Terminal Transferase Research Articles

Use of Ozone Therapy and Thymoquinone in the Prevention of Formaldehyde Toxicity by Inhalation: An Experimental Study.

The study determined the damage caused by formaldehyde (FA) exposure in blood and liver samples using biochemical markers. Histopathological analysis was performed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method and measurement of CD68 cell density. To what extent the antioxidant molecules thymoquinone (TQ) and ozone (O3) reversed the damage caused by FA exposure was investigated, both when used alone and combined. Fifty-six Sprague-Dawley male rats of eight to ten weeks of age were used in the experiment. The rats were divided into eight groups, with seven rats in each group: the untreated control group, the group treated with TQ (10 mg/kg/day), the group treated with O3 (150 μg/kg/day), the group treated with TQ+O3, the group exposed to FA (10 ppm 8 h/day), the group receiving FA+TQ, the group receiving FA+O3, and the group receiving FA+TQ+O3.Serum aspartate transaminase (AST), alanine transaminase (ALT), total antioxidant (TAS, U/mL), and total oxidant (TOS, nmol/mL) levels were analyzed. TAS and TOS levels, CD68 cell density, and apoptotic cells were determined in liver tissues. FA exposure caused an increase in serum AST and ALT levels of (p<0.05) experimental animals, a decrease in TAS levels in serum (p=0.03) and liver (p>0.05) and an increase in TOS levels (p>0.05), TUNEL positivity (p<0.001), and CD68 cell density (p=0.004). Administration of TQ and O3as antioxidants significantlyreversed biochemical and histopathological alterations in the serum and liver. TQ and ozone therapy suppressed oxidative stress caused by FA exposure and reversed the emerging histopathological deteriorations. Ozone therapy did not suppress the effects of TQ. Therefore, ozone therapy can be given as a supportive therapy along with the main therapeutic agents. We think TQ and ozone therapy may be useful to protect individuals exposed to FA.

Enzymatic Synthesis of TNA Protects DNA Nanostructures

AbstractXeno‐nucleic acids (XNAs) are synthetic genetic polymers with improved biological stabilities and offer powerful molecular tools such as aptamers and catalysts. However, XNA application has been hindered by a very limited repertoire of tool enzymes, particularly those that enable de novo XNA synthesis. Here we report that terminal deoxynucleotide transferase (TdT) catalyzes untemplated threose nucleic acid (TNA) synthesis at the 3’ terminus of DNA oligonucleotide, resulting in DNA‐TNA chimera resistant to exonuclease digestion. Moreover, TdT‐catalyzed TNA extension supports one‐pot batch preparation of biostable chimeric oligonucleotides, which can be used directly as staple strands during self‐assembly of DNA origami nanostructures (DONs). Such TNA‐protected DONs show enhanced biological stability in the presence of exonuclease I, DNase I and fetal bovine serum. This work not only expands the available enzyme toolbox for XNA synthesis and manipulation, but also provides a promising approach to fabricate DONs with improved stability under the physiological condition.

Enzymatic Synthesis of TNA Protects DNA Nanostructures.

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with improved biological stabilities and offer powerful molecular tools such as aptamers and catalysts. However, XNA application has been hindered by a very limited repertoire of tool enzymes, particularly those that enable de novo XNA synthesis. Here we report that terminal deoxynucleotide transferase (TdT) catalyzes untemplated threose nucleic acid (TNA) synthesis at the 3' terminus of DNA oligonucleotide, resulting in DNA-TNA chimera resistant to exonuclease digestion. Moreover, TdT-catalyzed TNA extension supports one-pot batch preparation of biostable chimeric oligonucleotides, which can be used directly as staple strands during self-assembly of DNA origami nanostructures (DONs). Such TNA-protected DONs show enhanced biological stability in the presence of exonuclease I, DNase I and fetal bovine serum. This work not only expands the available enzyme toolbox for XNA synthesis and manipulation, but also provides a promising approach to fabricate DONs with improved stability under the physiological condition.

Exposure to Ozone Downregulates Bcl-2 and Increases Executing Caspases-3 and -8 in the Hippocampus, Frontal Cortex, and Cerebellum of Rats.

