Telomerase Mechanism of Telomere Synthesis.

Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines.

Single-strand DNA breaks cause replisome disassembly.

DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the point of synthesis, yet eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or "nicks," which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, "runs off" the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates along double-stranded DNA beyond the break and is then ubiquitylated and removed from chromatin by the same pathway used during replication termination. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they suggest that CMG cannot be stored on dsDNA while cells resolve replication stress.

The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells.

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements.

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.

Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts.

DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.

TRAIP is a master regulator of DNA interstrand crosslink repair.

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink1; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination2. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.

Total iron binding capacity: an independent predictor of prognosis for pulmonary arterial hypertension in systemic lupus erythematosus.

OBJECTIVE: To investigate the role of parameters of iron metabolism in systemic lupus erythematosus (SLE) patients with pulmonary arterial hypertension (PAH). METHOD: This was a prospective observational study recruiting patients diagnosed with systemic lupus erythematosus-associated pulmonary arterial hypertension (SLE-PAH). Patients with other factors that might lead to PAH were excluded from the study. All patients were assessed for PAH every 1-3 months and were followed up for 6 months. The primary outcome was considered improved if the grade of risk stratification declined at the endpoint; otherwise, it was considered unimproved. RESULTS: In total, 29 patients with SLE-PAH were included in this study. The mean of serum ferritin was higher than normal, and total iron binding capacity (TIBC) decreased in 48% of patients. Correlation analyses showed that serum iron (SI) was negatively correlated with World Health Organization functional class (WHO-FC) (r = -0.409, p = 0.028), and positively correlated with Six-Minute Walk Test distance (6MWD) (r = 0.427, p = 0.021) and tricuspid annular plane systolic excursion (TAPSE) (r = 0.388, p = 0.037). Primary outcomes improved in 12 patients at the endpoint, and univariate logistic regression analyses indicated that TIBC was associated with improved primary outcomes in patients with SLE-PAH (odds ratio 12.00, 95% confidence interval 1.90-75.72). CONCLUSION: SI was negatively correlated with WHO-FC, and positively correlated with 6MWD and TAPSE. Furthermore, TIBC was associated with improved outcomes of patients with SLE-PAH, which could be an independent predictor of prognosis. Further research is needed to verify the findings.

Mouse model of Graves' orbitopathy induced by the immunization with TSHR A and IGF-1R α subunit gene.

PURPOSE: The study aimed to establish a mouse model of Graves' disease (GD) with Graves' orbitopathy (GO; GD + GO) that can represent the clinical disease characteristics. METHODS: A eukaryotic expression plasmid of insulin-like growth factor 1 receptor (IGF-1R) α subunit (pcDNA3.1/IGF-1Rα) and a thyrotropin receptor (TSHR) A subunit plasmid (pcDNA3.1/TSHR-289) were injected in female BALB/c mice followed by immediate electroporation to induce a GD + GO model. Grouping was performed according to the frequency of injection (2- to 4-week intervals) and type of injected plasmids: T: pcDNA3.1/TSHR-289( +), I: pcDNA3.1/IGF-1Rα( +), or co-injection T + I: pcDNA3.1/TSHR-289( +) and pcDNA3.1/IGF-1Rα( +). Serum TSH, T4, TSAb, TSBAb, body weight, and blood glucose levels were evaluated. Thyroid 99mTcO4- imaging and retrobulbar magnetic resonance imaging (MRI) were performed, and bilateral eye muscle volumes were measured. Immunohistochemistry and hematoxylin-eosin staining were performed on the relevant tissues, and semi-quantitative analysis was performed. RESULTS: A total of 60% of mice (3/5, one mouse died) in the T group developed GD + GO. In the T + I group, 83.3% of mice (5/6) developed GD + GO. Mice in the I group did not develop GD. Compared with the control group, serum T4, TSAb, and TSBAb of the mice in the GD + GO model groups were increased to varying degrees (P < 0.05), and serum TSH and body weight were significantly lower compared to the control group (P < 0.05). The thyroid uptake capacity of 99mTcO4- and the volume of eye muscle of mice in the GD + GO group were significantly higher compared to the control group (P < 0.05). The thyroid and retrobulbar muscles of these mice showed varying inflammatory infiltration and interstitial muscle edema. The severity of GD + GO in the co-injection group was not related to injection frequency; however, GD and ocular signs in co-injection mice were more severe compared to the T group. CONCLUSIONS: We successfully induced a GD + GO mouse model by a repeated co-injection of pcDNA3.1/IGF-1Rα and pcDNA3.1/TSHR-289 plasmids. Injection of pcDNA3.1/IGF-1Rα alone failed to induce GD. Co-injection of two plasmids induced more severe GD + GO than pcDNA3.1/TSHR-289( +) alone.

