Exonuclease-iii -propelled DNAzyme cascade for sensitive and reliable cervical cancer related miRNA analysis.

MicroRNAs (miRNAs) can serve as biomarkers for early-diagnosis, therapy, and postoperative care of cervical cancer. Sensitive and reliable quantification of miRNA remains a huge challenge due to its low expressing levels and background interference. Herein, we propose a novel exonuclease-III (Exo-III)-propelled DNAzyme cascade for sensitive and high-efficient miRNA analysis. This method involves the engineering of compact DNAzyme hairpin probes, including the H1 probe and H2 probe. The H1 probe is designed with exposed analyte recognition subunits that can specifically recognize target miRNA. This recognition triggers two processes: Exo-iii-assisted target regeneration and successive substrate cleavage catalyzed by DNAzyme. The unique character of Exo-III that catalyzes removal of mononucleotides from the blunt or recessed 3'-OH termini of dsDNA confers the approach with a minimal background signal. The multiple signal cycles provided an abundant signal amplification and consequently, the method exhibited a low limit of detection of 3.12 fM, and a better specificity over several homologous miRNAs. In summary, this powerful Exo-III driven DNAzyme cascaded system offers broader and more adaptable methods for comprehending the activities of miRNA in various biological occurrences.

Distinct fibroblast subpopulations associated with bone, brain or intrapulmonary metastasis in advanced non-small-cell lung cancer.

BACKGROUND: Bone or brain metastases may develop in 20-40% of individuals with late-stage non-small-cell lung cancer (NSCLC), resulting in a median overall survival of only 4-6 months. However, the primary lung cancer tissue's distinctions between bone, brain and intrapulmonary metastases of NSCLC at the single-cell level have not been underexplored. METHODS: We conducted a comprehensive analysis of 14 tissue biopsy samples obtained from treatment-naïve advanced NSCLC patients with bone (n = 4), brain (n = 6) or intrapulmonary (n = 4) metastasis using single-cell sequencing originating from the lungs. Following quality control and the removal of doublets, a total of 80 084 cells were successfully captured. RESULTS: The most significant inter-group differences were observed in the fraction and function of fibroblasts. We identified three distinct cancer-associated fibroblast (CAF) subpopulations: myofibroblastic CAF (myCAF), inflammatory CAF (iCAF) and antigen-presenting CAF (apCAF). Notably, apCAF was prevalent in NSCLC with bone metastasis, while iCAF dominated in NSCLC with brain metastasis. Intercellular signalling network analysis revealed that apCAF may play a role in bone metastasis by activating signalling pathways associated with cancer stemness, such as SPP1-CD44 and SPP1-PTGER4. Conversely, iCAF was found to promote brain metastasis by activating invasion and metastasis-related molecules, such as MET hepatocyte growth factor. Furthermore, the interaction between CAFs and tumour cells influenced T-cell exhaustion and signalling pathways within the tumour microenvironment. CONCLUSIONS: This study unveils the direct interplay between tumour cells and CAFs in NSCLC with bone or brain metastasis and identifies potential therapeutic targets for inhibiting metastasis by disrupting these critical cell-cell interactions.

Predictive value of coagulation function, alpha-fetoprotein and placental growth factor in patients with perilous placenta previa.

OBJECTIVE: To analyze the predictive value of coagulation function, alpha-fetoprotein (AFP) and placental growth factor (PIGF) for postpartum hemorrhage in patients with perilous placenta previa (PPP). METHODS: The clinical data of 104 PPP patients were retrospectively analyzed. The patients were divided into a hemorrhage group (n=68) and a non-hemorrhage group (n=36). A total of 55 healthy pregnant women were recruited as controls. The coagulation function, AFP and PIGF were compared between the three groups. Multivariate logistic regression was performed to determine independent risk factors for hemorrhage. RESULTS: PT, TT, APTT, FIB and AFP were significantly higher while PIGF was lower in the PPP group than the control group (all P<0.05). Placental adhesion (OR 3.924, 95% CI 1.389-11.083, P=0.01), anterior placenta (OR 4.583, 95% CI 1.589-13.22, P=0.005), AFP (OR 0.208, 95% CI 0.068-0.635, P=0.006) and PIGF (OR 3.963, 95% CI 1.385-11.34, P=0.01) were independent risk factors for hemorrhage. CONCLUSION: Coagulation function, AFP and PIGF could predict postpartum hemorrhage in PPP patients.

Real-world data on efficacy and safety of osimertinib in non-small cell lung cancer patients with EGFR T790M mutation detected by first and repeat rebiopsy.

