With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.

Metabolic diseases caused by gene and unhealthy living habits are increasing, which seriously threaten the life of people worldwide. Moreover, the microbiome has been shown to play an active role in the prevention and treatment of metabolic diseases. However, reliable evidence on renalase gene (Rnls), as a common gene related to metabolic diseases, is still lacking with regard to the influence on the microbiome. Hence, we investigated the effect of a normal diet (ND) and a high-fat diet (HFD) on the gut microbiota of Rnls knockout (Rnls^−/−) and wild-type (Rnls^+/+) mice. At the end of the 8-week experiment, DNA samples were extracted from fresh feces, and the composition of microbiota was profiled. The species in Rnls^+/+-ND group were Bifidobacterium pseudolongum and Lactobacillus reuteri. Conversely, the species in Rnls^−/−-ND group belonged to the genera Lactobacillus and Turicibacter. The HFD changed the ratio of Firmicutes/Bacteroidetes; while the bacteria in the Rnls^+/+-HFD and Rnls^−/−-HFD groups were different. Overall, this study not only revealed the composition of microbiota in Rnls^−/− mice, but also indicated that Rnls and the bacteria related to Rnls may be new candidates in the prevention and diagnosis of metabolic diseases at an early stage.

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injury. However, its molecular mechanisms are unknown. To clarify these mechanisms, in this study, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cAMP response element-binding protein 1 (CREB1) was markedly phosphorylated (as pCREB1) and CREB-binding protein (CBP) was recruited to pCREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes such as Pgc-1α involved in mitochondrial biogenesis. The foregoing results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes in a manner dependent on cold stimulation frequency and duration. This information may serve as an impetus for further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.