Quantitative evaluation of bronchoalveolar lavage for the treatment of Severe mycoplasma pneumoniae pneumonia in children-A new complementary index: Bronchial Insufflation Sign Score.

OBJECTIVE: The aim of this study was to investigate the value of Broncoplasma Insufflation Sign in lung ultrasound signs in assessing the efficacy of bronchoalveolar lavage in Severe mycoplasma pneumoniae pneumonia in children. METHODS: Forty-seven children with Severe mycoplasma pneumoniae pneumonia were treated with medication and bronchial lavage. Laboratory and imaging results were collected, and lung ultrasonography was performed before bronchoalveolar lavage and 1, 3, and 7 days after lavage to record changes in Bronchial Insufflation Sign and changes in the extent of solid lung lesions. Factors affecting the effectiveness of bronchoalveolar lavage were analyzed using logistic regression and other factors. RESULTS: Bronchial Insufflation Sign Score and the extent of lung solid lesions were the factors affecting the effectiveness of bronchoalveolar lavage treatment. The smaller the area of lung solid lesions and the higher the Bronchial Insufflation Sign Score, the more effective the results of bronchoalveolar lavage treatment were, and the difference was statistically significant, with a difference of p < 0.05. The Bronchial Insufflation Sign Score had the highest sensitivity and specificity for the prediction of the efficacy of bronchoalveolar lavage treatment in the first 7 days after the treatment. CONCLUSION: Bronchial Insufflation Sign Score combined with the extent of solid lung lesions can assess the efficacy of bronchoalveolar lavage in the treatment of Severe mycoplasma pneumoniae pneumonia in children; lung ultrasound is a timely and effective means of assessing the efficacy of bronchoalveolar lavage.

A Cluster Randomized Trial of a Family Health History Platform to Identify and Manage Patients at Increased Risk for Colorectal Cancer.

BACKGROUND: Obtaining comprehensive family health history (FHH) to inform colorectal cancer (CRC) risk management in primary care settings is challenging. OBJECTIVE: To examine the effectiveness of a patient-facing FHH platform to identify and manage patients at increased CRC risk. DESIGN: Two-site, two-arm, cluster-randomized, implementation-effectiveness trial with primary care providers (PCPs) randomized to immediate intervention versus wait-list control. PARTICIPANTS: PCPs treating patients at least one half-day per week; patients aged 40-64 with no medical conditions that increased CRC risk. INTERVENTIONS: Immediate-arm patients entered their FHH into a web-based platform that provided risk assessment and guideline-driven decision support; wait-list control patients did so 12 months later. MAIN MEASURES: McNemar's test examined differences between the platform and electronic medical record (EMR) in rates of increased risk documentation. General estimating equations using logistic regression models compared arms in risk-concordant provider actions and patient screening test completion. Referral for genetic consultation was analyzed descriptively. KEY RESULTS: Seventeen PCPs were randomized to each arm. Patients (n = 252 immediate, n = 253 control) averaged 51.4 (SD = 7.2) years, with 83% assigned male at birth, 58% White persons, and 33% Black persons. The percentage of patients identified as increased risk for CRC was greater with the platform (9.9%) versus EMR (5.2%), difference = 4.8% (95% CI: 2.6%, 6.9%), p < .0001. There was no difference in PCP risk-concordant action [odds ratio (OR) = 0.7, 95% CI (0.4, 1.2; p = 0.16)]. Among 177 patients with a risk-concordant screening test ordered, there was no difference in test completion, OR = 0.8 [0.5,1.3]; p = 0.36. Of 50 patients identified by the platform as increased risk, 78.6% immediate and 68.2% control patients received a recommendation for genetic consultation, of which only one in each arm had a referral placed. CONCLUSIONS: FHH tools could accurately assess and document the clinical needs of patients at increased risk for CRC. Barriers to acting on those recommendations warrant further exploration. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT02247336 https://clinicaltrials.gov/ct2/show/NCT02247336.