BACKGROUND: Osimertinib could effectively target epidermal growth factor receptor (EGFR) T790M resistance mutations in non-small cell lung cancer (NSCLC), indicating that rebiopsy may be particularly important. However, the clinical benefit of repeat rebiopsy in T790M-negative patients with NSCLC detected by the first rebiopsy is still unclear, and data on the efficacy and safety of osimertinib in patients with NSCLC who are T790M-positive patients on a repeat rebiopsy remain rare. METHODS: We retrospectively collected the clinical data of advanced NSCLC patients with common EGFR mutation who were treated with 1/2-generation (1/2G) EGFR-tyrosine kinase inhibitors (TKIs) in first-line therapy in our center from January 2018 to December 2020. The detection rate of T790M by first and repeat rebiopsy was recorded, and we also analyzed the efficacy and safety of osimertinib for T790M-positive patients. RESULTS: Among 190 common EGFR-mutant patients who received 1/2G EGFR-TKIs with advanced NSCLC in the first-line treatment, 141 patients developed progressive disease. In total, 110 of 141 accepted the first rebiopsy, with a T790M prevalence of 50.9% (56/110). In total, 43 T790M-positive patients who received osimertinib were included in first rebiopsy group. Of 54 T790M-negative patients detected by the first rebiopsy, 28 underwent repeated rebiopsy in subsequent clinical treatment, and 10 (35.7%) T790M-positive cases were confirmed. In total, eight T790M-positive patients treated with osimertinib were included in repeat rebiopsy group. Overall, 66 (60%) of 110 patients acquired a T790M mutation. In patients with the T790M mutation discovered by the first and repeat rebiopsy, osimertinib resulted in median progression-free survival of 7 (95% confidence interval [CI]: 5.3-8.7) and 6 (95% CI: 4.7-7.3) months, respectively (p = .656). The median overall survival since osimertinib initiation for T790M-positive patients at first rebiopsy was 20 (95% CI: 15.1-24.9) months and 19 (95% CI: 16.9-21.1) months, for those at repeated rebiopsy (p = .888). Adverse events of grade 3 or higher were similar in the two groups (25.6% vs. 12.5%, p = .616). There was no treatment-related death in the two groups. CONCLUSIONS: Repeat rebiopsy can increase the T790M mutation positivity rate. Osimertinib showed similar efficacy and safety in T790M-positive patients whether detected by the first or repeat rebiopsy.

Efficacy and safety of apatinib as second or later-line therapy in extensive-stage small cell lung cancer: a prospective, exploratory, single-arm, multi-center clinical trial.

Background: A paucity of strategies exist for extensive-stage small cell lung cancer (ES-SCLC) patients who fail the first-line chemotherapy. Apatinib is a tyrosine kinase inhibitor (TKI) that selectively inhibits vascular endothelial growth factor receptor-2 (VEGFR-2), which has been demonstrated to have active anti-tumor activity in ES-SCLC when used only or combined with PD-1 inhibitors or chemotherapy with good tolerance. However, the efficacy and safety of apatinib monotherapy is unclear in second-line or beyond treatment of ES-SCLC. Methods: In this prospective, exploratory, single-arm, multi-center study, eligible patients were aged 18 years or older with histologically confirmed ES-SCLC, and had progressed on, or were intolerant to previous systemic treatment. Patients received apatinib 500 mg (orally qd, every 4 weeks a cycle). The efficacy was assessed after 1 cycle and then every 2 cycles based on computed tomography imaging per the Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1). The primary endpoint was progression-free survival (PFS). The adverse events (AEs) were assessed per the National Cancer Institute Common Terminology Criteria for Adverse Events 4.0 (NCI-CTCAE 4.0). This study is registered in the Chinese Clinical Trial Registry, number ChiCTR-OPC-17013964. Results: From 28 July 2017 to 21 June 2019, 62 patients were screened for eligibility, among whom 57 patients were available for efficacy and safety analysis. The objective response rate (ORR) was 14.3% and disease control rate (DCR) was 79.6%. The median PFS was 5.6 months [95% confidence interval (CI): 3.3-8.0 months] and the median overall survival (OS) was 11.2 months (95% CI: 7.5-24.0 months). Among the participants who received apatinib as second-line treatment, the median PFS and OS were 6.1 months (95% CI: 2.6-7.6 months) and 12.0 months (95% CI: 7.9 months to not reached), respectively. The most common AEs of all grades were anemia (36.8%), hypertension (33.3%), fatigue (31.6%), blood bilirubin increased (22.8%), elevated transaminase (19.3%), and hand-foot syndrome (17.54%). Grade 3 AEs included 2 (3.5%) cases of hypertension and 1 (1.8%) case of fatigue. No grade 4/5 AEs were observed. Conclusions: Apatinib showed encouraging anti-tumor activity in pretreated ES-SCLC patients with tolerable toxicities. Further larger scale studies are warranted to demonstrate the efficacy of apatinib.

