Fecal gene detection based on next generation sequencing for colorectal cancer diagnosis.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies worldwide. Given its insidious onset, the condition often already progresses to advanced stage when symptoms occur. Thus, early diagnosis is of great significance for timely clinical intervention, efficacy enhancement, and prognostic improvement. Featuring high throughput, fastness, and rich information, next generation sequencing (NGS) can greatly shorten the detection time, which is a widely used detection technique at present. AIM: To screen specific genes or gene combinations in fecal DNA that are suitable for diagnosis and prognostic prediction of CRC, and to establish a technological platform for CRC screening, diagnosis, and efficacy monitoring through fecal DNA detection. METHODS: NGS was used to sequence the stool DNA of patients with CRC, which were then compared with the genetic testing results of the stool samples of normal controls and patients with benign intestinal disease, as well as the tumor tissues of CRC patients. Specific genes or gene combinations in fecal DNA suitable for diagnosis and prognostic prediction of CRC were screened, and their significances in diagnosing CRC and predicting patients' prognosis were comprehensively evaluated. RESULTS: High mutation frequencies of TP53, APC, and KRAS were detected in the stools and tumor tissues of CRC patients prior to surgery. Contrastively, no pathogenic mutations of the above three genes were noted in the postoperative stools, the normal controls, or the benign intestinal disease group. This indicates that tumor-specific DNA was detectable in the preoperative stools of CRC patients. The preoperative fecal expression of tumor-associated genes can reflect the gene mutations in tumor tissues to some extent. Compared to the postoperative stools and the stools in the two control groups, the pathogenic mutation frequencies of TP53 and KRAS were significantly higher for the preoperative stools (χ 2 = 7.328, P < 0.05; χ 2 = 4.219, P < 0.05), suggesting that fecal TP53 and KRAS genes can be used for CRC screening, diagnosis, and prognostic prediction. No significant difference in the pathogenic mutation frequency of the APC gene was found from the postoperative stools or the two control groups (χ 2 = 0.878, P > 0.05), so further analysis with larger sample size is required. Among CRC patients, the pathogenic mutation sites of TP53 occurred in 16 of 27 preoperative stools, with a true positive rate of 59.26%, while the pathogenic mutation sites of KRAS occurred in 10 stools, with a true positive rate of 37.04%. The sensitivity and negative predictive values of the combined genetic testing of TP53 and KRAS were 66.67% (18/27) and 68.97%, respectively, both of which were higher than those of TP53 or KRAS mutation detection alone, suggesting that the combined genetic testing can improve the CRC detection rate. The mutation sites TP53 exon 4 A84G and EGFR exon 20 I821T (mutation start and stop positions were both 7579436 for the former, while 55249164 for the latter) were found in the preoperative stools and tumor tissues. These "undetected" mutation sites may be new types of mutations occurring during the CRC carcinogenesis and progression, which needs to be confirmed through further research. Some mutations of "unknown clinical significance" were found in such genes as TP53, PTEN, KRAS, BRAF, AKT1, and PIK3CA, whose clinical values is worthy of further exploration. CONCLUSION: NGS-based fecal genetic testing can be used as a complementary technique for the CRC diagnosis. Fecal TP53 and KRAS can be used as specific genes for the screening, diagnosis, prognostic prediction, and recurrence monitoring of CRC. Moreover, the combined testing of TP53 and KRAS genes can improve the CRC detection rate.

Effects of different surgical treatments on children with ankyloglossia: protocol for a systematic review and meta-analysis.

INTRODUCTION: Ankyloglossia is a situation where the tongue tip cannot go beyond the mandibular incisor because the frenulum linguae is short. It could affect children's health by interfering with their ability to talk, breast feeding and dental development. The most effective measure to control ankyloglossia is the surgical method. However, which surgical procedure is the best one is still controversial. Thus, this protocol aims to assess the effectiveness of different surgical interventions in children with ankyloglossia. METHODS AND ANALYSIS: PubMed, EMBASE, Cochrane Library, Web of Science and OVID will be searched for relevant information from inception to 31 May 2022. Observational studies in English that investigate the association between surgical methods and ankyloglossia will be included in this protocol. This protocol follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols. The Critical Appraisal Checklist for Analytical Cross-Sectional Studies and the Newcastle-Ottawa Quality Assessment Scale for longitudinal studies will be used to assess the included studies. The improvement of breast feeding and nipple pain will be the primary outcome. STATA V.15.1 will do the statistical analysis in the meta-analysis. Subgroup and meta-regression will be carried out based on the characteristics of included studies. ETHICS AND DISSEMINATION: This systematic review and meta-analysis will summarise relevant information on the effects of different surgical treatments on patients with ankyloglossia. The results will be disseminated through peer-reviewed publications. The data included in this study will be extracted from the published original studies. Thus, ethical approval and informed consent will not be required. PROSPERO REGISTRATION NUMBER: CRD42022323350.

