A Novel Aptamer LL4A Specifically Targets Vemurafenib-Resistant Melanoma through Binding to the CD63 Protein.

Melanoma is a highly aggressive tumor with a poor prognosis, and half of all melanoma patients harbor BRAF mutations. A BRAF inhibitor, vemurafenib (PLX4032), has been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) to treat advanced melanoma patients with BRAFV600E mutation. However, the efficacy of vemurafenib is impeded by adaptive resistance in almost all patients. In this study, using a cell-based SELEX (systematic evolution of ligands by exponential enrichment) strategy, we obtained a DNA aptamer (named LL4) with high affinity and specificity against vemurafenib-resistant melanoma cells. Optimized truncated form (LL4A) specifically binds to vemurafenib-resistant melanoma cells with dissociation constants in the nanomolar range and with excellent stability and low toxicity. Meanwhile, fluorescence imaging confirmed that LL4A significantly accumulated in tumors formed by vemurafenib-resistant melanoma cells, but not in control tumors formed by their corresponding parental cells in vivo. Further, a transmembrane protein CD63 was identified as the binding target of aptamer LL4A using a pull-down assay combined with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. CD63 formed a supramolecular complex with TIMP1 and β1-integrin, activated the nuclear factor кB (NF-кB) and mitogen-activated protein kinase (MAPK) signaling pathways, and contributed to vemurafenib resistance. Potentially, the aptamer LL4A may be used diagnostically and therapeutically in humans to treat targeted vemurafenib-resistant melanoma

Effect of ligustrazine injection on the change of oxidative stress system during renal ischemia reperfusion injury in rabbits

目的  通过由兔肾脏缺血/再灌注损伤（IRI）导致的氧化应激体系中的变化，研究中药川芎嗪对IRI干预下的作用机制。方法  建立持续性阻断兔双侧肾动脉血流1h，再灌注5h的肾IRI动物模型。日本大耳兔32只，随机分成3组（n=10）：假手术组(sham，S组)，缺血/再灌注组(ischemia-reperfusion，IR组)，川芎嗪干预缺血/再灌注组（ligustrazine+ ischemia-reperfusion，LZ组），另外两只作为补充实验中意外死亡之用。于缺血前、缺血1h、再灌注1h、3h和5h依次经颈总动脉抽血用以检测超氧化物歧化酶（SOD）活力、黄嘌呤氧化酶（XO）活力以及丙二醛（MDA）含量。在实验结束后取兔肾组织依次检测SOD、XO活力以及MDA含量，并对其进行电镜观察。结果  随着肾缺血和再灌注时间的增加，IR组和LZ组血浆中的XO活力和MDA含量逐渐呈上升趋势，但同时间点LZ组较于IR组明显降低（均P<0.01）；SOD活力随着肾缺血和再灌注时间的增加而逐渐呈下降趋势，但同时间点下，LZ组较于IR组均明显偏高（均P<0.01）。IR组和LZ组相较于S组，肾组织XO活力、MDA含量均明显升高，SOD活力均明显降低（均P<0.01）；而LZ组肾组织中的XO活力、MDA含量均显著低于IR组，SOD活力均显著高于IR组，差异有非常显著性（均P<0.01），LZ组肾组织细胞的超微结构异常改变较IR组显著减轻。结论  川芎嗪能够使氧自由基水平降低，氧化应激损伤减轻，具有保护肾缺血/再灌注损伤的作用。Objective: To investigate the change of oxidative stress system during renal ischemia-reperfusion injury, and to study the mechanism of Ligustrazine on IRI under the intervention. Methods: Establish the IRI animal model by persistent blocking rabbits’ bilateral renal artery blood flow for 1 hour, then reperfusion for another 5 hours. Japanese big ear rabbits, 32, were randomly divided into three groups (n=10): sham operation group (group S), ischemia/reperfusion group (group IR), the effect of ligustrazine on ischemia/ reperfusion group (LZ group), the other two being added in the experiment for accidental death. In 1 hour before ischemia, ischemia, reperfusion 1h, 3h and 5h in turn to check the enzyme activity of superoxide dismutase (SOD), xanthine oxidase (XO) and the content of malondialdehyde (MDA) by getting the blood from common carotid artery. At the end of the experiment, the rabbit’s kidney was used to check the enzyme activity of XO, SOD and the content of MDA, then to observe the morphological changes under the electron microscopy. Results: With the increase of renal ischemia and reperfusion time, XO activity and MDA content of IR group and LZ group in plasma gradually up, they were significantly lower in LZ group than in IR group at same time point (all P<0.01), the activity of SOD in plasma was shown a time-dependent decline in both IR group and LZ group, whereas it was significant higher in LZ group compared with IR group at same time point during ischemia reperfusion (all P<0.01). SOD activity gradually decreased with the increase of renal ischemia and reperfusion time, but at the same time point, LZ group compared with IR group were significantly higher (P<0.01), however, for LZ group, as compared with IR group, activity of XO, content of MDA were increased remarkably while activity of SOD was decreased significantly in kidney tissue (all P<0.01) the abnormal changes of ultrastructure were mitigated significantly. Conclusion: Ligustrazine may attenuate renal ischemia-reperfusion injury by dropping oxygen free radical generation and enhancing oxygen free radical scavenge so that it can antagonize oxidative stress