Exploration of immune-related cells and ceRNA in squamous cell lung cancer.

ABSTRACT: The treatment for squamous cell lung cancer (SqCLC) is limited, and the prognosis of SqCLC is poor. In this article, we aimed to analyze and identify immune-related cells and competition endogenous RNA (ceRNA) that influence the prognosis of SqCLC. SqCLC and lung adenocarcinoma data were downloaded from TCGA-GDC. A total of 22 types of immune cell fractions were estimated using CIBERSORT. R software was used to identify any significantly different transcriptome data, including mRNA, LncRNA, and miRNA. The univariate cox regression method was applied to screen for prognosis-related lncRNA, miRNA, mRNA and tumor-infiltrating immune cells. There were 504 patients included in this study. There was a higher proportion of memory activated CD4+ T cells and CD8+ T cells in younger women. Follicular helper T (Tfh) cells were predictive of a good prognosis and reflected immune activation in SqCLC. The SFTA1P/NKX2-1-AS1, hsa-mir-503, GREM2 ceRNA axes and NKX2-1-AS1, hsa-mir-96, PROK2 ceRNA axes were found to be important for the immune function, pathogenesis, and prognosis of SqCLC. Collectively, the immune-related ceRNA and tumor-infiltrating immune cells in SqCLC are likely important determinants of SqCLC pathogenesis, prognosis, and immune status.

Neutrophil-to-Lymphocyte Ratios Are Closely Associated With the Severity and Course of Non-mild COVID-19.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is spreading worldwide. Measuring the prevention and control of the disease has become a matter requiring urgent focus. Objective: Based on coronavirus disease 2019 (COVID-19) clinical data from Wuhan, we conducted an in-depth analysis to clarify some of the pathological mechanisms of the disease and identify simple measures to predict its severity early on. Methods: A total of 230 patients with non-mild COVID-19 were recruited, and information on their clinical characteristics, inflammatory cytokines, and T lymphocyte subsets was collected. Risk factors for severity were analyzed by binary logistic regression, and the associations of neutrophil-to-lymphocyte ratios (N/LRs) with illness severity, disease course, CT grading, inflammatory cytokines, and T lymphocyte subsets were evaluated. Results: Our results showed that the N/LRs were closely related to interleukin (IL)-6 and IL-10 (P < 0.001, P = 0.024) and to CD3+ and CD8+ T lymphocytes (P < 0.001, P = 0.046). In particular, the N/LRs were positively correlated with the severity and course of the disease (P = 0.021, P < 0.001). Compared to the values at the first test after admission, IL-6 and IL-10 were significantly decreased and increased, respectively, as of the last test before discharge (P = 0.006, P < 0.001). More importantly, through binary logistic regression, we found that male sex, underlying diseases (such as cardiovascular disease), pulse, and N/LRs were all closely related to the severity of the disease (P = 0.004, P = 0.012, P = 0.013, P = 0.028). Conclusions: As a quick and convenient marker of inflammation, N/LRs may predict the disease course and severity level of non-mild COVID-19; male sex, cardiovascular disease, and pulse are also risk factors for the severity of non-mild COVID-19.

Risk-adapted Treatment Strategy For COVID-19 Patients.

BACKGROUND: There are no clear expert consensus or guidelines on how to treat 2019 coronavirus disease (COVID-19). The objective of this study is to investigate the short-term effect of risk-adapted treatment strategy on patients with COVID-19. METHODS: We collected the medical records of 55 COVID-19 patients for analysis. We divided these patients into mild, moderate and severe groups, and risk-adapted treatment approaches were given according to the illness severity. RESULTS: Twelve patients were in mild group and 22 were in moderate group (non-severe group, n=34), and 21 patients were in severe group. At the end of the first two weeks after admission, clinical manifestations had completely despeared in 31(91.2%)patients in non-severe group, and 18(85.7%) patients in severe group (p=0.85). Both groups had a satisfied chest CT imaging recovery, which includes 22(64.7%) patients in non-severe group and 12(57.1%) patients in severe group recovered at least 50% of the whole leisions in the first week, and 28(82.4%) and 16(76.2%) recovered at least 75% in the second week, respectively. There were no significant differences in SARS-CoV-2 nucleic acid negativity (p=0.92). There were also no significant differences in the levels of SARS-CoV-2-IgM and IgG antibody production between the two groups (p=0.13, 0.62). There were 45 cases were discharged from the hospital, and no patients died at the time of this clinical analysis. CONCLUSIONS: Risk-adapted treatment strategy was associated with significant clinical manifestations alleviation and clinical imaging recovery. In severe COVID-19 patients, early and short-term use of low-dose methylprednisolone was beneficial and did not delay SARS-CoV-2 nucleic acid clearance and influence IgG antibody production.

