Development and validation of a nomogram of all-cause mortality in adult Americans with diabetes.

This study aimed to develop and validate a predictive model of all-cause mortality risk in American adults aged ≥ 18 years with diabetes. 7918 participants with diabetes were enrolled from the National Health and Nutrition Examination Survey (NHANES) 1999-2016 and followed for a median of 96 months. The primary study endpoint was the all-cause mortality. Predictors of all-cause mortality included age, Monocytes, Erythrocyte, creatinine, Nutrition Risk Index (NRI), neutrophils/lymphocytes (NLR), smoking habits, alcohol consumption, cardiovascular disease (CVD), urinary albumin excretion rate (UAE), and insulin use. The c-index was 0.790 (95% CI 0.779-0.801, P < 0.001) and 0.792 (95% CI: 0.776-0.808, P < 0.001) for the training and validation sets, respectively. The area under the ROC curve was 0.815, 0.814, 0.827 and 0.812, 0.818 and 0.829 for the training and validation sets at 3, 5, and 10 years of follow-up, respectively. Both calibration plots and DCA curves performed well. The model provides accurate predictions of the risk of death for American persons with diabetes and its scores can effectively determine the risk of death in outpatients, providing guidance for clinical decision-making and predicting prognosis for patients.

MSTN modification in goat mediated by TALENs and performance analysis.

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. It can inhibit the proliferation of myoblasts and serve as an important candidate gene for animal breed improvement. Mutations of the MSTN gene can cause extensive skeletal muscle hyperplasia and hypertrophy, resulting in "double muscle" symptoms. This leads to reduction of animal fat differentiation and increase of muscle content, thereby meeting the demand for quality consumption of animal meat in the market. In order to obtain a double-muscle phenotype using mutant MSTN gene in cloned goat, the goat MSTN gene was target-modified by TALENs. In this study, the TALENs expression vector was designed and constructed in the first exon sequence of the goat MSTN gene, which was then transfected into the goat fetal fibroblasts. The resistant cell lines were obtained by puromycin selection, and the cell lines with the MSTN gene mutations were analyzed by PCR and gene sequencing, thereby identifying the mutation type(s). The MSTN gene mutant cell lines were used as the nuclear donor cells in somatic cell nuclear transfer procedures in goats, and The morphological structure of the muscle tissue of the goats with MSTN gene mutations was analyzed by tissue section. The body weight of the cloned goats were monitored at different months of age, which provided the growth trend of their weight at different developmental stages. The results show that a total of 109 MSTN gene mutant cell lines were obtained. The mutation efficiency was 79.0% (109/138), of which 46 were biallelic mutations, accounting for 33.3% (46/138) of the total cell lines. Four MSTN gene mutant cell lines (1 biallelic homozygous mutation, 3 non-homozygous mutations) with good growth status were selected for somatic cell nuclear transfer in 12 recipients, of which 4 were pregnant by B-ultrasound at 30 days, indicating the a 33.3% (4/12) pregnancy rate. Two cloned goats were born at the end of the pregnancy. Sequencing analysis showed that there was no mutation in one allele of the M-1 cloned lamb, and the other allele harbored a 3 bp-deletion. The M-2 cloned lamb harbored a 1 bp base insertion in one allele of the MSTN gene, and a deletion of 13 bp in the other allele, resulting in mutations in both alleles and the loss of the protein-coding sequence of MSTN after the mutation site. In addition, the muscle fibers of cloned M-1 goats are tightly arranged and thick, and their monthly body weight is higher than that of normal wild-type goats. However, it is still consistent with the growth trend of normal wild-type goats and the M-1 goats can develop into healthy adults. In summary, this study showed that goat fetal fibroblasts with the multiple MSTN gene mutations were successfully obtained by TALENs technology, and cloned goats with mutant MSTN genes could be generated by somatic cell nuclear transfer method, thereby providing a technical foundation for the cultivation of the "double muscle" phenotype goats, and serving as a reference method for the preparation of other transgenic animals in the future.

Human lactoferrin efficiently targeted into caprine beta-lactoglobulin locus with transcription activator-like effector nucleases.

OBJECTIVE: To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF) with transcription activator-like effector nucleases. METHODS: TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG) locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT). RESULTS: The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23%) were confirmed pregnant at 30 d. In second round SCNT, 7 (53%), 4 (31%), and 3 (23%) recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. CONCLUSION: This finding signifies the combined use of TALENs and SCNT can generate bi-allelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.