Fetal cardiac and neonatal cerebral hemodynamics and oxygen metabolism in transposition of the great arteries.

OBJECTIVES: Hemodynamic abnormalities and brain development disorders have been reported previously in fetuses and infants with transposition of the great arteries and intact ventricular septum (TGA-IVS). A ventricular septal defect (VSD) is thought to be an additional risk factor for adverse neurodevelopment, but literature describing this population is sparse. The objectives of this study were to assess fetal cardiac hemodynamics throughout pregnancy, to monitor cerebral hemodynamics and oxygen metabolism in neonates, and to compare these data between patients with TGA-IVS, those with TGA-VSD and age-matched controls. METHODS: Cardiac hemodynamics were assessed in TGA-IVS and TGA-VSD fetuses and compared with healthy controls matched for gestational age (GA) during three periods: ≤ 22 + 5 weeks (GA1), 27 + 0 to 32 + 5 weeks (GA2) and ≥ 34 + 5 weeks (GA3). Left (LVO), right (RVO) and combined (CVO) ventricular outputs, ductus arteriosus flow (DAF, sum of ante- and retrograde flow in systole and diastole), diastolic DAF, transpulmonary flow (TPF) and foramen ovale diameter were measured. Aortic (AoF) and main pulmonary artery (MPAF) flows were derived as a percentage of CVO. Fetal middle cerebral artery and umbilical artery (UA) pulsatility indices (PI) were measured and the cerebroplacental ratio (CPR) was derived. Bedside optical brain monitoring was used to measure cerebral hemoglobin oxygen saturation (SO2 ) and an index of microvascular cerebral blood flow (CBFi ), along with peripheral arterial oxygen saturation (SpO2 ), in TGA-IVS and TGA-VSD neonates. Using hemoglobin (Hb) concentration measurements, these parameters were used to derive cerebral oxygen delivery and extraction fraction (OEF), as well as an index of cerebral oxygen metabolism (CMRO2i ). These data were acquired in the early preoperative period (within 3 days after birth and following balloon atrial septostomy) and compared with those of age-matched healthy controls, and repeat measurements were collected before discharge when vital signs were stable. RESULTS: LVO was increased in both TGA groups compared with controls throughout pregnancy. Compared with controls, TPF was increased and diastolic DAF was decreased in TGA-IVS fetuses throughout pregnancy, but only during GA1 and GA2 in TGA-VSD fetuses. Compared with controls, DAF was decreased in TGA-IVS fetuses throughout pregnancy and in TGA-VSD fetuses at GA2 and GA3. At GA2, AoF was higher in TGA-IVS and TGA-VSD fetuses than in controls, while MPAF was lower. At GA3, RVO and CVO were higher in the TGA-IVS group than in the TGA-VSD group. In addition, UA-PI was lower at GA2 and CPR higher at GA3 in TGA-VSD fetuses compared with TGA-IVS fetuses. Within 3 days after birth, SpO2 and SO2 were lower in both TGA groups than in controls, while Hb, cerebral OEF and CMRO2i were higher. Preoperative SpO2 was also lower in TGA-VSD neonates than in those with TGA-IVS. From preoperative to predischarge periods, SpO2 and OEF increased in both TGA groups, but CBFi and CMRO2i increased only in the TGA-VSD group. During the predischarge period, SO2 was higher in TGA-IVS than in TGA-VSD neonates, while CBFi was lower. CONCLUSIONS: Fetal cardiac and neonatal cerebral hemodynamic/metabolic differences were observed in both TGA groups compared with controls. Compared to those with TGA-IVS, fetuses with TGA-VSD had lower RVO and CVO in late gestation. A higher level of preoperative hypoxemia was observed in the TGA-VSD group. Postsurgical cerebral adaptive mechanisms probably differ between TGA groups. Patients with TGA-VSD have a specific physiology that warrants further study to improve neonatal care and neurodevelopmental outcome. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Cardiovascular magnetic resonance myocardial feature tracking for the determination of left atrial strain in hypertensive left ventricular hypertrophy and hypertrophic cardiomyopathy.

