Background It is currently unclear whether erythropoietin (EPO) has value in predicting the prognosis of acute aortic syndrome (AAS). Methods A real-world analysis was conducted on the relationship between EPO and AAS outcomes. The primary end points were all-cause mortality and aortic-related mortality. The major adverse cardiac and cerebral events included all-cause mortality, acute myocardial infarction, stroke, and secondary procedures. Serum EPO concentration was examined using the Quantikine Human EPO ELISA. Results A total of 254 AAS patients were recruited, of whom 73 died. The median time of follow-up was 18.1 months. In Cox regression analysis, log EPO (hazard ratio [HR]: 2.491, P < 0.001), Stanford type A (HR: 3.003, P < 0.001), and treatment with surgery/thoracic endovascular aortic repair (TEVAR; HR: 0.283, P < 0.001) were independent predictors of all-cause mortality. In Kaplan-Meier curves, patients with EPO ≤16 IU/mL had significantly higher survival and event-free rates. Nomograms indicated that log EPO had the greatest contribution to AAS prognosis. In subgroup analysis, log EPO was an important prognostic indicator for patients with type A AAS and those treated without surgery/TEVAR. In correlation analyses, EPO was positively correlated with C-reactive protein, interleukin (IL)-4, IL-6, and monocytes and negatively correlated with albumin. The maximum diameter of the ascending aorta was positively correlated with EPO in AAS and aortic dissection (AoD) patients. The false lumen diameter and false lumen area were positively correlated with EPO in AoD patients. Conclusion The serum EPO level is closely associated with the adverse outcomes of AAS and has good prognostic value.

Previous studies have reported that mitochondrial DNA copy number (mtDNA-CN) of blood was associated with a series of aging-related diseases. However, it remains unknown whether mtDNA-CN can be a potential biomarker of acute aortic syndromes (AASs). The mtDNA-CN in blood of 190 male patients with AAS and 207 healthy controls were detected by standardized real-time quantitative PCR-based assay. The mtDNA sequencing data of blood and myocardial muscle in 134 individuals were used to analyze mtDNA somatic mutations in blood. mtDNA-CN in peripheral blood was negatively correlated with age of individuals. Further analysis based on next-generation sequencing data demonstrated numbers and heteroplasmy of mtDNA mutations were positively correlated with age. Remarkably, mtDNA-CN of patients with AAS was lower than that of healthy controls. Logistic regression also showed that mtDNA-CN was independently associated with risk of AAS. During follow-up, patients with the lowest mtDNA-CN quartile had a hazard ratio of 2.543 for all-cause-mortality and 1.964 for composite end points compared with the other patients. Moreover, multivariate Cox regression indicated that lowest mtDNA-CN quartile was independently associated with all-cause mortality in patients with AAS. Our study demonstrated a negative correlation between mtDNA-CN and age. Moreover, lower mtDNA-CN in peripheral blood was significantly associated with higher risk and worse prognosis of AAS. It provided crucial evidence supporting the potential of mtDNA-CN as a novel biomarker of AAS.

Background: Secreted frizzled-related protein 5 (SFRP5) is a novel adipokine that has been found to be closely associated with metabolic and cardiovascular diseases. We investigated serum SFRP5 levels during the acute phase and their predictive value for the prognosis of acute aortic dissection (AAD). Methods: In total, 152 AAD patients and 164 controls were enrolled in this study. Serum SFRP5 levels were measured using an enzyme-linked immunosorbent assay (ELISA). AAD patients were divided into high-SFRP5 and low-SFRP5 groups based on the optimal cutoff value and followed up for prognosis. The primary endpoint was all-cause mortality, and the secondary endpoint focused on AAD-related events (including AAD-related mortality and unplanned reoperations). Results: Serum SFRP5 levels were significantly higher in AAD patients than in non-AAD controls, regardless of whether they had Stanford type A or B AD. Multivariate logistic regression analysis revealed an independent association between SFRP5 and the presence of AAD (adjusted OR 1.267, 95 % CI 1.152–1.394; p < 0.001). The receiver operating characteristic curve demonstrated that the optimal cutoff value for SFRP5 to predict the presence of AAD was 10.26 ng/mL (AUC 0.7241, sensitivity 49.34 %, specificity 87.20 %). Notably, serum SFRP5 levels of patients in the death group were significantly higher than those in the survival group. Compared with patients in the low-SFRP5 group, those in the high-SFRP5 group exhibited a significantly increased risk of all-cause mortality (HR 9.540, 95 % CI 2.803–32.473; p < 0.001) and AAD-related events (HR 6.915, 95 % CI 2.361–20.254; p < 0.001) during the follow-up period. Conclusion: Serum SFRP5 levels were significantly elevated in the acute phase of AAD, and high serum SFRP5 levels were independently associated with poor AAD prognosis. These results suggest that serum SFRP5 level during the acute phase may be an effective biomarker and therapeutic target for the prognosis of AAD.