Gut microbiota and its metabolites – molecular mechanisms and management strategies in diabetic kidney disease

Diabetic kidney disease (DKD) is one of the major microvascular complications of diabetes mellitus and is also one of the serious risk factors in cardiovascular events, end-stage renal disease, and mortality. DKD is associated with the diversified, compositional, and functional alterations of gut microbiota. The interaction between gut microbiota and host is mainly achieved through metabolites, which are small molecules produced by microbial metabolism from exogenous dietary substrates and endogenous host compounds. The gut microbiota plays a critical role in the pathogenesis of DKD by producing multitudinous metabolites. Nevertheless, detailed mechanisms of gut microbiota and its metabolites involved in the occurrence and development of DKD have not been completely elucidated. This review summarizes the specific classes of gut microbiota-derived metabolites, aims to explore the molecular mechanisms of gut microbiota in DKD pathophysiology and progression, recognizes biomarkers for the screening, diagnosis, and prognosis of DKD, as well as provides novel therapeutic strategies for DKD

Identification of ULK1 as a novel mitophagy-related gene in diabetic nephropathy

BackgroundAccumulating evidence indicates that mitophagy is crucial for the development of diabetic nephropathy (DN). However, little is known about the key genes involved. The present study is to identify the potential mitophagy-related genes (MRGs) in DN.MethodsFive datasets were obtained from the Gene Expression Omnibus (GEO) database and were split into the training and validation set. Then the differentially expressed MRGs were screened and further analyzed for GO and KEGG enrichment. Next, three algorithms (SVM-RFE, LASSO and RF) were used to identify hub genes. The ROC curves were plotted based on the hub genes. We then used the CIBERSORT algorithm to assess the infiltration of 22 types of immune cells and explore the correlation between hub genes and immune cells. Finally, the Nephroseq V5 tool was used to analyze the correlation between hub genes and GFR in DN patients.ResultsCompared with the tubulointerstitium, the expression of MRGs was more noticeably varied in the glomeruli. Twelve DE-MRGs were identified in glomerular samples, of which 11 genes were down-regulated and only MFN1 was up-regulated. GO and KEGG analysis indicated that several enrichment terms were associated with changes in autophagy. Three genes (MFN1, ULK1 and PARK2) were finally determined as potential hub genes by three algorithms. In the training set, the AUROC of MFN1, ULK1 and PARK2 were 0.839, 0.906 and 0.842. However, the results of the validation set demonstrated that MFN1 and PARK2 had no significant difference in distinguishing DN samples from healthy controls, while the AUROC of ULK1 was 0.894. Immune infiltration analysis using CIBERSORT showed that ULK1 was positively related to neutrophils, whereas negatively related to M1 and M2 macrophages. Finally, ULK1 was positively correlated with GFR in Nephroseq database.ConclusionsULK1 is a potential biomarker for DN and may influence the development of diabetic nephropathy by regulating mitophagy

