Unraveling the Significance of MET Focal Amplification in Lung Cancer: Integrative NGS, FISH, and IHC Investigation.

MET amplification (METamp) represents a promising therapeutic target in non-small cell lung cancer, but no consensus has been established to identify METamp-dependent tumors that could potentially benefit from MET inhibitors. In this study, an analysis of MET amplification/overexpression status was performed in a retrospectively recruited cohort comprising 231 patients with non-small cell lung cancer from Shanghai Chest Hospital (SCH cohort) using 3 methods: fluorescence in situ hybridization (FISH), hybrid capture-based next-generation sequencing, and immunohistochemistry for c-MET and phospho-MET. The SCH cohort included 130 cases known to be METamp positive by FISH and 101 negative controls. The clinical relevance of these approaches in predicting the efficacy of MET inhibitors was evaluated. Additionally, next-generation sequencing data from another 2 cohorts including 22,010 lung cancer cases were utilized to examine the biological characteristics of different METamp subtypes. Of the 231 cases, 145 showed MET amplification/overexpression using at least 1 method, whereas only half of them could be identified by all 3 methods. METamp can occur as focal amplification or polysomy. Our study revealed that the inconsistency between next-generation sequencing and FISH primarily occurred in the polysomy subtype. Further investigations indicated that compared with polysomy, focal amplification correlated with fewer co-occurring driver mutations, higher protein expressions of c-MET and phospho-MET, and higher incidence in acquired resistance than in de novo setting. Moreover, patients with focal amplification presented a more robust response to MET inhibitors compared with those with polysomy. Notably, a strong correlation was observed between focal amplification and programmed cell death ligand-1 expression, indicating potential therapeutic implications with combined MET inhibitor and immunotherapy for patients with both alterations. Our findings provide insights into the molecular complexity and clinical relevance of METamp in lung cancer, highlighting the role of MET focal amplification as an oncogenic driver and its feasibility as a primary biomarker to further investigate the clinical activity of MET inhibitors in future studies.

EGFR and ERBB2 Exon 20 Insertion Mutations in Chinese Non-small Cell Lung Cancer Patients: Pathological and Molecular Characterization, and First-Line Systemic Treatment Evaluation.

BACKGROUND: Data from studies looking at both EGFR and ERBB2 exon 20 insertion mutations (-20ins) in the same cohort of patients with non-small cell lung cancer (NSCLC) are limited. OBJECTIVE: The purpose of this study was to analyze EGFR/ERBB2-20ins in all-stage NSCLC patients to reveal their histological and molecular features, and to retrospectively evaluate the results of first-line real-world systemic treatments in patients with advanced-stage disease. PATIENTS AND METHODS: We collected 13,920 formalin-fixed paraffin-embedded NSCLC specimens. Clinicopathological features were recorded and DNA-based next-generation sequencing was performed. First-line systemic treatment data were obtained via chart review. RESULTS: In total, 414 (2.97%) EGFR-20ins cases and 666 (4.78%) ERBB2-20ins cases were identified. Both were more common in women, non-smokers, and patients with adenocarcinoma. The incidence of EGFR/ERBB2-20ins in adenocarcinoma is inversely proportional to the degree of invasion; 77 and 26 variants were detected in EGFR-20ins and ERBB2-20ins cases, respectively. The most common concurrently mutated genes were TP53 and RB1. In invasive adenocarcinoma, lepidic components were more common in EGFR/ERBB2-20ins-alone cases than in those with other concurrent mutated genes. In EGFR-/ERBB2-20ins patients, there was no significant difference in progression-free survival (PFS) or treatment response to first-line systemic treatments in this study. There was no significant difference in PFS or treatment response among patients with different EGFR/ERBB2-20ins variants and those with or without concurrent mutated genes. CONCLUSIONS: EGFR/ERBB2-20ins is more common in early lung adenocarcinoma. EGFR-20ins had more variants. In both cohorts, the results for first-line systemic treatments showed no significant difference.

Prevalence of high blood pressure and its associated factors among students in Shenyang, China: A cross-sectional study.