AIM: To measure the left atrial (LA) function in patients with hypertrophic cardiomyopathy (HCM; with [OHCM] and without obstruction [NOHCM]) and hypertension-related left ventricular hypertrophy (H-LVH) using cardiovascular magnetic resonance imaging feature tracking (CMR-FT). MATERIALS AND METHODS: Patients who met the criteria for HCM (n=68), H-LVH (n=46), and 30 healthy controls participated. Left atrial strain was analysed using CMR-FT in cine images with two and four chambers. RESULTS: The strain rate and LA strain measurements showed that patients with HCM, and H-LVH had impaired conduit and reservoir functions (versus controls). These capacities were more severely impaired in OHCM than those seen in NOHCM and H-LVH. The LA volume parameters (LAVIpac, LAVImin and LAVImax) from the OHCM group were higher than both the NOHCM and H-LVH groups (all p<0.05). There were differences between the OHCM and H-LVH groups in terms of the parameters for LA reservoir function (εs), booster pump function (SRa), and conduit function (SRe, LA passive EF, εe; p<0.05). The strongest correlations included the associations between LA total EF and εs, εe and LA passive EF, and SRe and LA passive EF. CONCLUSION: CMR-FT can reliably identify LA dysfunction and deformation in the early stages of HCM and H-LVH.

[Comprehensive assessment of mismatch repair and microsatellite instability status in molecular classification of endometrial carcinoma].