Introduction Ozone (O3) is one of the most prevalent atmospheric pollutants, arising from a photochemical reaction between volatile organic compounds (VOC), nitrogen oxides (NOx), and sunlight.O3 triggers oxidative stress, resulting in lipid oxidation, inflammation, alterations in metabolic and cellular signaling, and potentially initiating cell death in vulnerable brain regions.Inflammation and oxidative stress are recognized for their ability to induce cell death, primarily through the apoptosis pathway, involving various proteins that participate in this process via two pathways: intrinsic and extrinsic. Objective This study aims to identify the expression of pro-apoptotic proteins and Bcl-2 in the frontal cortex, cerebellum, and hippocampus of rats exposed to O3 acutely. Methods Two groups of 20 Wistar rodents (250-300 g) were established. The control group (n=10) was exposed to unrestricted polluted air for 12 hours, while the experimental group (n=10) was exposed to 1 ppm of O3. After exposure, the animals were sacrificed for immunofluorescence and Western blot analysis. Using a t-test, the arbitrary units of pro-apoptotic proteins and Bcl-2 were compared between the two groups. Results Significant increases in caspase-8 and caspase-3 activation were found in the O3-exposedgroup compared to the control group, specifically in the frontal cortex, cerebellum, and hippocampus. Additionally, notable changes in Bcl-2 expression were observed in these brain regions. The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay further indicated significant differences in immunopositivity between the groups in the same areas. However, intrinsic apoptotic proteins such as Bax, VDAC1, and cytochrome-c did not show significant differences between the groups within these structures. Western blotanalyses aligned with the immunofluorescence results, showing statistically significant concentrations of caspase-8 in the cerebellum, caspase-3 in the hippocampus, and Bcl-2 in the frontal cortex in the O3exposed group. Conversely, proteins like Bax, cytochrome-c, and VDAC1 did not exhibit significant differences in all analyzed structures. Conclusions This study demonstrates that acute exposure to 1 ppm of ozone can trigger neuronal apoptosis in the frontal cortex, hippocampus, and cerebellum of rats, primarily through the activation of the extrinsic apoptosis pathway via caspase-8 and caspase-3. Additionally, it causes a reduction in Bcl-2 expression, an essential antiapoptotic protein. Despite not observing the activation of intrinsic pathway proteins like BAX, VDAC, or cytochrome-c, the study suggests that chronic O3 exposure might promote cell death by activating this pathway, requiring further long-term research.

Chlorogenic acid attenuates cardiac hypertrophy via up-regulating Sphingosine-1-phosphate receptor1 to inhibit endoplasmic reticulum stress.

Cardiac hypertrophy, an adaptive response of the heart to stress overload, is closely associated with heart failure and sudden cardiac death. This study aimed to investigate the therapeutic effects of chlorogenic acid (CGA) on cardiac hypertrophy and elucidate the underlying mechanisms. To simulate cardiac hypertrophy, myocardial cells were exposed to isoproterenol (ISO, 10μM). A rat model of ISO-induced cardiac hypertrophy was also established. The expression levels of cardiac hypertrophy markers, endoplasmic reticulum stress (ERS) markers, and apoptosis markers were measured using quantitative reverse transcription PCR and western blotting. The apoptosis level, size of myocardial cells, and heart tissue pathological changes were determined by terminal deoxynucleotidyl transferase dUTP nick-end labelling staining, immunofluorescence staining, haematoxylin and eosin staining, and Masson's staining. We found that CGA treatment decreased the size of ISO-treated H9c2 cells. Moreover, CGA inhibited ISO-induced up-regulation of cardiac hypertrophy markers (atrial natriuretic peptide, brain natriuretic peptide, and β-myosin heavy chain), ERS markers (C/EBP homologous protein, glucose regulatory protein 78, and protein kinase R-like endoplasmic reticulum kinase), and apoptosis markers (bax and cleaved caspase-12/9/3) but increased the expression of anti-apoptosis marker bcl-2 in a dose-dependent way (0, 10, 50, and 100μM). Knockdown of sphingosine-1-phosphate receptor 1 (S1pr1) reversed the protective effect of CGA on cardiac hypertrophy, ERS, and apoptosis in vitro (P<0.05). CGA also restored ISO-induced inhibition on the AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signalling in H9c2 cells, while S1pr1 knockdown abolished these CGA-induced effects (P<0.05). CGA (90mg/kg/day, for six consecutive days) protected rats against cardiac hypertrophy in vivo (P<0.05). CGA treatment attenuated ISO-induced ERS and cardiac hypertrophy by activating the AMPK/SIRT1 pathway via modulation of S1pr1.

In silico and in vitro biopesticidal activity of Melia dubia (Cav.) plant leaves extract phytocompounds against selected enzymes, proteins, cell lines and Callosobruchus maculatus (Fab.)

Most synthetic pesticides have negative impacts on both human health and the environment, which has led to a global economic and ecological emergency for insect pest control. Crude extracts of phytocompounds from Melia dubia Cav. were tested for their pesticidal effects on a certain enzyme cell line. An in silico molecular docking analysis of phytocompounds showed that the enzymes Glutathione S-Transferase (7RKA), Mytocontrial (5H3F), Acetylcholinesterase (7QAK), GABA receptor (7WGT), and DNA nucleotidylexotransferase (6GO4) interacted strongly with the phytocompounds. Compared to Dichlorvos and Malathion, the substances Guanosine (1) and Medroxyprogesterone (2) showed higher in-silico biopesticidal efficacy against Callosobruchus maculatus (Fab.). A silica gel column chromatographic technique was used to isolate Compounds 1 and 2. In vitro studies revealed that, after 96 hours, Guanosine (90.4% and LC50 value 5.63 mg/ml) caused the maximum toxicity of C. maculatus compared to the reference pesticides Dichlorvos (84.6% and LC50 value 5.94 mg/ml) and Malathion (92% and LC50 value 5.62 mg/ml). Comparing the results to the reference drugs, guanosine (1) showed potential as a pesticidal agent. Therefore, these particular compounds may provide alternative treatments that enhance the plants under investigation's historical use.