There is growing evidence that the prevalence of high blood pressure is increasing, and it may have serious consequences. However, research on the prevalence and influencing factors of high blood pressure among primary and secondary school students is still relatively scarce. This study aims to investigate the prevalence and influencing factors of high blood pressure among primary and secondary school students in Shenyang, in order to provide scientific evidence for the prevention and management of this disease. From April to May 2020, 4892 students aged 7 to 17 years were selected as the survey subjects, and on-site physical measurements and questionnaire surveys were conducted. The prevalence of high blood pressure was described. Restricted cubic spline was used to analyze the dose-response relationship between sleep duration, BMI and the risk of high blood pressure. Logistic regression was used to analyze the risk factors. Multiplicative and additive models were used to analyze the interaction between sleep duration and BMI. The results showed that the overall prevalence of high blood pressure among students aged 7 to 17 years in Shenyang was 9.9%, with a higher prevalence in females than males (12.1% vs 7.9%) and in urban areas than suburban areas (11.8% vs 7.7%). The prevalence was lowest in students with normal weight (8.3%) and highest in those who were obese (12.5%). The prevalence fluctuated to some extent among different age groups, but overall, it increased with age, with the lowest prevalence in primary school students (7.0%), 11.4% in mild school students, and the highest prevalence of 14.3% in high school students. Multivariable analysis showed that the risk of high blood pressure in female students was 1.90 times higher than that in male students (95% CI: 1.54-2.35), and the risk in suburban areas was 0.65 times lower than that in urban areas (95% CI: 0.52-0.81). Students with a BMI ≥ 21 kg/m2 had a 1.58 times higher risk than those with a BMI < 21 kg/m2(95% CI: 1.28-1.96), while those with a sleep time ≥ 8 hours had a 0.80 times lower risk than those with a sleep time < 8 hours (95% CI: 0.65-0.99). Exercise can significantly reduce the risk of high blood pressure, while using electronic devices for more than 0.5 hours significantly increases the risk of high blood pressure. BMI and sleep duration have no interaction effect on the risk of high blood pressure. To reduce the prevalence of high blood pressure, students should reduce the use of electronic devices, ensure adequate exercise, maintain a reasonable weight, and ensure sufficient sleep.

Meta-analysis and time trend prediction of the prevalence of hypertension in Chinese college students.

BACKGROUND: To understand the prevalence of hypertension among Chinese college students over the past decade (2010-2020) and predict its future trend, we aim to provide a basis for preventing and controlling hypertension among college students. METHODS: Databases such as Chinese National Knowledge Infrastructure, Wanfang database, PubMed, and Web of Science were searched, and publications on the prevalence of hypertension among Chinese college students from 2010 to 2020 were collected. Search for publications in both Chinese and English databases using keywords "hypertension," "prevalence," "disease status," "cross-sectional survey," "epidemiology," "China," "adolescents," and "college students." Publication screening, data extraction, and quality assessment were independently conducted by 2 researchers. Meta-analysis was performed using Stata 16, and trends in the prevalence of hypertension among college students were analyzed using R 4.2.0. RESULTS: A total of 37 publications were included in this analysis, which involved 233,603 Chinese college students. The Meta-analysis results showed significant heterogeneity among the studies (I2 = 98.9%, P < .05). Using a random-effects model, the overall prevalence of hypertension among college students was estimated to be 3.3% (95% CI = 2.9%-3.6%), with a higher prevalence among male students (6.2%, 95% CI = 5.4%-7.1%) than female students (1.1%, 95% CI = 0.9%-1.3%). The prevalence of hypertension is notably higher in northern regions than in southern regions. The prevalence of hypertension among college students showed an increasing trend from 2010 to 2020. Trend analysis predicted that the prevalence of hypertension among college students will reach 10% and 14.6% by 2030 and 2040, respectively. The risk of hypertension in male students was 4.63 times higher than that of female students (95% CI = 2.97-7.23). Compared normal weight students, overweight and obese students had 3.08 times (95% CI = 2.48-3.82) and 6.69 times (95% CI = 2.25-19.90) higher risk of hypertension, respectively. CONCLUSION: The prevalence of hypertension in Chinese college students was about 3.3%. The prevalence of hypertension in male college students was higher than that in females, and the prevalence in northern regions was generally higher than that in southern regions. The prevalence of hypertension among Chinese college students will reach 10.0% in the next 10 years and 14.6% in the next 20 years. Male and BMI ≥ 24 were risk factors for hypertension among college students.

