RESUMEN
Due to the high incidence of diabetes mellitus (DM) and poor response to the first-line treatment of DM-induced erectile dysfunction (DMED), new therapeutic strategies for DMED are needed. Adipose-derived stem cell (ADSC) transplantation is considered a promising treatment modality for DMED but is limited by poor survival and efficacy after transplantation. In this study, we aimed to increase the therapeutic effect of DMED by overexpressing the relaxin family peptide receptor 1 (RXFP1) using a clustered regularly interspaced short palindromic repeats activation (CRISPRa) system in ADSCs. Two lentiviruses carrying the CRISPRa system transfected ADSCs to overexpress RXFP1 (RXFP1-ADSCs). The intracavernous injection of ADSCs was performed in DMED rats induced by the intraperitoneal injection of streptozotocin. Four weeks after transplantation, we measured erectile function and collected specimens of the corpus cavernosum for follow-up detection. The results showed that ADSCs improved erectile function in diabetic rats, and the RXFP1-ADSCs were more significant. We detected reduced levels of oxidative stress, apoptosis and fibrosis together with relative normalization of endothelial and smooth muscle cell function in the penis after ADSC transplantation. RXFP1-ADSCs had more potent efficacy in the above alterations compared to negative control ADSCs due to the high levels of survival and paracrine capacity in RXFP1-ADSCs. The results revealed that RXFP1-ADSC transplantation could partially preserve erectile function in DMED rats associated with the regulation of oxidative stress, apoptosis, fibrosis and endothelial and smooth muscle cell dysfunction. RXFP1 may be the new target for the genetic modification of ADSCs, which benefits the management of DMED.
RESUMEN
Kidney renal clear cell carcinoma (KIRC) is a heterogeneous malignant tumor with high incidence, metastasis, and mortality. The imbalance of copper homeostasis can produce cytotoxicity and cause cell damage. At the same time, copper can also induce tumor cell death and inhibit tumor transformation. The latest research found that this copper-induced cell death is different from the known cell death pathway, so it is defined as cuproptosis. We included 539 KIRC samples and 72 normal tissues from the Cancer Genome Atlas (TCGA) in our study. After identifying long non-coding RNAs (lncRNAs) significantly associated with cuproptosis, we clustered 526 KIRC samples based on the prognostic lncRNAs and obtained two different patterns (Cuproptosis.C1 and C2). C1 indicated an obviously worse prognostic outcome and possessed a higher immune score and immune cell infiltration level. Moreover, a prognosis signature (CRGscore) was constructed to effectively and accurately evaluate the overall survival (OS) of KIRC patients. There were significant differences in tumor immune microenvironment (TIME) and tumor mutation burden (TMB) between CRGscore-defined groups. CRGscore also has the potential to predict medicine efficacy.
RESUMEN
Alkaline phosphatase (ALP) is widely expressed in human tissues. ALP plays an important role in the dephosphorylation of proteins and nucleic acids. Therefore, quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods. Terminal deoxynucleotidyl transferase (TdT) catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3'-OH end of single-stranded DNA in the absence of a template. In this study, we developed a highly sensitive and selective method based on TdT and endonuclease IV (Endo IV) to quantify ALP activity. After ALP hydrolyzes the 3'-PO4 end of the substrate and generates 3'-OH, TdT can effectively elongate the 3'-OH end with deoxynucleotide adenine triphosphate (dATP) and produce a poly A tail, which can be detected by the poly T probes. Endo IV digests the AP site in poly T probes to generate a fluorescent signal and a new 3'-OH end, leading to the generation of exponential fluorescence signal amplification. The substrate for TdT elongation was optimized, and a limit of detection of 4.3 × 10-3 U/L was achieved for ALP by the optimized substrate structure. This method can also detect ALP in the cell lysate of a single cell. This work has potential applications in disease diagnosis and biomedical detection.
RESUMEN
Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3'-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 3'-PO4 end of the substrate and generates 3'-OH,TdT can effectively elongate the 3'-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 3'-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10-3 U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.
RESUMEN
8-oxoguanine DNA glycosylase (OGG), which plays a crucial role in base excision repair (BER), is an important biomarker. The existing highly sensitive fluorescent methods always need complicated amplification design. The method with high sensitivity and simple design at the same time is urgently needed. Here, we developed a highly sensitive detection method for OGG detection with lambda exonuclease and the background signal suppression probe. Through probe structure design, the steric hindrance and competitive binding effects successfully suppressed the background signal. We achieved sensitive detection of OGG with a simple design, and the limit of detection was 5.0 × 10-4 U mL-1. Moreover, the method was highly selective and successfully applied to OGG detection in biological samples, which shows the potential clinical application value.