A Novel Postconditioning Approach Attenuates Myocardial Ischaemia-Reperfusion Injury in Rats.

Ischaemia-reperfusion injury (IRI) is the damage that occurs when blood flow is restored to a tissue or organ after a period of ischaemia. Postconditioning is a therapeutic strategy aimed at reducing the tissue damage caused by IRI. Postconditioning in rodents is a useful tool to investigate the potential mechanisms of postconditioning. Currently, there is no convenient approach for postconditioning rodents. Rats were subjected to a balloon postconditioning procedure. A balloon was used to control the flow in the vessel. This allowed for easy and precise manipulation of perfusion. Evans blue and triphenyltetrazolium chloride (TTC) double staining were used to determine the infarct size. Apoptosis in the myocardium was visualised and quantified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Western blotting was performed to assess the expression of key apoptotic proteins, i.e., B-cell lymphoma 2 (Bcl-2), Bcl-2 Associated X (Bax), and cleaved caspase-3. The balloon control approach to postconditioning provided accurate control of coronary blood flow and simplified the postconditioning manipulation. Infarct size reduction was observed in IRI rats after post-conditioning. There was a decrease in cardiac apoptosis in IRI rats after conditioning, as detected by TUNEL staining. IRI rats showed increased Bcl-2 levels and decreased Bax and cleaved caspase-3 levels in the myocardium. Postconditioning was successfully applied in rats using this novel approach. Postconditioning with this approach reduced infarct size and apoptosis in the area at risk.

Construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues

Fat mass and obesity-associated protein (FTO) is a crucial eraser of RNA N6- methyladenosine (m6A) modification, and abnormal FTO expression level is implicated in pathogenesis of numerous cancers. Herein, we demonstrate the construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues. When FTO is present, it specifically erases the methyl group in m6A, inducing the cleavage of demethylated DNA by endonuclease DpnII and the generation of a single-stranded DNA (ssDNA) with a 3′-hydroxyl group. Subsequently, terminal deoxynucleotidyl transferase (TdT) promotes the incorporation of dTTPs into the ssDNA to obtain a long polythymidine (T) DNA sequence. The resultant long poly (T) DNA sequence can act as a template to trigger hyperbranched strand displacement amplification (HSDA), yielding numerous DNA fragments that may be stained by SYBR Gold to produce an enhanced fluorescence signal. This biosensor processes ultrahigh sensitivity with a detection limit of 1.65 × 10−10 mg/mL (2.6 fM), and it can detect the FTO activity in a single MCF-7 cell. Moreover, this biosensor can screen the FTO inhibitors, evaluate enzyme kinetic parameters, and discriminate the FTO expression levels in the tissues of breast cancer patients and healthy persons.

Sodium tanshinone IIA sulfonate inhibits tumor growth via miR-138 upregulation in intermittent hypoxia-induced xenograft mice.

We studied the functions of sodium tanshinone IIA sulfonate (TSA) in inducing tumor growth in obstructive sleep apnea (OSA)-mimicking intermittent hypoxia (IH) xenograft mice and the underlying potential molecular mechanism. RNA sequencing was conducted to screen the differentially expressed microRNAs in cell lines exposed to IH with or without TSA treatment. As part of the 5-week in vivo study, we treated xenograft mice with 8-h IH once daily. TSA and miR-138 inhibitors or mimics were administrated appropriately. In addition, we performed real-time quantitative polymerase chain reaction (RT-PCR), Western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), microvessel density (MVD), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. RNA sequencing and RT-PCR results demonstrated that TSA increased the levels of miR-138 under IH conditions in vitro. TSA reduced the IH-stimulated high levels of hypoxia-induced factor-1α and vascular endothelial growth factor. Furthermore, IH contributed to high tumor migration, invasion, MVD, and low apoptosis. TSA attenuated IH-mediated tumor proliferation, migration, invasion, MVD, and increased apoptosis, whereas miR-138 inhibitor interrupted the effect of TSA on treating IH-induced tumor behaviors. OSA mimicking IH facilitates tumor growth and reduces miR-138 levels. TSA inhibits IH-induced tumor growth by upregulating the expression of miR-138.

PRMT6-FOXO3A ATTENUATES APOPTOSIS BY UPREGULATING PARKIN EXPRESSION IN INTESTINAL ISCHEMIA-REPERFUSION INJURY.