Background Disruptions in calcium homeostasis are associated with a wide range of diseases, and play a pivotal role in the development of cancer. However, the construction of prognostic models using calcium extrusion-related genes in colon adenocarcinoma (COAD) has not been well studied. We aimed to identify whether calcium extrusion-related genes serve as a potential prognostic biomarker in the COAD progression. Methods We constructed a prognostic model based on the expression of calcium extrusion-related genes (SLC8A1, SLC8A2, SLC8A3, SLC8B1, SLC24A2, SLC24A3 and SLC24A4) in COAD. Subsequently, we evaluated the associations between the risk score calculated by calcium extrusion-related genes and mutation signature, immune cell infiltration, and immune checkpoint molecules. Then we calculated the immune score, stromal score, tumor purity and estimate score using the Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm. The response to immunotherapy was assessed using tumor immune dysfunction and exclusion (TIDE). Finally, colorectal cancer cells migration, growth and colony formation assays were performed in RKO cells with the overexpression or knockdown SLC8A3, SLC24A2, SLC24A3, or SLC24A4. Results We found that patients with high risk score of calcium extrusion-related genes tend to have a poorer prognosis than those in the low-risk group. Additionally, patients in high-risk group had higher rates of KRAS mutations and lower MUC16 mutations, implying a strong correlation between KRAS and MUC16 mutations and calcium homeostasis in COAD. Moreover, the high-risk group showed a higher infiltration of regulatory T cells (Tregs) in the tumor microenvironment. Finally, our study identified two previously unreported model genes (SLC8A3 and SLC24A4) that contribute to the growth and migration of colorectal cancer RKO cells. Conclusions Altogether, we developed a prognostic risk model for predicting the prognosis of COAD patients based on the expression profiles of calcium extrusion-related genes, Furthermore, we validated two previously unreported tumor suppressor genes (SLC8A3 and SLC24A4) involved in colorectal cancer progression.

Background Omicron variant of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global threat to public health. Numerous asymptomatic and mild cases had been admitted in shelter hospitals to quickly win the fight against Omicron pandemic in Shanghai. However, little is known about influencing factors for deterioration and length of stay (LOS) in hospitals among these non-severe cases. Methods This study included 12,555 non-severe cases with COVID-19 in largest shelter hospital of Shanghai, aiming to explore prognostic factors and build effective models for prediction of LOS. Results Data showed that 75.0% of participants were initially asymptomatic. In addition, 94.6% were discharged within 10 days, only 0.3% with deterioration in hospitals. The multivariate analysis indicated that less comorbidities (OR = 1.792, P = 0.012) and booster vaccination (OR = 0.255, P = 0.015) was associated with the decreased risk of deterioration. Moreover, age (HR = 0.991, P < 0.001), number of symptoms (HR = 0.969, P = 0.005), time from diagnosis to admission (HR = 1.013, P = 0.001) and Cycle threshold (CT) values of N gene (HR = 1.081, P < 0.001) were significant factors associated with LOS. Based on these factors, a concise nomogram model for predicting patients discharged within 3 days or more than 10 days was built in the development cohort. In validation cohort, 0.75 and 0.73 of Areas under the curve (AUC) in nomograms, similar with AUC in models of simple machine learning, showed good performance in estimating LOS. Conclusion Collectively, this study not only provides important evidence to deeply understand clinical characteristics and risk factors of short-term prognosis in Shanghai Omicron outbreaks, but also offers a concise and effective nomogram model to predict LOS. Our findings will play critical roles in screening high-risk groups, providing advice on duration of quarantine and helping decision-makers with better preparation in outbreak of COVID-19.