Objective: To explore the concordance and causes of different mismatch repair (MMR) and microsatellite instability (MSI) detection results in endometrial carcinoma (EC) molecular typing. Methods: A total of 214 EC patients diagnosed from January 2021 to April 2023 were selected at the Department of Pathology, Peking University Third Hospital. The immunohistochemistry (IHC) results of MMR protein were reviewed. Tumor specific somatic mutations, MMR germline mutations, microsatellite scores and tumor mutation burden (TMB) were detected by next-generation sequencing (NGS) with multi-gene panel. Methylation-specific PCR was used to detect the methylation status of MLH1 gene promoter in cases with deficient MLH1 protein expression. In cases with discrepant results between MMR-IHC and MSI-NGS, the MSI status was detected again by PCR (MSI-PCR), and the molecular typing was determined by combining the results of TMB and MLH1 gene promoter methylation. Results: (1) In this study, there were 22 cases of POLE gene mutation subtype, 55 cases of mismatch repair deficient (MMR-d) subtype, 29 cases of p53 abnormal subtype, and 108 cases of no specific molecular profile (NSMP). The median age at diagnosis of MMR-d subtype (54 years old) and the proportion of aggressive histological types (40.0%, 22/55) were higher than those of NSMP subtype [50 years old and 12.0% (13/108) respectively; all P<0.05]. (2) Among 214 patients, MMR-IHC test showed that 153 patients were mismatch repair proficient (MMR-p), 49 patients were MMR-d, and 12 patients were difficult to evaluate directly. MSI-NGS showed that 164 patients were microsatellite stable (MSS; equal to MMR-p), 48 patients were high microsatellite instability (MSI-H; equal to MMR-d), and 2 patients had no MSI-NGS results because the effective sequencing depth did not meet the quality control. The overall concordance between MMR-IHC and MSI-NGS was 94.3% (200/212). All the 12 discrepant cases were MMR-d or subclonal loss of MMR protein by IHC, but MSS by NGS. Among them, 10 cases were loss or subclonal loss of MLH1 and (or) PMS2 protein. Three discrepant cases were classified as POLE gene mutation subtype. In the remaining 9 cases, 5 cases and 3 cases were confirmed as MSI-H and low microsatellite instability (MSI-L) respectively by MSI-PCR, 6 cases were detected as MLH1 gene promoter methylation and 7 cases demonstrated high TMB (>10 mutations/Mb). These 9 cases were classified as MMR-d EC. (3) Lynch syndrome was diagnosed in 27.3% (15/55) of all 55 MMR-d EC cases, and the TMB of EC with MSH2 and (or) MSH6 protein loss or associated with Lynch syndrome [(71.0±26.2) and (71.5±20.1) mutations/Mb respectively] were significantly higher than those of EC with MLH1 and (or) PMS2 loss or sporadic MMR-d EC [(38.2±19.1) and (41.9±24.3) mutations/Mb respectively, all P<0.01]. The top 10 most frequently mutated genes in MMR-d EC were PTEN (85.5%, 47/55), ARID1A (80.0%, 44/55), PIK3CA (69.1%, 38/55), KMT2B (60.0%, 33/55), CTCF (45.5%, 25/55), RNF43 (40.0%, 22/55), KRAS (36.4%, 20/55), CREBBP (34.5%, 19/55), LRP1B (32.7%, 18/55) and BRCA2 (32.7%, 18/55). Concurrent PTEN, ARID1A and PIK3CA gene mutations were found in 50.9% (28/55) of MMR-d EC patients. Conclusions: The concordance of MMR-IHC and MSI-NGS in EC is relatively high.The discordance in a few MMR-d EC are mostly found in cases with MLH1 and (or) PMS2 protein loss or MMR protein subclonal staining caused by MLH1 gene promoter hypermethylation. In order to provide accurate molecular typing for EC patients, MLH1 gene methylation, MSI-PCR, MMR gene germline mutation and TMB should be combined to comprehensively evaluate MMR and MSI status.

Knockdown of Long Noncoding RNA CCAT2 Suppresses Malignant Phenotype in Human Laryngeal Squamous Cell Carcinoma.

This study aimed to explore the biological role and mechanism underlying the effects of colon cancer-associated transcript 2 (CCAT2), a long noncoding RNA (lncRNA) in human laryngeal squamous cell carcinoma (LSCC). CCAT2 expression levels in clinical LSCC samples and TU-212 cell line were evaluated by quantitative real-time PCR. The correlation of CCAT2 expression level with clinical-pathological characteristics of patients and their prognosis was analyzed. The functional role of CCAT2 in human LSCC was assessed by Cell Counting Kit-8, Transwell assay, flow cytometric analysis, and LSCC xenograft experiment in vivo. The expression of potential targeted proteins was detected by Western blotting and immunohistochemistry. We found that expression of CCAT2 was significantly elevated in LSCC tissues and TU-212 cells (p<0.05). Survival analysis showed that LSCC patients with high expression of CCAT2 had a shorter 5-year overall survival rate than those with low expression (p<0.05). In addition, CCAT2 silencing with short hairpin RNA significantly decreased the proliferative and invasive potential of TU-212 cells (p<0.05) and promoted their apoptosis. In Nude mice, CCAT2 knockdown suppressed the growth of tumor and decreased its volume and weight in comparison with the controls (p<0.05). In TU-212 cells, CCAT2 silencing with short hairpin RNA significantly down-regulated the expression of ß-catenin and CDK8 (p<0.05). Thus, knockdown of CCAT2 suppresses proliferation and invasion of the cells and inhibits Wnt/ß-catenin signaling pathway in LSCC, which indicates novel therapeutic targets and prognostic indicators in patients with LSCC.