Co-exposure to multiple vitamins and the risk of all-cause mortality in patients with diabetes.

Objective: Although the effect of vitamins on the risk of mortality in diabetic patients has been reported, most studies focus on individual vitamins. However, humans are often exposed to multiple vitamins simultaneously in daily life. Therefore, it is worth exploring the effects of co-exposure to multiple vitamins on the risk of mortality in diabetic patients. Methods: This study included diabetic patients aged ≥20WD years who participated in NHANES from 2003 to 2006. An unsupervised K-means clustering method was used to cluster eight vitamins in serum into several patterns of co-exposure to multiple vitamins, and the Cox proportional hazards model was used to evaluate the impact of different patterns of co-exposure to multiple vitamins on the risk of all-cause mortality in diabetic patients. Results: Three patterns of co-exposure to multiple vitamins were generated based on K-means clustering, namely, low-level, moderate-level, and high-level. Among the 484 diabetic patients, with a median follow-up of 13.7 years, a total of 211 deaths occurred. After adjusting for covariates, the individual vitamins had varying effects on the risk of all-cause mortality in diabetic patients. Compared to the low-level group of co-exposure to multiple vitamins, the high-level group significantly reduced the risk of all-cause mortality in diabetic patients, with a HR of 0.42 (95% CI: 0.20, 0.87). Subgroup analysis demonstrated that high levels of co-exposure to multiple vitamins significantly reduced the risk of all-cause mortality in males, individuals aged ≥ 60 years, and non-Hispanic White people with diabetes compared to the low-level group, with HR of 0.42 (95% CI: 0.18, 0.98), 0.53 (95% CI: 0.26, 0.98), and 0.26 (95% CI: 0.12, 0.58) respectively. Conclusion: While individual vitamins had different effects on the risk of all-cause mortality in patients with diabetes, high-level co-exposure to multiple vitamins significantly reduced the risk of all-cause mortality in patients with diabetes, with differences observed among genders, ages, and race. This suggests that when developing vitamin intervention strategies for patients with diabetes, consideration should be given not only to the dosage of individual vitamins but also to the variations between different population groups.

Prevalence and associated factors of myopia among school students in Shenyang, China: a cross-sectional study.

Background: In recent years, the prevalence of myopia has increased significantly and caused great concern. Nevertheless, an estimate of myopia in the student population in Shenyang, Liaoning Province, China is still lacking. This study aims to determine the prevalence of myopia among students in Shenyang and investigate the associated factors affecting myopia development. Methods: Standard logarithmic visual acuity chart and automatic computerized optometry under non-ciliary muscle paralysis were used to test the students' naked visual acuity of their right and left eyes. The included students were organized to fill in questionnaires on WeChat to collect the factors affecting myopia. Results: A total of 34,644 students with a median age of 11.9 years were examined, including 17,563 males and 17,081 females. The overall prevalence of myopia was 60%, with a prevalence of 45% for mild myopia, 13% for moderate myopia, and 1.9% for high myopia. The sex, high educational stage, family history of myopia, doing homework after school or reading and writing for more than 2 h were associated with a higher risk of myopia, while doing eye exercises twice a day or more, going outdoors during recess, reading and writing with eyes more than one foot from books, and sleeping more than 8 h a day were associated factors for preventing myopia. The associated factors influencing myopia vary among different subgroups. Conclusion: The prevalence of myopia in Shenyang is at a high level. In addition to sex, high educational stage and genetic factors, environmental factors including length of eye usage, eye exercises, outdoor activities, eye working distance, and sleep duration are associated with myopia prevalence. Therefore, it is recommended that the occurrence and development of myopia can be prevented by controlling the above environmental factors.

Effects of N-acetyl-l-cysteine on chronic heat stress-induced oxidative stress and inflammation in the ovaries of growing pullets.