Intestinal ischemia-reperfusion injury (IIRI) is a serious disease with high morbidity and mortality. This study aims to investigate the potential regulatory mechanisms involving protein arginine methyltransferase 6 (PRMT6), Forkhead box O3a (FoxO3a), and Parkin in IIRI and elucidate their roles in mediating cell apoptosis. The IIRI animal model was established and confirmed using hematoxylin and eosin staining. Oxygen-glucose deprivation and reperfusion (OGD/R) cell model was established to mimic ischemic injury in vitro . Transient transfection was used to overexpress or knock down genes. Cell death or apoptosis was assessed by propidium iodide staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and flow cytometry. The expression of proteins was detected by western blot. The histopathology observed by hematoxylin and eosin staining suggested that the IIRI animal model was successfully established. Our findings revealed that IIRI resulted in increased Bax and decreased Bcl-2 levels. In vitro experiments showed that overexpression of Parkin decreased OGD/R injury and suppressed elevation of Bax/Bcl-2. PRMT6 regulated the methylation level of FoxO3a. Moreover, FoxO3a directly binds to Parkin, and FoxO3a overexpression reduced OGD/R-induced cell death and regulation of Parkin. Overexpression of PRMT6 can attenuate the downregulation of Parkin and elevation of Bax/Bcl-2 caused by OGD/R. Knockdown of PRMT6 promoted apoptosis in intestinal epithelial cells of OGD/R group, while PRMT6 overexpression exhibited the opposite effect. Notably, the levels of PRMT6, FoxO3a, and Parkin were decreased in IIRI mouse intestinal tissue. Knocking out PRMT6 causes a significant decrease in the lifespan of mice. Altogether, our results demonstrated that PRMT6 upregulated the expression of Parkin by regulating FoxO3a methylation level, attenuating the apoptosis induced by IIRI.

Genotoxicity Assessment of Quinoin, a Ribosome Inactivating Protein from Quinoa Seeds, in the Teleost Danio rerio.

Ribosome inactivating proteins (RIPs) are N-glycosylases found in various plants that are able to specifically and irreversibly inhibit protein translation, thereby leading to cell death. Their cytotoxic properties have attracted attention in the medical field in the context of developing new anticancer therapies. Quinoin is a novel toxic enzyme obtained from quinoa seeds and classified as a type 1 RIP (Chenopodium quinoa Willd.). Recently, quinoin was found to be cytotoxic to normal fibroblasts and keratinocytes in vitro, as well as to several tumor cell lines. The aim of this study was to evaluate the in vitro and in vivo genotoxicity of quinoin in a zebrafish model. We evaluated its ability to induce DNA fragmentation, genomic instability, and reactive oxygen species (ROS) generation by means of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction, randomly amplified polymorphic DNA (RAPD) Polymerase Chain Reaction (PCR) technique, and dichlorofluorescine (DCF) assay, respectively. Quinoin was found to cause genomic damage in zebrafish, as shown by DNA fragmentation, polymorphic variations leading to genomic instability, and oxidative stress. Interestingly, longer quinoin treatment caused less damage than shorter treatments. This study demonstrated ROS-mediated genotoxicity of quinoin toward the zebrafish genome. The reduced damage observed after longer quinoin treatment could indicate the activation of detoxification mechanisms, activation of repair mechanisms, or the loss of protein activity due to enzymatic digestion. In order to clarify the genotoxic actions of quinoin, further investigations of the response pathways to DNA damage are needed. Overall, the ability of quinoin to cause breaks and instability in DNA, together with its clear cytotoxicity, make it an interesting candidate for the development of new drugs for cancer treatment.

Trophectoderm cells of human mosaic embryos display increased apoptotic levels and impaired differentiation capacity: a molecular clue regarding their reproductive fate?