Responses of manure decomposition to nitrogen addition: Role of chemical composition.

Understanding the interactions among organic manure chemical composition, decomposition and nitrogen (N) fertilization is critical for sustainable agriculture management. Six organic manures were incubated in a cultivated black soil with or without N addition for one year, and carbon dioxide (CO2) emissions from these organic manures were monitored. Chemical compositions of the organic manures were determined by elemental analysis, proximate chemical analysis, and carbon (C)-13 nuclear magnetic resonance spectroscopy, and evaluated after cupric-oxide oxidation for lignin biomarkers. During the experimental period, 19-44% of manure C was decomposed without N addition, which decreased to 17-35% with N addition, except for the composted furfural residue with rice dregs. However, during different decomposition stages, N effect changed from stimulation to inhibition, or behaved as increasing inhibition. During stage 1 (days 0-100) when N stimulation effect reached a maximum, CO2 emissions from manure had positive relationships with labile C fraction indicators, including total sugars, soluble polyphenols, and lignin cinnamyl/vanillyl ratio regardless of N addition. N effect on manure decomposition was related to the C/N ratio and labile organic C content. During stage 2 (days 101-267), N effect shifted to inhibition, with CO2 emissions from manure negatively related to lignin vanillyl-units content. The magnitude of N inhibition increased linearly with the aromaticity of dissolved organic C, and was strengthened by nitrate in manure. Finally, N inhibition effect reached a maximum during stage 3 (days 268-365), increasing with higher aromatic C in manure. Critical factors for manure decomposition shifted from total sugars, soluble polyphenols, and lignin cinnamyl-units to recalcitrant lignin vanillyl-units and aromatic C fraction, which mediated the type and magnitude of N effect on decomposition. Our results suggested that the potential for enhancing soil C sequestration with organic manures would magnify under combined application with N fertilizer in the long term.

Extreme rainfall and snowfall alter responses of soil respiration to nitrogen fertilization: a 3-year field experiment.

Extreme precipitation is predicted to be more frequent and intense accompanying global warming and may have profound impacts on soil respiration (Rs) and its components, that is, autotrophic (Ra) and heterotrophic (Rh) respiration. However, how natural extreme rainfall or snowfall events affect these fluxes are still lacking, especially under nitrogen (N) fertilization. In this study, extreme rainfall and snowfall events occurred during a 3-year field experiment, allowing us to examine their effects on the response of Rs, Rh, and Ra to N supply. In normal rainfall years of 2011/2012 and 2012/2013, N fertilization significantly stimulated Rs by 23.9% and 10.9%, respectively. This stimulation was mainly due to the increase of Ra because of N-induced increase in plant biomass. In the record wet year of 2013/2014, however, Rs was independent on N supply because of the inhibition effect of the extreme rainfall event. Compared with those in other years, Rh and Ra were reduced by 36.8% and 59.1%, respectively, which were likely related to the anoxic stress on soil microbes and decreased photosynthates supply. Although N supply did not affect annual Rh, the response ratio (RR) of Rh flux to N fertilization decreased firstly during growing season, increased in nongrowing season and peaked during spring thaw in each year. Nongrowing season Rs and Rh contributed 5.5-16.4% to their annual fluxes and were higher in 2012/2013 than other years due to the extreme snowfall inducing higher soil moisture during spring thaw. The RR of nongrowing season Rs and Rh decreased in years with extreme snowfall or rainfall compared to those in normal years. Overall, our results highlight the significant effects of extreme precipitation on responses of Rs and its components to N fertilization, which should be incorporated into models to improve the prediction of carbon-climate feedbacks.

[Comparison of the effect of different diagnostic criteria of subclinical hypothyroidism and positive TPO-Ab on pregnancy outcomes].