Objective To investigate the risk factors associated with post-implantation syndrome (PIS) in Stanford type B aortic dissection (TBAD) patients undergoing thoracic endovascular aortic repair (TEVAR) and the relationship of PIS with short-term outcomes. Methods A case-control trial was conducted on the clinical data of patients undergoing TEVAR treatment for TBAD in our department from December 2013 to December 2022. They were divided into 2 groups based on the occurrence of PIS. Independent risk factors were analyzed according to the different clinical characteristics between the 2 groups. The overall 30-day mortality following TEVAR was assessed in both groups. Kaplan-Meier survival curves were utilized to evaluate the 30-day postoperative survival. Logistic regression models were constructed to investigate the relationship between PIS and overall mortality occurring within 30 d after TEVAR. Results A total of 373 TBAD patients, with an average age of 57.9±12.1 years and a male ratio of 77.5% (289 cases), were enrolled in this study. There were 106 cases (28.4%) experiencing PIS following TEVAR. Significantly larger proportions of implanted stents ≥2, operation duration ≥2 h and implantation of polyethylene terephthalate (PET)-coverted stents were observed in the PIS group than the non-PIS group (P < 0.05). Multivariate logistic regression analysis suggested that implanted stents ≥2 (OR=1.886, 95%CI: 1.049~3.389, P=0.034), operation duration ≥2 h (OR=1.938, 95%CI: 1.147~3.275, P=0.013) and implantation of PET-coverted stents (OR=2.131, 95%CI: 1.263~3.597, P=0.005) were independent risk factors for PIS after TEVAR. Within 30 d after TEVAR, 15 (4.0%) cases dead, including 10 (9.4%) from the PIS group and 5 (1.9%) from the non-PIS group. Kaplan-Meier survival curve indicated a significantly lower survival in the PIS group (Log-rank P=0.000 7). The results of multivariate logistic regression model indicated that cystatin C (OR=1.486, 95%CI: 1.053~2.097, P=0.024) and PIS (OR=5.628, 95%CI: 1.836~17.248, P=0.002) were independent risk factors for 30-day mortality after TEVAR in TBAD patients. Conclusion Implanted stents ≥2, operation duration ≥2 h and implantation of PET-coverted stents are closely associated with the occurrence PIS in TBAD patients after TEVAR. Cystatin C level and PIS are primary risk factors for poor 30-day prognosis after TEVAR.

Background Hemorrhage is a potential and serious adverse drug reaction, especially for geriatric patients with long-term administration of rivaroxaban. It is essential to establish an effective model for predicting bleeding events, which could improve the safety of rivaroxaban use in clinical practice. Methods The hemorrhage information of 798 geriatric patients (over the age of 70 years) who needed long-term administration of rivaroxaban for anticoagulation therapy was constantly tracked and recorded through a well-established clinical follow-up system. Relying on the 27 collected clinical indicators of these patients, conventional logistic regression analysis, random forest and XGBoost-based machine learning approaches were applied to analyze the hemorrhagic risk factors and establish the corresponding prediction models. Furthermore, the performance of the models was tested and compared by the area under curve (AUC) of the receiver operating characteristic (ROC) curve. Results A total of 112 patients (14.0%) had bleeding adverse events after treatment with rivaroxaban for more than 3 months. Among them, 96 patients had gastrointestinal and intracranial hemorrhage during treatment, which accounted for 83.18% of the total hemorrhagic events. The logistic regression, random forest and XGBoost models were established with AUCs of 0.679, 0.672 and 0.776, respectively. The XGBoost model showed the best predictive performance in terms of discrimination, accuracy and calibration among all the models. Conclusion An XGBoost-based model with good discrimination and accuracy was built to predict the hemorrhage risk of rivaroxaban, which will facilitate individualized treatment for geriatric patients.