Biopsy-tract haemocoagulase injection reduces major complications after CT-guided percutaneous transthoracic lung biopsy.

AIM: To determine whether the injection of haemocoagulase into the biopsy tract can reduce pneumothorax and pulmonary haemorrhage after computed tomography (CT)-guided percutaneous transthoracic lung biopsy (PTLB). MATERIALS AND METHODS: A retrospective study was performed involving patients with undiagnosed pulmonary lesions scheduled for PTLB between January 2020 and March 2021. Patients were assigned to the haemocoagulase group or the non-haemocoagulase group. After CT-guided biopsies were performed with a 17 G coaxial system, patients in the haemocoagulase group received a haemocoagulase injection (0.2-0.5 units) in the biopsy tract as the sheath was withdrawn. Postoperative image studies were performed to evaluate complications, including pneumothorax and pulmonary haemorrhage. Factors, including the patient's position, lesion location, and pathological results, were evaluated to determine their associations with the complications. RESULTS: A total of 100 patients were included, with 44 men and a mean age of 53 years old. The overall incidences of pneumothorax and pulmonary haemorrhage were 15% and 13%, respectively. The incidences of pneumothorax and pulmonary haemorrhage were statistically significantly lower in the haemocoagulase group (8% and 6%, respectively) than in the non-haemocoagulase group (22% and 20%, respectively; p=0.04 and 0.03, respectively). There was no statistically significant difference in haemoptysis between the haemocoagulase (6%) and non-haemocoagulase (2%) groups (p=0.23). There were also no statistically significant associations of pneumothorax or pulmonary haemorrhage with the patients' positions, lesion location, or pathological results. CONCLUSION: Biopsy tract haemocoagulase injection reduced the incidences of postoperative pneumothorax and pulmonary haemorrhage after PTLB.

Hypoperfusion intensity ratio correlates with clinical outcome of endovascular thrombectomy in acute ischaemic stroke patients with late therapeutic window.

AIM: To evaluate the prognostic value of the hypoperfusion intensity ratio (HIR) on 90-day clinical outcome in acute ischaemic stroke (AIS) patients with late therapeutic window. MATERIALS AND METHODS: One hundred and sixty-eight consecutive AIS patients with anterior-circulation large-vessel occlusion who underwent endovascular thrombectomy during the late window were enrolled retrospectively. Clinical data, Alberta Stroke Program Early Computed Tomography Score (ASPECTS) based on unenhanced computed tomography (CT), and perfusion parameters included ischaemic core, hypoperfusion volume, mismatch volume between core and penumbra, and the HIR were assessed and compared between patients with or without favourable outcomes (defined as modified Rankin Scale score of 0-2). Statistical analysis included binary logistic regression and receiver operating characteristic (ROC) analyses. RESULTS: A favourable outcome was achieved in 76 (45.2%) patients. In univariable analysis, age, National Institutes of Health Stroke Scale (NIHSS) score at admission, ASPECTS score, HIR, ischaemic core, and hypoperfusion volume were significantly associated with functional outcome (p<0.05). In multivariate analyses, age (OR 0.95; 95% CI 0.92-0.99), NIHSS score at admission (OR 0.89, 95% CI 0.84-0.96) and HIR (OR 0.018, 95% CI 0.003-0.113) remained as independent outcome predictors (p<0.01). The optimal threshold of HIR was 0.36 (sensitivity 70.7%, specificity 61.8%). The combination of age, NIHSS score at admission, and HIR yield good performance for outcome prediction with an area under the ROC curve of 0.815 (sensitivity 88.2%, specificity 64.1%), significantly higher than individual variable (p<0.05). CONCLUSION: Low HIR was a predictor for favourable outcome in AIS patients with late therapeutic window. Integrating HIR with clinical variables improved the ability for outcome classification.