The aims of this study were to investigate the effects of supplemental N-acetyl-l-cysteine (NAC) on chronic heat stress-induced oxidative stress and inflammation in the ovaries of growing pullets. A total of 120, 12-wk-old, Hy-Line Brown hens were randomly separated into 4 groups with 6 replicates of 5 birds in each group for 21 d. The 4 treatments were as follows: the CON group and CN group were supplemented with basal diet or basal diet with 1 g/kg NAC, respectively; and the HS group and HSN group were heat-stressed groups supplemented with basal diet or basal diet with 1 g/kg NAC, respectively. The results indicated that the ovaries suffered pathological damage due to chronic heat stress and that NAC effectively ameliorated these changes. Compared with the HS group, antioxidant enzyme activities (including SOD, GSH-Px, CAT, and T-AOC) were enhanced, while the MDA contents and the expression levels of HSP70 were decreased in the HSN group. In addition, NAC upregulated the expression levels of HO-1, SOD2, and GST by upregulating the activity of Nrf2 at different time points to mitigate oxidative stress caused by heat exposure. Simultaneously, NAC attenuated chronic heat stress-induced NF-κB pathway activation and decreased the expression levels of the proinflammatory cytokines IL-8, IL-18, TNF-α, IKK-α, and IFN-γ. Cumulatively, our results indicated that NAC could ameliorate chronic heat stress-induced ovarian damage by upregulating the antioxidative capacity and reducing the secretion of proinflammatory cytokines.

Antioxidative and anti-inflammatory effects of vitamin C on the liver of laying hens under chronic heat stress.

In this study, we investigated the therapeutic effect and mechanism of action of vitamin C on chronic heat stress (CHS)-induced liver oxidative damage and inflammation in laying hens. The thermoneutral control group (TN group) was kept at a constant temperature of 22 ± 1°C, while the chronic heat stress group (CHS group) and the vitamin C supplemented group (HSV group) were exposed to heat stress (HS) (36 ± 1°C, 8 h/d). The TN and HS groups were fed the basic diet at will, and the HSV group was supplemented with 300 mg/kg of vitamin C on top of the basic diet. The experimental results showed a significant improvement in body weight and feed intake in the HSV group compared to the HS group. A significantly lower pH and higher HCO 3 - and PCO2 levels were observed in the HSV group compared to the CHS group. As laying hens were supplemented with vitamin C, serum malondialdehyde (MDA) level was declined, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were increased, and total antioxidant capacity (T-AOC) was increased. Further, CHS induced an increase in the expression of inflammation-related genes and a decrease in the expression of antioxidant-related genes. In contrast, the addition of vitamin C reversed the effects of CHS, resulting in an increase in the expression of antioxidant-related genes and a decrease in the expression of inflammation-related genes. In conclusion, vitamin C can effectively alleviate CHS-induced acid-base imbalance in body fluids of laying hens and the oxidative damage and inflammatory response caused to the liver. Therefore, vitamin C can be used clinically as an effective drug to alleviate chronic heat stress in laying hens. This experiment provides clinical evidence and theoretical basis for the use of vitamin C as an effective drug to alleviate chronic heat stress in laying hens.

Inhibition of Hepatic AMPK Pathway Contributes to Free Fatty Acids-Induced Fatty Liver Disease in Laying Hen.

Metabolism-associated fatty liver disease (MAFLD) is one of the most common causes of liver disease; however, the underlying processes remain unknown. This study aimed to investigate the changes of free fatty acids (FFA) on the expression of genes related to the AMP-activated protein kinase (AMPK) signaling pathway in the primary hepatocytes of laying hens. The primary hepatocytes of laying hens were treated with FFA (containing a 2:1 ratio of oleic and palmitic acids) for 24 h. FFA significantly increased lipid droplet accumulation, decreased glycogen synthesis, increased the levels of triglycerides (TG), total cholesterol (TC), reactive oxygen species (ROS), malondialdehyde (MDA), and glucose content in the supernatant (GLU) in the primary hepatocytes of laying hens, and decreased the levels of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD), as well as mitochondrial membrane potential (MMP). The results of the PCR array combined with Western blotting experiments showed that the activity of AMPK was inhibited. Inhibition of AMPK signaling pathway decreases the expression of genes involved in fatty acid oxidation, increases the expression of genes involved in lipid synthesis, decreases the expression of genes involved in glycogen synthesis, increases the expression of genes involved in glycolysis, increases the expression of genes involved in oxidative stress, and increases the expression of genes involved in cell proliferation and apoptosis. Taken together, our results suggest that FFA can affect the homeostasis of the AMPK signaling pathway by altering energy metabolic homeostasis, inducing oxidative stress, and adjusting the onset of cell proliferation and apoptosis.