A recent letter published in this journal provided important evidence for systematically understanding clinical characteristics and outcomes of 1965 patients infected by Omicron in Shanghai.1Wang B. Yu Y. Yu Y. et al.Clinical features and outcomes of hospitalized patients with COVID-19 during the Omicron wave in Shanghai, China.J Infect. 2022; Google Scholar Omicron variant of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly resulted in numerous asymptomatic and mild cases during pandemic of Corona virus disease 2019 (COVID-19) in Shanghai.2Ao Y. Li J. Wei Z. et al.Clinical and virological characteristics of SARS-CoV-2 Omicron BA.2.2 variant outbreaks during April to May, 2022, Shanghai, China.J Infect. 2022; 85: 573-607Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar Considering higher infectivity and lower severity of Omicron, shelter hospitals providing isolation for non-severe cases were used to effectively cut down further spread of virus.3Zhang X. Zhang W. Chen S. Shanghai's life-saving efforts against the current omicron wave of the COVID-19 pandemic.Lancet. 2022; 399: 2011-2012Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar In shelter hospitals, there were many patients combined with cardiovascular disease (CVD), who might have different clinical features and prognostic outcomes. According to previous studies in Wuhan, CVD patients infected with SARS-CoV-2 were more likely to have severe pulmonary edema, multiple organ failure and high rate of mortality.4Ssentongo P. Ssentongo A.E. Heilbrunn E.S. Ba D.M. Chinchilli VM. Association of cardiovascular disease and 10 other pre-existing comorbidities with COVID-19 mortality: a systematic review and meta-analysis.PLoS One. 2020; 15e0238215Crossref Scopus (296) Google Scholar, 5Semenzato L. Botton J. Drouin J. et al.Chronic diseases, health conditions and risk of COVID-19-related hospitalization and in-hospital mortality during the first wave of the epidemic in France: a cohort study of 66 million people.Lancet Reg Health Eur. 2021; 8: 44Google Scholar However, little is known about Omicron infection combined with CVD. We aimed to comprehensively analyze clinical characteristics and outcomes of CVD patients infected with Omicron in shelter hospital. In this study, a total of 1589 COVID-19 patients with confirmed diagnoses of hypertension, coronary heart disease (CHD) or chronic heart failure (CHF) in Shelter Hospital of National Convention and Exhibition Centre, the largest shelter hospital in China, were enrolled during April 14 to May 8 2022. Children under 18 years and pregnant women were excluded. Informed consent has been obtained from all the participants. This study was approved by the Ethics Committee of Chinese PLA General Hospital (S2022-769-01). RT-qPCR targeting nucleocapsid protein (N) and open reading frame 1ab (ORF lab) gene was daily detected for patients from admission to discharge. Primary outcomes were deterioration, which meant admission into designated hospitals or ICU. The second outcomes were length of stay (LOS) in hospital, which meant the days from admission to discharge in shelter hospital. Basic information, including gender, age, ethnicity, comorbidity, vaccination status, initial symptoms, was recorded and verified carefully. If some data were missing or unclear, we re-obtained them by directly communicating with patients within 24 hours. Continuous variables were described as median and interquartile range (IQR) and categorical variables were summarized as percentages and numbers. Fisher's exact probability test was performed to compare difference of proportions for categorical variables. The Mann-Whitney U test was performed to analyze the difference between two independent groups of continuous variables. Spearman's correlation was performed to evaluate correlations between stay length in hospital and other variables. In the entire cohort, median age was 57 years (IQR, 49–65). Distribution diagram in Fig. 1A revealed that age of adult patients was concentrated in range of 45-75 years old, in which 1264 patients accounted for 79.5%. The most common comorbidity of CVD was hypertension (1363, 85.8%), followed by CHD (397, 25.0%) and CHF (92, 5.8%) (Fig. 1B). 66.8% of participants were initially diagnosed with asymptomatic infection. Cough was the most common symptom (23.2%), followed by expectoration (14.7%), fatigue (8.3%) and fever (6.0%) (Fig. 1C). Similarly, a recent study showed that clinical symptoms of patients with Omicron infection were more complex and needed to be differentiated from influenza.6Wang M. Liu Z. Wang Z. et al.Clinical characteristics of 1139 mild cases of the SARS-CoV-2 Omicron variant infected patients in Shanghai.J Med Virol. 2022; Crossref Scopus (2) Google Scholar The median LOS was 5 days (IQR, 4–7). Distribution curve in Fig. 1D demonstrated that most of participants (98.0%) were discharged within 2 weeks, 77.9% discharged within 7 days. These findings can be explained by lower pathogenicity of Omicron variants7Obermeyer F. Jankowiak M. Barkas N. et al.Analysis of 6.4 million SARS-CoV-2 genomes identifies mutations associated with fitness.Science. 2022; 376: 1327-1332Crossref PubMed Scopus (64) Google Scholar and exclusion of severe cases from shelter hospitals on admission. Eventually, only 8 participants (0.5%) had deteriorated and were transferred to designated hospitals for further treatment. Results showed that CVD Patients with deterioration were significantly older than those without deterioration (67.5 vs. 57.0, P=0.005, Fig. 1E). In addition, percentage of deterioration among patients with CHF was remarkably higher than that without CHF (3.3% vs. 0.3%, P=0.002, Fig. 1F). These results suggested that patients who were older or with CHF should be treated more cautiously due to the higher risk of deterioration. In contrary, percentage of vaccinated patients in deterioration group was significantly lower than that in non-deterioration group (37.5% vs. 75.3%, P=0.040, Fig. 1G). Moreover, booster vaccination effectively reduced the rate of deterioration (0.1% vs. 1.3%, P=0.050, Fig. 1H). Therefore, accelerating coverage of vaccination, especially booster vaccination, might be an effective strategy to fight against COVID-19 pandemic in the future. Further, data showed that LOS in shelter hospital was positively correlated with age (Spearman r=0.136, P<0.001, Fig. 2A). Consistently, a previous study has revealed a similar trend that adults gradually recover slower from the infections as they age.1Wang B. Yu Y. Yu Y. et al.Clinical features and outcomes of hospitalized patients with COVID-19 during the Omicron wave in Shanghai, China.J Infect. 2022; Google Scholar In contrary, an inversely proportional relationship was observed between LOS and CT values of N gene on admission, semi-quantitative proxies of viral load (Spearman r=-0.096, P<0.001, Fig. 2B). Previous studies have demonstrated that Ct value is associated with biochemical and hematological markers,8Shi F. Wu T. Zhu X. et al.Association of viral load with serum biomakers among COVID-19 cases.Virology. 2020; 546: 122-126Crossref PubMed Scopus (52) Google Scholar disease severity9Liu Y. Yan L.M. Wan L. et al.Viral dynamics in mild and severe cases of COVID-19.Lancet Infect Dis. 2020; 20: 656-657Abstract Full Text Full Text PDF PubMed Scopus (1113) Google Scholar in COVID-19 patients. Compared with asymptomatic cases, those with cough, expectoration, fatigue, fever or myalgia had longer LOS in shelter hospital (both P<0.005, Fig. 2C). In addition, vaccinated patients had shorter LOS than unvaccinated patients (P=0.047, Fig. 2D). All of above characteristics could be used to predict the length of stay, manage public resource allocation and adjust pandemic control strategies faster. Taken together, this study provides important evidence to comprehensively understand clinical characteristics of CVD patients infected with Omicron. Milder symptoms, shorter hospital duration and better outcomes underline that pandemic control strategies for CVD patients should be improved in time. By analyzing risk factors of short-term prognosis, including deterioration and LOS, it was observed that older or unvaccinated CVD patients were more likely to have longer LOS and deterioration. Therefore, accelerating vaccination among CVD patients might play a contributing role better preparation during the COVID-19 future epidemic. All the authors declare no conflicts of interest. We thank all health workers working in Shelter Hospital of National Convention and Exhibition Centre. This study was supported by the National Social Science Foundation of China (2021-SKJJ-C-041).