Implementation-effectiveness trial of systematic family health history based risk assessment and impact on clinical disease prevention and surveillance activities.

BACKGROUND: Systematically assessing disease risk can improve population health by identifying those eligible for enhanced prevention/screening strategies. This study aims to determine the clinical impact of a systematic risk assessment in diverse primary care populations. METHODS: Hybrid implementation-effectiveness trial of a family health history-based health risk assessment (HRA) tied to risk-based guideline recommendations enrolling from 2014-2017 with 12 months of post-intervention survey data and 24 months of electronic medical record (EMR) data capture. SETTING: 19 primary care clinics at four geographically and culturally diverse U.S. healthcare systems. PARTICIPANTS: any English or Spanish-speaking adult with an upcoming appointment at an enrolling clinic. METHODS: A personal and family health history based HRA with integrated guideline-based clinical decision support (CDS) was completed by each participant prior to their appointment. Risk reports were provided to patients and providers to discuss at their clinical encounter. OUTCOMES: provider and patient discussion and provider uptake (i.e. ordering) and patient uptake (i.e. recommendation completion) of CDS recommendations. MEASURES: patient and provider surveys and EMR data. RESULTS: One thousand eight hundred twenty nine participants (mean age 56.2 [SD13.9], 69.6% female) completed the HRA and had EMR data available for analysis. 762 (41.6%) received a recommendation (29.7% for genetic counseling (GC); 15.2% for enhanced breast/colon cancer screening). Those with recommendations frequently discussed disease risk with their provider (8.7%-38.2% varied by recommendation, p-values ≤ 0.004). In the GC subgroup, provider discussions increased referrals to counseling (44.4% with vs. 5.9% without, P < 0.001). Recommendation uptake was highest for colon cancer screening (provider = 67.9%; patient = 86.8%) and lowest for breast cancer chemoprevention (0%). CONCLUSIONS: Systematic health risk assessment revealed that almost half the population were at increased disease risk based on guidelines. Risk identification resulted in shared discussions between participants and providers but variable clinical action uptake depending upon the recommendation. Understanding the barriers and facilitators to uptake by both patients and providers will be essential for optimizing HRA tools and achieving their promise of improving population health. TRIAL REGISTRATION: Clinicaltrials.gov number NCT01956773 , registered 10/8/2013.

Genome-wide association study for reproduction-related traits in Chinese domestic goose.

1. This study measured six reproduction traits in a Sichuan white goose population (209 individuals), including fertility, qualified egg rate, plasma concentrations of progesterone (P), follicle-stimulating hormone (FSH), prolactin (PRL) and oestrogen (E2).2. Whole-genome resequencing data from the same goose population (209 individuals) were used in a genome-wide association study (GWAS) utilising a mixed linear model to investigate the genes and genetic markers associated with reproduction traits. The frequency of the selected SNPs and haplotypes were determined using the Matrix-Assisted Laser Desorption Ionisation Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) method.3. In total, 42 SNPs significantly associated with these traits were identified. A haplotype block was constructed based on five SNPs that were significantly associated with qualified egg rate, with individuals having the haplotype CCTTAAGGAA having the lowest qualified egg rate.4. In conclusion, these results provided potential markers for marker-assisted selection to improve goose reproductive performance and a basis for elucidating the genetics of goose reproduction.

[Treatment and prognosis analysis of perineural invasion on sinonasal adenoid cystic carcinoma].