Identification and therapeutic evaluation of ALK rearrangements in non-small-cell lung cancer.

This study aimed to present a comprehensive assessment of anaplastic lymphoma kinase (ALK) rearrangements evaluated by DNA/RNA-based next-generation sequencing (NGS) and Ventana immunohistochemistry (IHC) in patients with non-small-cell lung cancer (NSCLC) and to evaluate the therapeutic outcomes of ALK tyrosine kinase inhibitor (TKI) treatment. We investigated ALK gene fusions in 14,894 patients with NSCLC using Ventana IHC and NGS, including 12,533 cases detected via DNA-based NGS and 2,361 cases using RNA-based NGS. The overall percentage agreement (OPA), positive percentage agreement (PPA), and negative percentage agreement (NPA) were calculated when comparing the results between NGS and IHC. The therapeutic responses to ALK-TKIs were also evaluated. In total, 3.50% (439/12,533) of specimens were NGS ALK-positive (NGS-p) in the DNA-based NGS cohort and 3.63% (455/12,533) were IHC ALK-positive (IHC-p). The OPA of NGS was 99.60%, whereas its PPA and NPA were 92.75 and 99.86%, respectively. In the adenocarcinoma (ADC) subcohort, the PPA was 95.69%. In the RNA-based NGS cohort, 2.20% (52/2,361) of specimens were NGS-p and 2.63% (62/2,361) were IHC-p. The OPA of NGS was 99.49%; its PPA and NPA were 82.26 and 99.96%, respectively. Thirteen patients with discordant results received ALK-TKI treatment. In the seven NGS-p/IHC-negative (IHC-n) patients, the overall response rate (ORR) was 85.4% (6/7) and the disease control rate (DCR) was 100%. In the six NGS-negative/IHC-p patients, the ORR was 66.7% (4/6) and the DCR was 100%. In summary, a high concordance of ALK gene fusion detected via NGS and IHC was observed in this study. DNA-based NGS had a higher OPA, PPA, and PPA in the ADC subcohort, whereas RNA-based NGS had a higher NPA. Overall, the results suggest that the combination of NGS and IHC can improve the accuracy of ALK fusion detection; hence, a result determination algorithm for clinical detection of ALK gene fusion was also proposed.

Gut Microbiota and Serum Metabolite Potential Interactions in Growing Layer Hens Exposed to High-Ambient Temperature.

Emerging evidence has revealed the dysbiosis of gut microbiota contributes to development of metabolic diseases in animals. However, the potential interaction between gut microbiota and host metabolism in growing hens under metabolic disorder induced by chronic heat exposure (CHE) remains inconclusive. The aim of our study was to examine the potential association among the cecal microbiota community, physiological indicators, and serum metabolite profiles in CHE hens. One hundred and eighty Hy-Line Brown hens were randomly allocated into three groups: thermoneutral control (TN), heat stress (HS), and pair-fed (PF). The experiment lasted for 5 weeks, with the first 2 weeks serving as the adaptation period. Results showed that the expression level of heat shock protein 70 (HSP70) in both serum and cecal tissues was significantly increased in the HS group. Serum parameters analysis also revealed that CHE caused physiological function damage and metabolic disorders. These results suggest the experiment was successful, inducing chronic heat stress. 16S rRNA sequencing analysis showed that the CHE can clearly induce dysbiosis of the gut microbial community reflected in the increment of the F/B ratio. Besides, serum untargeted metabolomics revealed the relative concentrations of 40 metabolites were significantly altered in the HS group compared with the TN group. Pathway analysis showed that these metabolites were mainly involving the increased proteolysis rather than lipolysis, and this tendency could be a specific metabolic adaptation of the poultry. The pair-feed experiment showed that the above changes induced by CHE were partly independent from the reduction of feed intake. Mantel correlation analysis between gut microorganisms and physiological indicators showed that the phylum Firmicutes and Euryarchaeota have a potential interaction with a serum lipid parameter. Random forest analysis showed that both genus Faecalibacterium and Methanobrevibacter were important predictors of the CHE-induced lipid metabolism disorder. Taken together, our findings may contribute to a better understanding of the metabolic mechanisms underlying the energy metabolism imbalance caused by the CHE and provide novel insights into the host-microbes interactions and its effects on the metabolic adaptation of hens under chronic heat exposure.