Are there cell lineage-related differences in the apoptotic rates and differentiation capacity of human blastocysts diagnosed as euploid, mosaic, and aneuploid after preimplantation genetic testing for aneuploidy (PGT-A) based on concurrent copy number and genotyping analysis? Trophectoderm (TE) cells of mosaic and aneuploid blastocysts exhibit significantly higher levels of apoptosis and significantly reduced differentiation capacity compared to those of euploid blastocysts. Embryos diagnosed as mosaic after PGT-A can develop into healthy infants, yet understanding the reasons behind their reproductive potential requires further research. One hypothesis suggests that mosaicism can be normalized through selective apoptosis and reduced proliferation of aneuploid cells, but direct evidence of these mechanisms in human embryos is lacking. Additionally, data interpretation from studies involving mosaic embryos has been hampered by retrospective analysis methods and the high incidence of false-positive mosaic diagnoses stemming from the use of poorly specific PGT-A platforms. Prospective cohort study performing colocalization of cell-lineage and apoptotic markers by immunofluorescence (IF). We included a total of 64 human blastocysts donated to research on Day 5 or 6 post-fertilization (dpf) by 43 couples who underwent in vitro fertilization treatment with PGT-A at IVI-RMA Valencia between September 2019 and October 2022. A total of 27 mosaic blastocysts were analyzed. The study consisted of two phases: Phase I (caspase-3, n = 53 blastocysts): n = 13 euploid, n = 22 mosaic, n = 18 aneuploid. Phase II (terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), n = 11 blastocysts): n = 2 euploid, n = 5 mosaic, n = 4 aneuploid. Following donation for research, vitrified blastocysts were warmed, cultured until re-expansion, fixed, processed for IF, and imaged using confocal microscopy. For each blastocyst, the following cell counts were conducted: total cells (DAPI+), TE cells (GATA3+), inner cell mass (ICM) cells (GATA3-/NANOG+), and apoptotic cells (caspase-3+ or TUNEL+). The incidence of apoptosis was calculated for each blastocyst by dividing the number of caspase-3+ cells (Phase I) or TUNEL+ cells (Phase II) by the number of TE or ICM cells. Statistical analysis was performed according to data type and distribution (P < 0.05 was considered statistically significant). Phase I: Mosaic blastocysts displayed a similar number of total cells (49.6 ± 15 cells at 5 dpf; 58.8 ± 16.9 cells at 6 dpf), TE cells (38.8 ± 13.7 cells at 5 dpf; 49.2 ± 16.2 cells at 6 dpf), and ICM cells (10.9 ± 4.2 cells at 5 dpf; 9.7 ± 7.1 cells at 6 dpf) compared to euploid and aneuploid blastocysts (P > 0.05). The proportion of TE cells retaining NANOG expression increased gradually from euploid blastocysts (9.7% = 63/651 cells at 5 dpf; 0% = 0/157 cells at 6 dpf) to mosaic blastocysts (13.1% = 104/794 cells at 5 dpf; 3.4% = 12/353 cells at 6 dpf) and aneuploid blastocysts (27.9% = 149/534 cells at 5 dpf; 4.6% = 19/417 cells at 6 dpf) (P < 0.05). At the TE level, caspase-3+ cells were frequently observed (39% = 901/2310 cells). The proportion of caspase-3+ TE cells was significantly higher in mosaic blastocysts (44.1% ± 19.6 at 5 dpf; 43% ± 16.8 at 6 dpf) and aneuploid blastocysts (45.9% ± 16.1 at 5 dpf; 49% ± 15.1 at 6 dpf) compared to euploid blastocysts (26.6% ± 16.6 at 5 dpf; 17.5% ± 14.8 at 6 dpf) (P < 0.05). In contrast, at the ICM level, caspase-3+ cells were rarely observed (1.9% = 11/596 cells), and only detected in mosaic blastocysts (2.6% = 6/232 cells) and aneuploid blastocysts (2.5% = 5/197 cells) (P > 0.05). Phase II: Consistently, TUNEL+ cells were only observed in TE cells (32.4% = 124/383 cells). An increasing trend was identified toward a higher proportion of TUNEL+ cells in the TE of mosaic blastocysts (37.2% ± 21.9) and aneuploid blastocysts (39% ± 41.7), compared to euploid blastocysts (23% ± 32.5), although these differences did not reach statistical significance (P > 0.05). The observed effects on apoptosis and differentiation may not be exclusive to aneuploid cells. Additionally, variations in aneuploidies and unexplored factors related to blastocyst development and karyotype concordance may introduce potential biases and uncertainties in the results. Our findings demonstrate a cell lineage-specific effect of aneuploidy on the apoptotic levels and differentiation capacity of human blastocysts. This contributes to unravelling the biological characteristics of mosaic blastocysts and supports the concept of clonal depletion of aneuploid cells in explaining their reproductive potential. This work was funded by grants from Centro para el Desarrollo Tecnológico Industrial (CDTI) (20190022) and Generalitat Valenciana (APOTIP/2019/009). None of the authors has any conflict of interest to declare. N/A.

Chronic infection control relies on T cells with lower foreign antigen binding strength generated by N-nucleotide diversity.

The breadth of pathogens to which T cells can respond is determined by the T cell receptors (TCRs) present in an individual's repertoire. Although more than 90% of the sequence diversity among TCRs is generated by terminal deoxynucleotidyl transferase (TdT)-mediated N-nucleotide addition during V(D)J recombination, the benefit of TdT-altered TCRs remains unclear. Here, we computationally and experimentally investigated whether TCRs with higher N-nucleotide diversity via TdT make distinct contributions to acute or chronic pathogen control specifically through the inclusion of TCRs with lower antigen binding strengths (i.e., lower reactivity to peptide-major histocompatibility complex (pMHC)). When T cells with high pMHC reactivity have a greater propensity to become functionally exhausted than those of low pMHC reactivity, our computational model predicts a shift toward T cells with low pMHC reactivity over time during chronic, but not acute, infections. This TCR-affinity shift is critical, as the elimination of T cells with lower pMHC reactivity in silico substantially increased the time to clear a chronic infection, while acute infection control remained largely unchanged. Corroborating an affinity-centric benefit for TCR diversification via TdT, we found evidence that TdT-deficient TCR repertoires possess fewer T cells with weaker pMHC binding strengths in vivo and showed that TdT-deficient mice infected with a chronic, but not an acute, viral pathogen led to protracted viral clearance. In contrast, in the case of a chronic fungal pathogen where T cells fail to clear the infection, both our computational model and experimental data showed that TdT-diversified TCR repertoires conferred no additional protection to the hosts. Taken together, our in silico and in vivo data suggest that TdT-mediated TCR diversity is of particular benefit for the eventual resolution of prolonged pathogen replication through the inclusion of TCRs with lower foreign antigen binding strengths.

