Comparison of the effects of Liraglutide, Tirzepatide, and Retatrutide on diabetic kidney disease in db/db mice.

PURPOSE: To assess and compare the therapeutic efficacy of Liraglutide, Tirzepatide, and Retatrutide in treating diabetic kidney disease (DKD) in db/db mice. METHODS: Db/db mice were administered intraperitoneal injections of Liraglutide (10 nmol/kg), Tirzepatide (10 nmol/kg), and Retatrutide (10 nmol/kg) for 10 weeks. Subsequently, we assessed the effectiveness of these three drugs in controlling blood glucose levels, reducing weight, and improving serum biochemical indicators and DKD. Additionally, we measured and compared the renal inflammation and fibrosis indexes. Meanwhile, the content of intestinal metabolite butyrate was compared to reflect the regulatory effects of these three drugs on gut microbiota. RESULTS: Retatrutide demonstrated superior effectiveness in reducing weight and improving renal function in db/db mice compared to Liraglutide and Tirzepatide. Additionally, it markedly suppressed the expression of pro-inflammatory cytokines (TNF-α, caspase-1, and NLRP3) and pro-fibrotic factors (fibronectin, α-SMA, and collagen I) in the kidneys of mice. Furthermore, Retatrutide substantially enhanced liver function, reduced triglyceride levels, cholesterol levels, low-density lipoprotein cholesterol, elevated high-density lipoprotein cholesterol, and increased the content of intestinal metabolite butyrate in db/db mice when compared to the other two drugs. Unfortunately, despite its ability to lower blood glucose levels, Retatrutide did not outperform the other two drugs. In contrast, Tirzepatide exhibited better effects on lowering blood glucose, weight loss, lipid reduction, and improvement of DKD compared to Liraglutide. CONCLUSIONS: Retatrutide and Tirzepatide were significantly effective in improving DKD, controlling blood glucose and body weight. Retatrutide was the most effective in improving DKD and body weight, while Tirzepatide was the most effective in controlling blood glucose. Inhibiting the expression of inflammatory factors and fibrosis mediators and regulating intestinal microbiota may be the potential mechanisms of these two drugs to delay the progression of DKD.

Suppression of the METTL3-m6A-integrin ß1 axis by extracellular acidification impairs T cell infiltration and antitumor activity.

The acidic metabolic byproducts within the tumor microenvironment (TME) hinder T cell effector functions. However, their effects on T cell infiltration remain largely unexplored. Leveraging the comprehensive The Cancer Genome Atlas dataset, we pinpoint 16 genes that correlate with extracellular acidification and establish a metric known as the "tumor acidity (TuAci) score" for individual patients. We consistently observe a negative association between the TuAci score and T lymphocyte score (T score) across various human cancer types. Mechanistically, extracellular acidification significantly impedes T cell motility by suppressing podosome formation. This phenomenon can be attributed to the reduced expression of methyltransferase-like 3 (METTL3) and the modification of RNA N6-methyladenosine (m6A), resulting in a subsequent decrease in the expression of integrin ß1 (ITGB1). Importantly, enforced ITGB1 expression leads to enhanced T cell infiltration and improved antitumor activity. Our study suggests that modulating METTL3 activity or boosting ITGB1 expression could augment T cell infiltration within the acidic TME, thereby improving the efficacy of cell therapy.

Extracellular acidosis restricts one-carbon metabolism and preserves T cell stemness.

The accumulation of acidic metabolic waste products within the tumor microenvironment inhibits effector functions of tumor-infiltrating lymphocytes (TILs). However, it remains unclear how an acidic environment affects T cell metabolism and differentiation. Here we show that prolonged exposure to acid reprograms T cell intracellular metabolism and mitochondrial fitness and preserves T cell stemness. Mechanistically, elevated extracellular acidosis impairs methionine uptake and metabolism via downregulation of SLC7A5, therefore altering H3K27me3 deposition at the promoters of key T cell stemness genes. These changes promote the maintenance of a 'stem-like memory' state and improve long-term in vivo persistence and anti-tumor efficacy in mice. Our findings not only reveal an unexpected capacity of extracellular acidosis to maintain the stem-like properties of T cells, but also advance our understanding of how methionine metabolism affects T cell stemness.