目的 构建水下爆炸致兔急性心肺损伤模型,通过观察各组模型动脉血氧分压及心肺组织湿干重比值的差异,评估乌司他丁的治疗效果.通过分析乌司他丁治疗下模型血清中的炎性细胞因子水平的变化,初步探讨乌司他丁治疗水下爆炸急性心肺损伤的可能机制.方法 将40只新西兰雄兔随机分为正常对照组、爆炸损伤组、低剂量乌司他丁组和高剂量乌司他丁组.利用水下爆炸装置对爆炸损伤组、低剂量乌司他丁组和高剂量乌司他丁组造成急性心肺损伤,并在致伤后即刻对低剂量乌司他丁组和高剂量乌司他丁组分别进行2.5万U/kg和10万U/kg的乌司他丁治疗.检测实验兔在爆炸后4 h、12 h、24 h的动脉血氧分压(PaO2)及血清肿瘤坏死因子α(TNF-α)、白细胞介素8(IL-8)和中性粒细胞弹性蛋白酶(NE)的水平.爆炸24 h后测量家兔的心肺组织湿干重比值.结果 动脉血氧分压检测结果 提示:爆炸损伤组实验兔在致伤后4 h、12 h、24 h的PaO2分别为(84.97±6.41)、(79.13±9.92)、(78.24±3.53)mmHg,与正常对照组4 h(92.15±4.13)、12 h(89.39±7.54)、24 h(90.24±9.33)相比均明显下降(P=0.042,P=0.018,P=0.002),而低剂量和高剂量乌司他丁组并未观测到PaO2下降.与对照组相比,爆炸损伤组动物的心肌组织和肺组织的湿干重比值均显著升高(P=0.037,P=0.028).高剂量乌司他丁组的心肌组织湿干重比值明显低于爆炸损伤组(P=0.047).炎性细胞因子检测结果 提示:与爆炸损伤组相比,低剂量乌司他丁组的血清TNF-α含量在爆炸后12 h和24 h均明显降低(P=0.018,P=0.013);高剂量乌司他丁组的血清TNF-α含量也在爆炸后24 h低于爆炸损伤组(P=0.004).低剂量乌司他丁组和高剂量乌司他丁组的血清NE含量均在爆炸后24 h显著低于爆炸损伤组(P=0.007,P=0.036).高剂量乌司他丁组的血清IL-8在爆炸后4 h、12 h、24 h均显著低于爆炸损伤组(P=0.024,P=0.007,P=0.011).结论 成功建立了靶向致伤心肺的水下爆炸伤动物模型,初步证明了乌司他丁对水下爆炸致急性心肺损伤的治疗价值,为未来海上战伤救治提供决策依据.