USP4 regulates TUT1 ubiquitination status in concert with SART3

The ubiquitin system plays pivotal roles in diverse cellular processes, including signal transduction, transcription and translation, organelle quality control, and protein degradation. Recent investigations have revealed the regulatory influence of ubiquitin systems on RNA metabolism. Previously, we reported that the deubiquitinating enzyme, ubiquitin specific peptidase 15 (USP15), promotes deubiquitination of terminal uridylyl transferase 1 (TUT1), a key regulator within the U4/U6 spliceosome, thereby instigating significant alterations in global RNA splicing [1]. In this study, we report that ubiquitin specific peptidase 4 (USP4), a homologous protein to USP15, also exerts control over the ubiquitination status of TUT1. Analogous to USP15, the expression of USP4 results in a reduction of TUT1 ubiquitination. Furthermore, squamous cell carcinoma antigen recognized by T-cells 3 (SART3) collaborates in enhancing the deubiquitinating activity of USP4 towards TUT1. A crucial revelation is that USP4 orchestrates the subnuclear relocation of TUT1 from the nucleolus to the nucleoplasm and facilitates the stability of U6 small nuclear RNA (snRNA). Notably, USP4 has a more profound effect on TUT1 redistribution compared to USP15. Our findings suggest that USP4 intricately modulates the ubiquitination status of TUT1, thereby exerting pronounced effects on the spliceosome functions.

Mechanism of resveratrol in protecting ovary in poor ovarian response mice by regulating Hippo signaling pathway

This study observed the protective effect of resveratrol(Res) on ovarian function in poor ovarian response(POR) mice by regulating the Hippo signaling pathway and explored the potential mechanism of Res in inhibiting ovarian cell apoptosis. Female mice with regular estrous cycles were randomly divided into a blank group, a model group, and low-and high-dose Res groups(20 and 40 mg·kg~(-1)), with 20 mice in each group. The blank group received an equal volume of 0.9% saline solution by gavage, while the model group and Res groups received suspension of glycosides of Triptergium wilfordii(GTW) at 50 mg·kg~(-1) by gavage for two weeks to induce the model. After modeling, the low-and high-dose Res groups were continuously treated with drugs by gavage for two weeks, while the blank group and the model group received an equal volume of 0.9% saline solution by gavage. Ovulation was induced in all groups on the day following the end of treatment. Finally, 12 female mice were randomly selected from each group, and the remaining eight female mice were co-housed with male mice at a ratio of 1∶1. Changes in the estrous cycle of mice were observed using vaginal cytology smears. The number of ovulated eggs, ovarian wet weight, ovarian index, and pregnancy rate of mice were measured. The le-vels of anti-Mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in serum were determined using enzyme-linked immunosorbent assay(ELISA). Ovarian tissue morphology and ovarian cell apoptosis were observed using hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining, respectively. The protein expression levels of yes-associated protein(YAP) 1 and transcriptional coactivator with PDZ-binding motif(TAZ) were detected by immunohistochemistry(IHC), while the changes in protein expression levels of mammalian sterile 20-like kinase(MST) 1/2, large tumor suppressor(LATS) 1/2, YAP1, TAZ, B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were determined by Western blot. The results showed that compared with the blank group, the model group had an increased rate of estrous cycle disruption in mice, a decreased number of normally developing ovarian follicles, an increased number of blocked ovarian follicles, increased ovarian granulosa cell apoptosis, decreased ovulation, reduced ovarian wet weight and ovarian index, increased serum FSH and LH levels, decreased AMH and E_2 levels, decreased protein expression levels of YAP1 and TAZ in ovarian tissues, increased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and decreased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Additionally, the number of embryos per litter significantly decreased after co-housing. Compared with the model group, the low-and high-dose Res groups exhibited reduced estrous cycle disruption rates in mice, varying degrees of improvement in the number and morphology of ovarian follicles, reduced numbers of blocked ovarian follicles, improved ovarian granulosa cell apoptosis, increased ovulation, elevated ovarian wet weight and ovarian index, decreased serum FSH and LH levels, increased AMH and E_2 levels, elevated protein expression levels of YAP1 and TAZ in ovarian tissues, decreased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and increased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Furthermore, the number of embryos per litter increased to varying degrees after co-housing. In conclusion, Res effectively inhibits ovarian cell apoptosis in mice and improves ovarian responsiveness. Its mechanism may be related to the regulation of key molecules in the Hippo pathway.

Effect of 2.45 GHz Microwave Radiation on the Inner Ear: A Histopathological Study on 2.45 GHz Microwave Radiation and Cochlea.