A novel method for extracting circulating cell-free DNA from whole blood samples and its utility in the non-invasive prenatal test.

OBJECTIVE: We verified a magnetic bead-based, simple, and fast method for circulating cell-free DNA (cfDNA) extraction from whole blood samples(CEWB) and characterised its utility in non-invasive prenatal testing (NIPT). METHOD: We extracted cfDNA from both plasma and whole blood of the patients using CEWB and compared it to that extracted using a Qiagen extraction kit; droplet digital polymerase chain reaction test was used to calculate the fragment size bias. In all, 304 samples were used for NIPT. RESULTS: The CEWB group (mean ± standard deviation [SD]: 4.34 ± 0.41 ng/ml plasma) reported less DNA weight yield than the Qiagen group (4.90 ± 0.50 ng/ml plasma). There was no significant difference between the CEWB group and the Qiagen group in the gene fragments (136 bp: p = 0.064 and 420 bp: p = 0.534). In a parallel cohort study to characterise the utility of the CEWB method in NIPT, the treatment group extracted by CEWB showed a sensitivity of 100%, a specificity of 99.65%, and a positive predictive value of 95%. CONCLUSIONS: This study demonstrated that CEWB achieves an acceptable yield of DNA without contamination from genomic DNA. Subsequent clinical experiments in a parallel cohort indicated its utility for NIPT.

Knockdown of DEAD-box RNA helicase 52 (DDX52) suppresses the proliferation of melanoma cells in vitro and of nude mouse xenografts by targeting c-Myc.

The ATP-dependent protein DEAD-box RNA helicase 52 (DDX52) is an important regulator in RNA biology and has been implicated in the development of prostate and lung cancer. However, its biological functions and clinical importance in malignant melanoma (MM) are still unclear. Understanding the potential mechanism underlying the regulation of MM progression by DDX52 might lead to novel therapeutic strategies. The aim of the present study was to investigate the role of DDX52 in the regulation of MM progression and its clinical relevance. DDX52 expression in normal and MM tissues was evaluated by GEO analysis and immunohistochemistry. The effects of DDX52 on cell growth were evaluated in MM cells with downregulated DDX52 expression. In this study, we found that DDX52 was markedly overexpressed in MM tissues compared with nontumor tissues and was associated with shorter overall survival in patients; therefore, DDX52 might be a prognostic marker in MM. Downregulation of DDX52 expression in the MM cell lines A2058 and MV3 markedly inhibited cell proliferation and colony formation. Additionally, knockdown of DDX52 in MM cells caused significant regression of established tumors in nude mice and delayed the onset time. Moreover, downregulation of DDX52 markedly suppressed c-Myc mRNA and protein expression, and an RNA immunoprecipitation assay confirmed the association between DDX52 and c-Myc. Restoration of c-Myc expression partly rescued the effects of DDX52 deficiency in MM cells. In conclusion, our study found that DDX52 mediated oncogenesis by promoting the transcriptional activity of c-Myc and could be a therapeutic target in MM.

A novel WSSV responsive plasma protein from Litopenaeus vannamei contributes to hemocytes anti-apoptosis.

Litopenaeus vannamei, as an important marine aquaculture species, has attracted more and more attentions in past several years. More recently people got its genome fine mapping, which unveiled a gene treasure. In this study, we have identified a novel trypsin-like protein which came from previous WSSV-infected shrimp plasma iTRAQ data. This protein is a 39 kDa protein with 363 amino acids. It contains a conserved trypsin-domain and could be strongly induced with WSSV infection. Interestingly, knockdown of this protein made shrimps vulnerable to WSSV infection. Further exploration unveiled that this fragility was probably due to the fact that knockdown of this protein could cause shrimp hemocytes apoptosis, which indicated that this protein played key roles in preventing shrimp hemocytes from apoptosis. To further explore how LvTLAP protected shrimp hemocytes from apoptosis, GST pull down assay was applied to screen LvTLAP interacting protein in shrimp plasma. L. vannamei growth and transformation-dependent-like protein (LvGTD-like protein) was identified as a LvTLAP interacting protein, which played proapoptotic roles in cells. Thus, a possible explanation for LvTLAP anti-apoptosis activity was that this protein could block LvGTD-like protein proapoptotic activity to protect shrimp hemocytes from death. In general, our study has uncovered a novel WSSV responsive shrimp plasma protein, which played key roles in shrimp hemocytes anti-apoptosis and shrimp against WSSV infection.