Background: Acute myocardial infarction (AMI) is a rare complication of acute organophosphorus pesticide poisoning. Although chlorpyrifos has been widely used as an organophosphate insecticide, a few cases of AMI complicated by chlorpyrifos poisoning have been reported thus far. Hence, a suitable treatment strategy remains to be explored. Case Presentation: Based on the clinical manifestations, medical history, results of an auxiliary examination, and serum biomarkers, a 65-year-old male farmer with complaints of nausea, vomiting, chest tightness, and pain was clearly diagnosed as having a severe chlorpyrifos self-poisoning with acute non-ST-segment elevation MI. Because the patient and his family confirmedly refused a coronary intervention, conservative treatment was used instead. It should be noted that there were some conflicts of the management for chlorpyrifos poisoning and AMI. Although rapid atropinization would contribute to the relief of muscarinic symptoms, it would also lead to an increased heart rate and myocardial oxygen consumption in AMI. Furthermore, the reduction of platelet aggregation, which is necessary for coronary recanalization of an AMI patient, is known to aggravate the gastrointestinal injury caused by poisoning. In this case, these conflicts were properly addressed, which led to an excellent effect and prognosis of the patient. Conclusions: To our knowledge, this is the first case report of acute chlorpyrifos poisoning with AMI. It is emphasized that patients with chest pain or coronary heart disease should be treated with atropine more cautiously because of the possible AMI. Moreover, proper resolution of conflicts in the management for chlorpyrifos poisoning and AMI played contributing roles in patient improvement.

Next-generation sequencing-based methods have been commonly used for detecting mutations of mitochondrial genome (mtDNA). PCR amplification is a highly effective method of mtDNA enrichment before sequencing. However, it has been observed that highly variable sequencing depth within PCR amplicons severely reduces the coverage uniformity and accuracy of mutation calling. Therefore, it is necessary to develop an optimized PCR-based strategy for mtDNA sequencing. Herein, the effect of DNA quality on the efficiency of PCR amplification was analyzed and the effects of different primer-design methods, including the number of primer pairs, overlap length of amplicons, and modification of primers, on coverage uniformity and mutation calling in mtDNA sequencing were assessed. Results showed that DNA quality significantly affected the efficiency of PCR amplification. Importantly, over- and under-representation of coverage depth at overlap regions of amplicons were observed when amplicons were not modified and overlap was shorter than two sequencing fragment sizes (800 bp). Then, under-representation was overcome by increasing the overlap length of the amplicons, and over-representation was effectively reduced by 5'-block modification of primers and sticky-end ligation of amplicons. Moreover, findings indicated that these two optimized PCR-based sequencing strategies effectively improved mutation calling in primer-binding regions. Optimized PCR-based mtDNA enrichment and sequencing approaches have been established, which laid a foundation for accurate mutation detection of mtDNA in diseases.

Numerous studies have identified essential contributions of altered mitochondrial DNA (mtDNA) copy number and mutations in many common disorders, including cancer. To date, capture-based next-generation sequencing (NGS) has been widely applied to detect mtDNA mutations, although it lacks the ability to assess mtDNA copy number. The current strategy for quantifying mtDNA copy number relies mainly on real-time quantitative PCR, which is limited in degraded samples. A novel capture-based NGS approach was developed using both mtDNA and nuclear DNA probes to capture target fragments, enabling simultaneous detection of mtDNA mutations and copy number in different sample types. First, the impact of selecting reference genes on mtDNA copy number calculation was evaluated, and finally, 3 nuclear DNA fragments of 4000 bp were selected as an internal reference for detection. Then, the effective application of this approach was verified in DNA samples of formalin-fixed, paraffin-embedded specimens and body fluids, indicating the widespread applicability. This approach showed more accurate and stable results in detecting mtDNA copy number compared with real-time quantitative PCR in degraded DNA samples. Moreover, data indicated this approach had good reproducibility in detecting both mtDNA copy number and mutations among three sample types. Altogether, a versatile and cost-effective capture-based NGS approach has been developed for concurrent detection of mtDNA copy number and mutations, which has numerous applications in research and diagnosis.