The present study aims to determine the possible low dose-dependent adverse effects of 2.45 GHz microwave exposure and Wi-Fi frequency on the cochlea. Twelve pregnant female rats (n=12) and their male newborns were exposed to Wi-Fi frequencies with varying electric field values of 0.6, 1.9, 5, 10 V/m, and 15 V/m during the 21-day gestation period and 45 days after birth, except for the control group. Auditory brainstem response testing was performed before exposure and sacrification. After removal of the cochlea, histopathological examination was conducted by immunohistochemistry methods using caspase (cysteine-aspartic proteases, cysteine aspartates, or cysteine-dependent aspartate-directed proteases)-3, -9, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Kruskal-Wallis and Wilcoxon tests and multivariate analysis of variance were used. Auditory brainstem response thresholds in postexposure tests increased statistically significantly at 5 V/m and above doses. When the number of apoptotic cells was compared in immunohistochemistry examination, significant differences were found at 10 V/m and 15 V/m doses (F(5,15)=23.203, P=.001; Pillai's trace=1.912, η2=0.637). As the magnitude of the electric field increased, all histopathological indicators of apoptosis increased. The most significant effect was noted on caspase-9 staining (η2 c9=0.996), followed by caspase-3 (η2 c3=0.991), and TUNEL staining (η2 t=0.801). Caspase-3, caspase-9, and TUNEL-stained cell densities increased directly by increasing the electric field and power values. Apoptosis and immune activity in the cochlea depend on the electric field and power value. Even at low doses, the electromagnetic field in Wi-Fi frequency damages the inner ear and causes apoptosis.

Curcumin alleviates myocardial inflammation, apoptosis, and oxidative stress induced by acute pulmonary embolism by regulating microRNA-145-5P/insulin receptor substrate 1 axis.

The treatment of patients with acute pulmonary embolism (APE) is extremely challenging due to the complex clinical presentation and prognosis of APE related to the patient's hemodynamic status and insufficient arterial blood flow and right ventricular overload. Protective efficacy against cardiovascular diseases of curcumin, a common natural polyphenolic compound, which has antithrombotic properties and reduces platelet accumulation in the circulation by inhibiting thromboxane synthesis has been demonstrated. However, the direct effect of curcumin on APE has rarely been studied. Therefore, the present study aimed to investigate the therapeutic potential of curcumin in APE and associated myocardial injury to provide new insights into curcumin as a promising competitive new target for the treatment of APE. A suspension of 12 mg/kg microspheres was injected intravenously into rats. An APE rat model was built. Before modeling, intragastric 100 mg/kg curcumin was given, and/or lentiviral plasmid vector targeting microRNA-145-5p or insulin receptor substrate 1 (IRS1) was injected. Pulmonary artery pressure was measured to assess right ventricular systolic pressure (RVSP). Hematoxylin and eosin (H&E) staining was performed on liver tissues and myocardial tissues of APE rats. TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) staining and immunohistochemical (IHC) staining were conducted to measure apoptosis and CyPA-CD147 expression in the myocardium, respectively. Inflammatory indices interleukin-1beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were measured by ELISA in cardiac tissues. RT-qPCR and Western blot were performed to determine the expression levels of related genes. In addition, by dual luciferase reporter assay and RIP assay, the relationship between microRNA-145-5p and insulin receptor substrate 1 (IRS1) was confirmed. In results: curcumin improved APE-induced myocardial injury, reduced myocardial tissue edema, and thrombus volume. It attenuated APE-induced myocardial inflammation and apoptosis, as well as reduced lung injury and pulmonary artery pressure. Curcumin promoted microRNA-145-5p expression in APE rat myocardium. MicroRNA-145-5p overexpression protected against APE-induced myocardial injury, and microRNA-145-5p silencing abolished the beneficial effects of curcumin in APE-induced myocardial injury. IRS1 was targeted by microRNA-145-5p. IRS1 silencing attenuated APE-induced myocardial injury, and enhanced therapeutic effect of curcumin on myocardial injury in APE rats. In conclusion, curcumin alleviates myocardial inflammation, apoptosis, and oxidative stress induced by APE by regulating microRNA-145-5p/IRS1 axis.

Circ-phkb promotes cell apoptosis and inflammation in LPS-induced alveolar macrophages via the TLR4/MyD88/NF-kB/CCL2 axis