Molecular characterization of a novel white spot syndrome virus response protein (dubbed LvWRP) from Litopenaeus vannamei.

White spot syndrome, which is caused by white spot syndrome virus (WSSV), is a highly contagious disease of penaeid shrimp. However, there is currently incomplete understanding of the infection mechanism and pathogenesis of WSSV. In this study, a novel gene of a previously uncharacterized WSSV response protein (LvWRP) in Litopenaeus vannamei was identified and characterized. The LvWRP gene has an open reading frame (ORF) of 879 bp encoding a putative protein of 292 amino acids. Sequence analysis revealed that LvWRP shared 24.9% identity with an uncharacterized protein of Penaeus monodon nudivirus. Real-time qPCR analysis showed that LvWRP was ubiquitously expressed in shrimp tissues, with transcript levels induced in hemocytes upon immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and WSSV. In addition, RNA interference-mediated knockdown of LvWRP followed by WSSV challenge revealed significant decrease in the transcript levels of WSSV IE1 and VP28 genes coupled with a reduction in WSSV copies in shrimp hemocytes. Moreover, depletion of LvWRP followed by WSSV challenge significantly increased the transcript levels of Vago4 and Vago5 as well as increased the phosphorylation of STAT, while hemocytes apoptosis in terms of caspase 3/7 activity was decreased. These results suggest that LvWRP is important for WSSV replication in shrimp, and therefore one of the vital host factors in WSSV infection.

Quantitative serum proteomics analyses reveal shrimp responses against WSSV infection.

For the past decades, quantitative serum proteomics in mammals have been widely applied in biomarker screening. Various serum protein removal methods have been developed to effectively sequester serum abundant proteins. However, few methods have been found for the removal of arthropod serum abundant proteins. Here, gel filtration chromatography and ultracentrifugation methods were applied to remove hemocyanin from Litopeaneus vannamei serum. When shrimps were challenged with white spot syndrome virus (WSSV), a total of 486 serum proteins were identified using mass spectrometry, and 18 upregulated WSSV responsive proteins were identified with isobaric tags for relative and absolute quantification (iTRAQ). These results provide an effective method to remove hemocyanin from shrimp serum. With this method some previously unidentified WSSV responsive serum proteins were revealed, which would give us a better insight into the response of crustaceans to WSSV infection.

Transcriptome analyses reveal Litopenaeus vannamei hemocytes response to lipopolysaccharide.

Although vertebrate immunity has been well studied for the past decades, invertebrate immunity was much less explored. One possible reason was that in vitro culture system was not well established. In this study, Litopenaeus vannamei was applied as an invertebrate study model. Primary culture conditions for L. vannamei hemocytes were optimized to get relatively quiescent state cells. LPS was used as an immune stimulator and the responses of primary cultured hemocytes were transcriptomically analyzed. Our results showed that around 1,600 genes were upregulated and 800 genes were downregulated from LPS treated hemocytes. The altered genes could be classified into three categories: upregulated, downregulated, upregulated and then downregulated. Further qPCR validation showed that ubiquitin, ubiquitin-conjugating enzyme E2 C, ubiquitin-conjugating enzyme H1 and ubiquitin-conjugating enzyme H5b in ubiquitin-proteasome pathway were upregulated, cytochrome c oxidase 1, NADH dehydrogenase 1, Inosine-5'-monophosphate dehydrogenase 1b and phospholipid-transporting ATPase IA in mitochondria oxidation phosphorylation were downregulated. Our results showed that L. vannamei hemocyte inflammation responses share a lot of similarities with mammalian macrophage inflammation responses.