BACKGROUND:DNAs released from tumor cells into blood (circulating tumor DNAs, ctDNAs) carry tumor-specific genomic aberrations, providing a non-invasive means for cancer detection. In this study, we aimed to leverage somatic copy number aberration (SCNA) in ctDNA to develop assays to detect early-stage HCCs.METHODS:We conducted low-depth whole-genome sequencing (WGS) to profile SCNAs in 384 plasma samples of hepatitis B virus (HBV)-related HCC and cancer-free HBV patients, using one discovery and two validation cohorts. To fully capture the robust signals of WGS data from the complete genome, we developed a machine learning-based statistical model that is focused on detection accuracy in early-stage HCC.FINDINGS:We built the model using a discovery cohort of 209 patients, achieving an overall area under curve (AUC) of 0.893, with 0.874 for early-stage (Barcelona clinical liver cancer [BCLC] stage 0-A) and 0.933 for advanced-stage (BCLC stage B-D). The performance of the model was then assessed in two validation cohorts (76 and 99 patients) that only consisted of patients with stage 0-A HCC. Our model exhibited a robust predictive performance, with an AUC of 0.920 and 0.812 for the two validation cohorts. Further analyses showed the impact of tumor sample heterogeneity in model training on detecting early-stage tumors, and a refined model addressing the heterogeneity in the discovery cohort significantly increased model performance in validation.INTERPRETATION:We developed an SCNA-based, machine learning-driven model in the non-invasive detection of early-stage HCC in HBV patients and demonstrated its performance through strict independent validations.

BACKGROUND Somatic mutations of mitochondrial DNA (mtDNA) have been extensively identified mainly by traditional Sanger sequencing technology in various cancer types. However, low detection sensitivity of traditional methods greatly limits the comprehensive profiling of mtDNA somatic mutations in cancers, especially in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Moreover, the functional roles of mtDNA mutation in HBV-related hepatocarcinogenesis have not been systematically revealed. PATIENTS AND METHODS Next-generation sequencing (NGS) platform was applied to profile the somatic mtDNA mutations of HCC and paired paratumor (non-HCC) tissues from a large cohort of 156 HBV-HCC patients. RESULTS Our data revealed the common existence of mtDNA mutation in both inflammatory and cancer tissues with significantly different mutation pattern. The mutation density (mutation number/region length) of D-loop region was much higher than that of other regions in both HCC and non-HCC tissues. Unexpectedly, the average mutation number in D-loop region of HCC tissues was significantly less than that of non-HCC tissues. In contrast, the heteroplasmy level of D-loop region mutations was significantly increased in HCC tissues, implying that the D-loop mutations might be positively selected in HCC tissues. Furthermore, our results indicated that the patients with D-loop mutations had a significantly lower mtDNA copy number and were more likely to relapse. In vitro experiments demonstrated that proliferation, invasion and metastasis ability of HCC cells with D-loop region mutations were significantly higher than those without D-loop region mutations. CONCLUSION These results emphasize the critical contributing role of somatic mtDNA D-loop mutations in HBV-related hepatocarcinogenesis.

结直肠癌是我国第三大常见癌症,现有的结直肠癌早期筛查和术后监测手段均存在局限性.循环肿瘤DNA(circulating tumor DNA,ctDNA)通过肿瘤细胞凋亡、坏死或分泌等途径释放到外周血,可反映肿瘤组织相关变异信息.随着二代测序(next generation sequencing,NGS)技术快速发展,血浆中微量ctDNA的精准检测为结直肠癌的早期筛查提供机遇.同时,纵向监测结直肠癌患者的ctDNA可以较好地反映肿瘤负荷的动态变化.本文拟从ctDNA检测在结直肠癌早期筛查、个性化用药指导、疗效监测以及预后评估等方面的潜在临床价值进行综述.