Enhancement of docosahexaenoic acid synthesis by manipulation of antioxidant capacity and prevention of oxidative damage in Schizochytrium sp.

Oxygen-mediated cell damage is an important issue in aerobic fermentation. In order to counteract these problems, effect of ascorbic acid on cell growth and docosahexaenoic acid (DHA) production was investigated in Schizochytrium sp. Addition of 9g/L ascorbic acid resulted in 16.16% and 30.44% improvement in cell dry weight (CDW) and DHA yield, respectively. Moreover, the total antioxidant capacity (T-AOC) of cells decreased from 2.17 at 12h to 0 at 60h and did not recover, while ascorbic acid addition could extend the time of arrival zero with the reduced intracellular ROS. However, ROS levels still increased after 72h. Therefore, to further solve the problem of high ROS levels and low T-AOC of cells after 72h, a two-point addition strategy was proposed. With this strategy, DHA yield was further increased to 38.26g/L. This work innovatively investigated the feasibility of manipulating Schizochytrium sp. cultivation through ROS level and T-AOC.

Adaptive evolution of Schizochytrium sp. by continuous high oxygen stimulations to enhance docosahexaenoic acid synthesis.

Adaptive laboratory evolution (ALE) is an effective method in changing the strain characteristics. Here, ALE with high oxygen as a selection pressure was applied to improve the production capacity of Schizochytrium sp. Results showed that cell dry weight (CDW) of endpoint strain was 32.4% higher than that of starting strain. But slight lipid accumulation impairment was observed. These major performance changes were accompanied with enhanced isocitrate dehydrogenase enzyme activity and reduced ATP:citrate lyase enzyme activity. And a serious decrease of 62.6% in SDHA 140rpm→170rpm was observed in the endpoint strain. To further study the docosahexaenoic acid (DHA) production ability of evolved strain, fed-batch strategy was applied and 84.34g/L of cell dry weight and 26.40g/L of DHA yield were observed. In addition, endpoint strain produced greatly less squalene than starting strain. This work demonstrated that ALE may be a promising tool in modifying microalga strains.

Introduction of ω-3 Desaturase Obviously Changed the Fatty Acid Profile and Sterol Content of Schizochytrium sp.

ω-3 fatty acids play significant roles in brain development and cardiovascular disease prevention and have been widely used in food additives and the pharmaceutical industry. The aim of this study was to assess the feasibility of ω-3 desaturase for regulating fatty acid composition and sterol content in Schizochytrium sp. The exogenous ω-3 desaturase gene driven by ubiqutin promoter was introduced by 18S homologous sequence to the genome of Schizochytrium sp. Genetically modified strains had greater size and lower polar lipids than wild type strains. In addition, the introduction of ω-3 desaturase improved the ω-3/ω-6 ratio from 2.1 to 2.58 and converted 3% docosapentaenoic acid (DPA) to docosahexaenoic acid (DHA). Furthermore, squalene and sterol contents in lipid of the genetically modified strain reduced by 37.19 and 22.31%, respectively. The present study provided an advantageous genetically engineered Schizochytrium sp. for DHA production and effective metabolic engineering strategy for fatty acid producing microbes.

The roles of different salts and a novel osmotic pressure control strategy for improvement of DHA production by Schizochytrium sp.

The effects of different osmotic pressure, changed by six salts (NaCl, Na2SO4, (NH4)2SO4, KH2PO4 and MSG), on cell growth and DHA synthesis by Schizochytrium sp. were investigated. Six optimal mediums were obtained to study different osmotic pressure combinations at cell growth stage and DHA synthesis stage. Results showed that cultivated cell in higher osmotic pressure condition and fermented in lower osmotic pressure condition was benefit to enhance DHA synthesis. Combination 17-6 could get the maximum cell dry weight of 56.95 g/L and the highest DHA percentage in total fatty acids of 55.21%, while combination 17-B could get the highest lipid yield of 33.47 g/L with 42.10% DHA in total fatty acids. This was the first report about the enhancement of DHA production by osmotic regulation and this work provided two novel osmotic control processes for high lipid yield and high DHA percentage in total fatty acids.