Negative regulation of transcription coactivator p300 by orphan receptor TR3

p300 regulates the transcriptional activity of a variety of transcription factors by forming an activation complex and/or promoting histone acetylation. Here, we show a unique characteristic of orphan receptor TR3 in negatively regulating the function of p300. TR3 was found to interact with p300 and inhibited the acetylation of transcription factors induced by p300, resulting in the repression of their transcriptional activity. Further analysis revealed that both a conserved transcriptional adapter motif (TRAM) in p300 and a specific sequence FLELFIL in TR3 were critical for their interaction. TR3 binding completely covered the histone acetyltransferase (HAT) domain of p300 and resulted in suppression of the HAT activity, as the p300-induced histone H3 acetylation and transcription were inhibited with the presence TR3. Furthermore, an agonist of TR3, a natural octaketide isolated from Dothiorella sp. HTF3 of an endophytical fungus, was shown to be a potent compound for inhibiting p300 HAT activity (IC50=1.5 mu g/ml) in vivo. More importantly, this agonist could repress the transcriptional activity of transcription factors, and proliferation of cancer cells. Taken together, our results not only delineate a novel transcriptional repressor function for TR3, but also reveal its modulation on p300 HAT activity as the underlying mechanism

Synthesis and activity study of novel N,N-diphenylurea derivatives as IDO1 inhibitors

Indoleamine 2,3-dioxygenase 1 (IDO1) has attracted much attention in the field of cancer immunotherapy as an immunomodulatory enzyme. To identify potential IDO1 inhibitors, a novel series of compounds with N,N-diphenylurea and triazole structures were synthesized. The designed compounds underwent organic synthesis, and subsequent enzymatic activity experiments targeting IDO1 confirmed their activity at the molecular level. These experiments provided validation for the efficacy of the designed compounds in inhibiting IDO1, compound 3g exhibited an IC50 value of 1.73 ± 0.97 μM. Further molecular docking study further explained the binding mode and reaction potential of compound 3g with IDO1. Our research has resulted in a series of novel IDO1 inhibitors, which is beneficial to the development of drugs targeting IDO1 in numerous cancer diseases

FAST observations of an extremely active episode of FRB 20201124A: IV. Spin Period Search

We report the properties of more than 800 bursts detected from the repeating fast radio burst (FRB) source FRB 20201124A with the Five-hundred-meter Aperture Spherical radio telescope (FAST) during an extremely active episode on UTC September 25th-28th, 2021 in a series of four papers. In this fourth paper of the series, we present a systematic search of the spin period and linear acceleration of the source object from both 996 individual pulse peaks and the dedispersed time series. No credible spin period was found from this data set. We rule out the presence of significant periodicity in the range between 1 ms to 100 s with a pulse duty cycle $< 0.49\pm0.08$ (when the profile is defined by a von-Mises function, not a boxcar function) and linear acceleration up to $300$ m s$^{-2}$ in each of the four one-hour observing sessions, and up to $0.6$ m s$^{-2}$ in all 4 days. These searches contest theoretical scenarios involving a 1 ms to 100 s isolated magnetar/pulsar with surface magnetic field $<10^{15}$ G and a small duty cycle (such as in a polar-cap emission mode) or a pulsar with a companion star or black hole up to 100 M$_{\rm \odot}$ and $P_b>10$ hours. We also perform a periodicity search of the fine structures and identify 53 unrelated millisecond-timescale "periods" in multi-components with the highest significance of 3.9 $\sigma$. The "periods" recovered from the fine structures are neither consistent nor harmonically related. Thus they are not likely to come from a spin period. We caution against claiming spin periodicity with significance below $\sim$ 4 $\sigma$ with multi-components from one-off FRBs. We discuss the implications of our results and the possible connections between FRB multi-components and pulsar micro-structures.Comment: Accepted by Research in Astronomy and Astrophysics (RAA