H1 histamine receptor (H1HR) belongs to the family of rhodopsin-like G-protein-coupled receptors. Recent studies have shown that H1HR expression is increased in several types of cancer. However, its functional roles in tumor progression remain largely unknown, especially in hepatocellular carcinoma (HCC). We found that H1HR is frequently unregulated in HCC, which is significantly associated with both recurrence-free survival and overall survival in HCC patients. Functional experiments revealed that H1HR promoted both the growth and metastasis of HCC cells by inducing cell cycle progression, formation of lamellipodia, production of matrix metalloproteinase 2, and suppression of cell apoptosis. Activation of cyclic adenosine monophosphate-dependent protein kinase A was found to be involved in H1HR-mediated HCC cell growth and metastasis. In addition, we found that overexpression of H1HR was mainly due to the downregulation of miR-940 in HCC cells. Moreover, the H1HR inhibitor terfenadine significantly suppressed tumor growth and metastasis in an HCC xenograft nude mice model. Our findings demonstrate that H1HR plays a critical role in the growth and metastasis of HCC cells, which provides experimental evidence supporting H1HR as a potential drug target for the treatment of HCC.

Heteroplasmic mutations in mitochondrial DNA (mtDNA) play critical roles in mitochondria disease, aging, and cancer. Recently, next-generation sequencing (NGS) has been widely used to detect mtDNA mutations for diagnosis and monitoring of the above-mentioned diseases. However, little attention is paid on inherent cross-contamination generated during mtDNA capture and sequencing of mixed samples, which may seriously reduce the detection accuracy of mtDNA heteroplasmic mutations. In this study, a novel sequencing strategy based on a unique double-barcode design was established. The results showed that when single barcode-based analysis strategy was used, cross-contamination level of 20 DNA samples ranged from 0.27% to 11.90% on HiSeq 2500 and from 0.93% to 17.70% on HiSeq X ten, whereas double barcode-based strategy could effectively eliminate cross-contamination. Moreover, the data indicated that cross-contamination was mainly derived from capture process and was significantly affected by different NGS platforms. In addition, contamination level was negatively related to sequencing depth. Moreover, cross-contamination significantly increased the false-positive calling of mtDNA heteroplasmic mutations and remarkably affected the heteroplasmy level of mtDNA mutations. In contrast, cross-contamination had no notable effect on classification of mtDNA haplogroup. Taken together, our novel double barcode-based sequencing strategy is effective in eliminating cross-contamination, enhancing the detection accuracy of mtDNA NGS, and improving its application in diagnosis or monitoring of diseases associated with mtDNA mutations.

近年来,液体活检技术在新型肿瘤标志物研发领域异军突起,新型检测技术、新产品、新应用不断涌现.肿瘤液体活检是通过各种技术检测肿瘤释放到体液,尤其是外周血中的肿瘤细胞、肿瘤游离DNA以及外泌体等物质,实现肿瘤精准诊疗.因具备微创性、能够克服肿瘤异质性、便于实时动态监测等优势,液体活检在肿瘤早期诊断及精准治疗等方面临床应用前景广阔.然而,基础研究、检测技术、质控规范及临床应用方面的诸多问题也对肿瘤液体活检提出了挑战.

The invention discloses a free mitochondrial DNA mutation detection technique based on a bibasic sequencing technique. The free mitochondrial DNA mutation detection technique mainly comprises the following steps of (1) preparing a cfDNA single-chain sequencing library; (2) performing mtDNA capturing; and (3) performing data analysis. Compared with the prior art, the free mitochondrial DNA mutationdetection technique has the advantages that in accordance with the characteristics of high gragmentation degree and low mutation frequency of free mtDNA, free mtDNA sequence information is enriched in NGS, so that the low-frequency mutation detection false positive problem is solved, interference of NUMT to mutation detection is overcome, and pinpoint detection of free mitochondrial DNA mutationis realized.

Liver cancer is a malignant tumor with the characteristics of high morbidity and mortality.However,the routine detection methods for the liver cancer such as imaging tests and pathological detection are with low specificity and sensitivity.And it's also hard to detect the condition of patients dynamically.Cell-free DNA (cfDNA) is the DNA fragment that is away from the extracellular and exists in the blood,which contains genetic information from the organism.The related disease information could be reflected on the cfDNA in patients with liver cancer,indicating that cfDNA has great potential in the screening,diagnosis,treatment and prognosis of liver cancer.The clinical applications of cfDNA can make up for the deficiency of existing detection methods and achieve noninvasive and dynamic monitoring of liver cancer.This article will present a review on the progress of clinical applications of cfDNA in liver cancer.