Distinct mutational features across preinvasive and invasive subtypes identified through comprehensive profiling of surgically resected lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a heterogeneous disease. Our study aimed to understand the unique molecular features of preinvasive to invasive LUAD subtypes. We retrospectively analyzed the clinical, histopathological, and molecular data of 3,254 Chinese patients with preinvasive lesions (n = 252), minimally invasive adenocarcinomas (n = 479), and invasive LUAD (n = 2,523). Molecular data were elucidated using a targeted 68-gene next-generation sequencing panel. Our findings revealed four preinvasive lesion-predominant gene mutations, including MAP2K1 insertion-deletions (indels), BRAF non-V600E kinase mutations, and exon 20 insertions (20ins) in both EGFR and ERBB2, which we referred to as mutations enriched in AIS (MEA). The detection rate of MEA in invasive tumors was relatively lower. MAP2K1 missense mutations, which were likely passenger mutations, co-occurred with oncogenic driver mutations, while small indels were mutually exclusive from other genes regardless of the invasion level. BRAF non-V600E kinase-mutant invasive adenocarcinomas (IAC) had significantly higher mutation rates in tumor suppressor genes but lower frequency of co-occurring oncogenic driver mutations than non-kinase-mutant IAC, suggesting the potential oncogenic activity of BRAF non-V600E kinase mutations albeit weaker than BRAF V600E. Moreover, similar to the extremely low frequency of MAP2K1 indels in IAC, BRAF non-V600E kinase domain mutations co-occurring with TSC1 mutations were exclusively found in preinvasive lesions. Compared with EGFR L858R and exon 19 deletion, patients with preinvasive lesions harboring 20ins in either EGFR or ERBB2 were significantly younger, while those with IAC had similar age. Furthermore, our study demonstrated distinct mutational features for subtypes of oncogene mutations favored by different invasion patterns in adenocarcinomas. In conclusion, our data demonstrate distinct mutational features between preinvasive lesions and invasive tumors with MEA, suggesting the involvement of MEA in the early stages of tumorigenesis. Further pre-clinical studies are required to establish the role of these genes in the malignant transformation of LUAD.

PGK1 represses autophagy-mediated cell death to promote the proliferation of liver cancer cells by phosphorylating PRAS40.

Autophagy predominantly promotes cell survival by recycling cell components, while it kills cells in specific contexts. Cell death related to autophagy plays important roles in multiple physiological and pathological situations including tumorigenesis, and the mechanism needs to be defined further. PRAS40 was found to be crucial in various cancers, and phosphorylation was reported to be involved in autophagy inhibition in monocytes. However, the detailed role of PRAS40 in autophagy and the relationship to tumorigenesis remain largely unknown. Herein we screened the binding partners of PRAS40, and found that PRAS40 interacted with Phosphoglycerate kinase 1 (PGK1). PGK1 phosphorylated PRAS40 at Threonine 246, which could be inhibited by blocking the interaction. Both in vitro and in vivo results revealed that PRAS40 mediated PGK1-induced cell growth. By tracing the mechanism, we found that PGK1 suppressed autophagy-mediated cell death, in which PRAS40 was crucial. Thus PGK1 phosphorylates PRAS40 to repress autophagy-mediated cell death under normoxia, promoting cellular proliferation. The binding of PGK1 to PRAS40 was transferred to Beclin1 under hypoxia, resulting in the increase of Beclin1 phosphorylation. These results suggest a novel model of tumorigenesis, in which PGK1 switches between repressing autophagy-mediated cell death via PRAS40 and inducing autophagy through Beclin1 according to the environmental oxygen level. Our study is anticipated to be able to offer novel insights in understanding PGK1/PRAS40 signaling hyperactivated cancers.

Identification and Validation of Noncanonical RET Fusions in Non-Small-Cell Lung Cancer through DNA and RNA Sequencing.