BackgroundCircular RNAs (CircRNAs) have been associated with acute lung injury (ALI), but their molecular mechanisms remain unclear.MethodsThis study developed a rat model of lipopolysaccharide (LPS)-induced ALI and evaluated the modeling effect by hematoxylin and eosin staining, Masson’s trichrome staining, lung wet-to-dry weight ratio, terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL), and enzyme-linked immunosorbent assay (ELISA) detection of inflammatory factors (interleukin-1β, tumor necrosis factor alpha, and interleukin-6). Using lung tissues from a rat model of LPS-induced ALI, we then conducted circRNA sequencing, mRNA sequencing, and bioinformatics analysis to obtain differential circRNA and mRNA expression profiles as well as potential ceRNA networks. Furthermore, we performed quantitative real-time polymerase chain reaction (qRT-PCR) assays to screen for circ-Phkb in ALI rat lung tissues, alveolar macrophages, and LPS-induced NR8383 cells. We conducted induction with or without LPS with circ-Phkb siRNA and overexpression lentivirus in NR8383. Cell Counting Kit-8, C5-Ethynyl-2′-deoxyuridine (Edu), TUNEL, and cytometry were used to identify proliferation and apoptosis, respectively. We detected inflammatory factors using ELISA. Finally, we used Western blot to detect the apoptosis-related proteins and TLR4/MyD88/NF-kB/CCL2 pathway activation.ResultsOur results revealed that both circRNA and mRNA profiles are different from those of the Sham group. We observed a significant circ-Phkb upregulation in NR8383 cells and LPS-exposed rats. Apoptosis and inflammation were greatly reduced when circ-Phkb expression was reduced in NR8383 cells, cell proliferation was increased, and circ-Phkb overexpression was decreased.ConclusionsIn terms of mechanism, circ-Phkb suppression inhibits CCL2 expression via the TLR4/MyD88/NF-kB pathway in LPS-induced alveolar macrophages.

Acetylmelodorinol isolated from Sphaerocoryne affinis seeds inhibits cell proliferation and activates apoptosis on HeLa cells

BackgroundCervical cancer is a major global health concern with a high prevalence in low- and middle-income countries. Natural products, particularly plant-derived compounds, have shown immense potential for developing anticancer drugs. In this study, we aimed to investigate the anticancer properties of the pericarp and seeds of Sphaerocoryne affinis fruit on human cervical carcinoma cells (HeLa) and isolate the bioactive compound from the active fraction.MethodsWe prepared solvent fractions from the ethanol extracts of the pericarp and the seed portion by partitioning and assessing their cytotoxicity on HeLa cells. Subsequently, we collected acetylmelodorinol (AM), an anticancer compound, from the ethyl acetate fraction of seeds and determined its structure using nuclear magnetic resonance. We employed cytotoxicity assay, western blotting, Annexin V apoptosis assay, measurement of intracellular reactive oxygen species (ROS) levels, 4′,6-diamidino-2-phenylindole (DAPI) staining, and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, to evaluate the anticancer properties of AM on HeLa.ResultsThe solvent fractions from the seed displayed considerably higher cytotoxic activity against HeLa cells than those of the pericarp. We isolated and identified acetylmelodorinol as an anticancer compound from the ethyl acetate fraction from S. affinis seed extract. Treatment with acetylmelodorinol inhibited HeLa cell proliferation with an IC50 value of 2.62 ± 0.57 µg/mL. Furthermore, this study demonstrated that acetylmelodorinol treatment disrupted cell cycle progression by reducing the expression of cyclin E, CDK1/2, and AKT/mTOR pathways, increasing the intracellular ROS levels, reducing BCL-2/BCL-XL expression, causing DNA fragmentation and nuclear shrinkage, and triggering apoptosis through caspase 3 and 9 activation in a dose-and time-dependent manner.ConclusionIn contrast to previous reports, this study focuses on the inhibitory effects of AM on the AKT/mTOR pathway, leading to a reduction in cell proliferation in cervical cancer cells. Our findings highlight the promising potential of acetylmelodorinol as an effective treatment for cervical cancer. Additionally, this study establishes a foundation for investigating the molecular mechanisms underlying AM’s properties, fostering further exploration into plant-based cancer therapies.

RUNX1/miR-429 feedback loop promotes growth, metastasis, and epithelial-mesenchymal transition in oral squamous cell carcinoma by targeting ITGB1.

This study aimed to explore the role of miR-429 on the progression of oral squamous cell carcinoma (OSCC). OSCC cell lines were transfected with miR-429 mimic, pcDNA3.1-RUNX1, or pcDNA3.1-ITGB1, and their cell viability, apoptosis, migration, and invasion abilities were analyzed by cell counting, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, wound healing, and transwell assays, respectively. Furthermore, luciferase reporter assay, RNA pull-down, and ChIP were used to assess the regulation of miR-429, RUNX1, and ITGB1 expression in OSCC. Lastly, the biological role of the RUNX1/miR-429 feedback loop was explored in nude mice. The results revealed that miR-429 level was down-regulated, while RUNX1 and ITGB1 levels were up-regulated in OSCC tissues and that miR-429 was negatively correlated with RUNX1 and ITGB1 in OSCC tissues. Transfection of miR-429 mimic suppressed OSCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Moreover, we found that miR-429 participated in OSCC progression by directly targeting ITGB1. Additionally, we found that RUNX1 negatively regulated miR-429 expression by binding to its promoter. Our results also revealed that the RUNX1/miR-429 feedback loop regulated ITGB1 expression and that RUNX1 overexpression rescued the inhibitory effects of miR-429 mimic on OSCC cells. In addition, miR-429 mimic significantly suppressed tumor growth, inflammatory cell infiltration, EMT, and ITGB1 expression in vivo, which were inhibited by RUNX1 overexpression. Altogether, these results indicate that the RUNX1/miR-429 feedback loop promoted growth, metastasis, and EMT in OSCC by targeting ITGB1.