro Meanwhile, it also provides a rationale for lead optimization of this very promising scaffold of antituberculosis agents to prevent them from being metabolized, thus improving their exposure in vivo.

MicroRNA-365 alleviates morphine analgesic tolerance via the inactivation of the ERK/CREB signaling pathway by negatively targeting ß-arrestin2.

BACKGROUND: Morphine is widely used in clinical practice for a class of analgesic drugs, long-term use of morphine will cause the action of tolerance. MicroRNAs have been reported to be involved in morphine analgesic tolerance.. METHODS: Forty male SD rats were selected and randomly divided into 5 groups: the control group, morphine tolerance group, miR-365 mimic + morphine (miR-365 mimic) group, miR-365 inhibitor + morphine (miR-365 inhibitor) group and miR-365 negative control (NC) + morphine (miR-365 NC) group. After the administration of morphine at 0 d, 1 d, 3 d, 5 d and 7 d, behavioral testing was performed. A dual luciferase reporter gene assay was performed to confirm the relationship between miR-365 and ß-arrestin2, RT-qPCR was used to detect miR-365, ß-arrestin2, ERK and CREB mRNA expressions, western blotting was used to evaluate the protein expressions of ß-arrestin2, ERK, p-ERK, CREB and p-CREB, ELISA was used to detect the contents of IL-1ß, TNF-α and IL-18, while immunofluorescence staining was used to measure the GFAP expression. Intrathecal injection of mir365 significantly increased the maximal possible analgesic effect (%MPE) in morphine tolerant rats. ß-arrestin2 was the target gene of miR-365. RESULTS: The results obtained showed that when compared with the morphine tolerance group, there was an increase in miR-365 expression and a decrease in the ß-arrestin2, ERK, CREB protein expressions, contents of IL-1ß, TNF-α, IL-18 and GFAP expression in the miR-365 mimic group, while the miR-365 inhibitor group displayed an opposite trend. CONCLUSIONS: The results of this experiment suggest that by targeting ß-arrestin2 to reduce the contents of IL-1ß, TNF-α and IL-18 and by inhibiting the activation of ERK/CREB signaling pathway, miR-365 could lower morphine analgesic tolerance.