OBJECTIVE: To explore the effect of different diagnositic criteria of subclinical hypothyroidism using thyroid stimulating hormone (TSH) and positive thyroid peroxidase antibodies (TPO-Ab) on the pregnancy outcomes. METHODS: 3 244 pregnant women who had their antenatal care and delivered in Child and Maternity Health Hospital of Shannxi Province August from 2011 to February 2013 were recruited prospectively. According to the standard of American Thyroid Association (ATA), pregnant women with normal serum free thyroxine (FT4) whose serum TSH level> 2.50 mU/L were diagnosed as subclinical hypothyroidism in pregnancy (foreign standard group). According to the Guideline of Diagnosis and Therapy of Prenatal and Postpartum Thyroid Disease made by Chinese Society of Endocrinology and Chinese Society of Perinatal Medicine in 2012, pregnant women with serum TSH level> 5.76 mU/L, and normal FT4 were diagnosed as subclinical hypothyroidism in pregnancy(national standard group). Pregnant women with subclinical hypothyroidism whose serum TSH levels were between 2.50-5.76 mU/L were referred as the study observed group; and pregnant women with serum TSH level< 2.50 mU/L and negative TPO- Ab were referred as the control group. Positive TPO-Ab results and the pregnancy outcomes were analyzed. RESULTS: (1) There were 635 cases in the foreign standard group, with the incidence of 19.57% (635/3 244). And there were 70 cases in the national standard group, with the incidence of 2.16% (70/3 244). There were statistically significant difference between the two groups (P < 0.01). There were 565 cases in the study observed group, with the incidence of 17.42% (565/3 244). There was statistically significant difference (P < 0.01) when compared with the national standard group; while there was no statistically significant difference (P > 0.05) when compared with the foreign standard group. (2) Among the 3 244 cases, 402 cases had positive TPO-Ab. 318 positive cases were in the foreign standard group, and the incidence of subclinical hypothyroidism was 79.10% (318/402). There were 317 negative cases in the foreign standard group, with the incidence of 11.15% (317/2 842). The difference was statistically significant (P < 0.01) between them. In the national standard group, 46 cases had positive TPO-Ab, with the incidence of 11.44% (46/402), and 24 cases had negative result, with the incidence of 0.84% (24/2 842). There were statistically significant difference (P < 0.01) between them. In the study observed group, 272 cases were TPO-Ab positive, with the incidence of 67.66% (272/402), and 293 cases were negative, with the incidence of 10.31% (293/2 842), the difference was statistically significant (P < 0.01). (3) The incidence of miscarriage, premature delivery, gestational hypertension disease, gestational diabetes mellitus(GDM)in the foreign standard group had statistically significant differences (P < 0.05) when compared with the control group, respectively. While there was no statistically significant difference (P > 0.05) in the incidence of placental abruption or fetal distress. And the incidence of miscarriage, premature delivery, gestational hypertension disease, GDM in the national standard group had statistical significant difference (P < 0.05) compared with the control group, respectively. While there was no statistically significant difference (P > 0.05) in the incidence of placental abruption or fetal distress. This study observed group of pregnant women's abortion, gestational hypertension disease, GDM incidence respectively compared with control group, the difference had statistical significance (P < 0.05); but in preterm labor, placental abruption, and fetal distress incidence, there were no statistically significant difference (P > 0.05). (4) The incidence of miscarriage, premature delivery, gestational hypertension disease, GDM, placental abruption, fetal distress in the TPO-Ab positive cases of the national standard group showed an increase trend when compared with TPO-Ab negative cases, with no statistically significant difference (P > 0.05). The incidence of gestational hypertension disease and GDM in the TPO-Ab positive cases of the study observed group had statistical significance difference (P < 0.05) when compared with TPO-Ab negative cases; while the incidence of miscarriage, premature birth, placental abruption, fetal distress had no statistically significant difference (P > 0.05). The incidence of gestational hypertension disease and GDM in the TPO-Ab positive cases had statistically significance difference when compared with TPO-Ab negtive cases of foreign standard group (P < 0.05). CONCLUSIONS: (1) The incidence of subclinical hypothyroidism is rather high during early pregnancy and can lead to adverse pregnancy outcome. (2) Positive TPO-Ab result has important predictive value of the thyroid dysfunction and GDM. (3) Relatively, the ATA standard of diagnosis (serum TSH level> 2.50 mU/L) is safer for the antenatal care; the national standard (serum TSH level> 5.76 mU/L) is not conducive to pregnancy management.