Objective: To analyze the efficacy of sinonasal adenoid cystic carcinoma (ACC) with perineural invasion (PNI), and explore the prognostic value of PNI on sinonasal adenoid cystic carcinoma. Methods: The clinical data of 105 patients with sinonasal ACC admitted to Cancer Hospital, Chinese Academy of Medical Sciences from January 2000 to December 2016 were retrospectively reviewed. All patients were restaged according to American Joint Committee on Cancer 8th edition. Follow-up visits were conducted to obtain information of treatment failure and survival outcome. The Log rank test was used for univariate analysis of prognostic factors, and Cox regression model was used for multivariate prognostic analysis. Results: The maxillary sinus (n=59) was the most common primary site, followed by the nasal cavity (n=38). There were 93 patients with stage Ⅲ-Ⅳ. The treatment modalities included surgery alone (n=14), radiotherapy alone (n=13), preoperative radiotherapy plus surgery (n=10), and surgery plus postoperative radiotherapy (n=68). The median follow-up time was 91.8 months, the 5-year local control (LC), distant metastasis-free survival (DMFS), progression-free survival (PFS), and overall survival (OS) rates were 72.6%, 73.0%, 52.9% and 78.0%, respectively. There were 33 patients (31.4%) with PNI-positive. The 5-year DMFS, PFS, and OS rates of PNI-positive group were 53.7%, 29.4% and 56.5%, respectively, which were significantly inferior to those of PNI-negative group (80.8%, 63.0% and 86.8%, respectively, P<0.05), while there was no significant difference in the 5-year LC rate between both groups (64.5% vs 76.5%, P=0.273). The multivariate Cox regression analysis showed PNI was one of the poor prognostic factors of DMFS (HR=3.514, 95%CI: 1.557-7.932), PFS (HR=2.562, 95%CI: 1.349-4.866) and OS (HR=2.605, 95%CI: 1.169-5.806). Among patients with PNI-positive, the 5-year LC, PFS and OS rates of patients received surgery combined with radiotherapy were 84.9%, 41.3% and 72.7%, respectively, which were significantly higher than 23.3%, 10.0% and 26.7% of patients receiving surgery or radiotherapy alone (P<0.05). Conclusion: The presence of PNI increases the risk of distant metastasis in patients with sinonasal ACC. Compared with patients with PNI-negative, the prognosis of patients with PNI-positive is relatively poor, and surgery combined with radiotherapy for PNI-positive sinonasal ACC results in good clinical outcomes.

[Long-term outcomes and failure patterns of definitive radiotherapy for cervical esophageal carcinoma].

Objective: To evaluate the long-term outcomes, failure patterns and prognostic factors of definitive radiotherapy in patients with cervical esophageal carcinoma (CEC). Methods: We retrospectively reviewed the clinical data of 148 CEC patients who treated with definitive radiotherapy in Cancer Hospital of Chinese Academy of Medical Sciences from January 2001 to December 2017. The median radiation dose was 66 Gy (59.4-70 Gy) and 33.1% of patients received concurrent chemotherapy. The Kaplan-Meier method was used to calculate survival rates. The log rank test was used for survival comparison and univariate prognostic analysis. The Cox model was used for multivariate prognostic analysis. Results: The median follow-up time was 102.6 months. The median survival time, 2- and 5-year overall survival (OS) were 22.7 months, 49.9% and 28.3%. The median, 2- and 5-year progression-free survival were 12.6 months, 35.8% and 25.8%. The 2- and 5-year locoregional recurrence-free survival were 59.1% and 50.8%. The 2- and 5-year distant metastases-free survival were 74.6% and 65.9%. Multivariate analysis showed that EQD(2)>66 Gy was the only independent prognostic indicator for OS (P=0.040). The median survival time and 5-year OS rate significantly improved in patients who received EQD(2)>66 Gy than those who received≤66 Gy (31.2 months vs. 19.2 months, 40.1% vs. 19.1%, P=0.027). A total of 87 patients (58.8%) developed tumor progression. There were 50 (33.8%), 23 (15.5%) and 39 (26.4%) patients developed local, regional recurrence and distant metastases, respectively. Eleven patients (7.4%) underwent salvage surgery, and the laryngeal preservation rate for entire group was 93.9%. Conclusions: Definitive radiotherapy is an effective treatment for cervical esophageal carcinoma with the advantage of larynx preservation. Local recurrence is the major failure pattern. EQD(2)>66 Gy is associated with the improved overall survival.