RET fusion has emerged as a targetable driver in non-small-cell lung cancer. A comparative analysis on RET fusions at DNA [DNA sequencing (DNA-seq)] and RNA [RNA sequencing (RNA-seq)] levels was performed in this study. Archived tumor samples from 54 non-small-cell lung cancer patients with DNA-level noncanonical RET fusions were selected for RNA-seq. RNA-seq identified RET fusion transcripts in 41 of 44 samples passing quality control. In the subset of cases harboring RET 3'-end fusions and predicted to produce in-frame proteins (group A; n = 33), RNA-seq identified the same 3'-end fusions in 32 (96.9%). A total of 26 of 32 also had a reciprocal RET 5'-end fusion detected by DNA-seq that was not transcribed. In the subset with DNA-level out-of-frame RET fusions (group B; n = 9), RNA-seq identified in-frame RET fusion transcripts in 8 cases (88.9%). In the subset only identified with a RET 5'-end fusion by DNA-seq (group C; n = 2), RNA-seq detected the corresponding 3'-end fusion in one case. The discordant DNA- and RNA-level fusions observed in group B may be mediated by complex genomic rearrangement events and transcriptional or post-transcriptional processes. In conclusion, DNA-seq demonstrates a high concordance of 96.9% on detecting in-frame RET fusion, but shows a low concordance on detecting out-of-frame RET fusion and RET 5'-end fusion compared with RNA-seq.

SPINK1 contributes to proliferation and clonal formation of HT29 cells through Beclin1 associated enhanced autophagy.

We aimed to explore the molecular mechanism that were involved in SPINK1-induced proliferation and clonogenic survival of human colorectal carcinoma (CRC) HT29 cells. Initially, we generated HT29 cells either permanently silencing or overexpressing SPINK1 protein. The results showed that SPINK1 overexpression (OE) significantly stimulated the proliferation and clonal formation of HT29 cells at the varied time points. Secondly, we found SPINK1 OE enhanced the ratio of LC3II/LC3I and the level of autophagy-related gene 5 (ATG5), whereas SPINK1 knockdown (Kd) reversed the above outcome under normal culturing and/or fasting condition in the cells, indicating its role in autophagy enhancement. Moreover, LC3-GFP-transfected SPINK1-OE HT29 cells strengthened the fluorescence intensity compared with the untransfected control. Chloroquine (CQ) significantly decreased the level of autophagy in both control and SPINK1-OE HT29 cells. The autophagy inhibitors, CQ and 3-Methyladenine (3-MA), remarkably inhibited the proliferation and colony formation of SPINK1-OE HT29 cells, while ATG5 upregulation resulted in the growth of the cells, suggesting the important function of autophagy in cell's growth. Thirdly, SPINK1-induced autophagy was independently of mTOR signaling as p-RPS6 and p-4EBP1 were activated in SPINK1-OE HT29 cells. Instead, Beclin1 up and down regulation were clearly observed in SPINK1-OE and SPINK1 Kd HT29 cells, respectively. Moreover, Beclin1 silencing apparently reduced autophagy in SPINK1-OE HT29 cells, indicating that SPINK1-induced autophagy was closely associated with Beclin1. Collectively, SPINK1-promoted proliferation and clonal formation of HT29 cells were closely associated with Beclin1 associated enhanced autophagy. The above findings would open a new window for probing the role of SPINK1-related autophagic signaling in the pathogenesis of CRC.

Emerging Design Principle of Near-Infrared Upconversion Sensitizer Based on Mitochondria-Targeted Organic Dye for Enhanced Photodynamic Therapy.

Upconversion luminescent (UCL) triggered photodynamic therapy (PDT) affords superior outcome for cancer treatment. However, conventional UCL materials which all work by a multiphoton absorption (MPA) process inevitably need extremely high power density far over the maximum permissible exposure (MPE) to laser. Here, a one-photon absorption molecular upconversion sensitizer Cy5.5-Br based on frequency upconversion luminescent (FUCL) is designed for PDT. The unusual super heavy atom effect (SHAE) in Cy5.5-Br strongly enhances its spin-orbit coupling (0.23 cm-1 ), triplet quantum yield (11.1 %) and triplet state lifetime (18.8 µs) while the potential hot-band absorption of Cy5.5-Br is well maintained. Importantly, Cy5.5-Br can efficiently target the tumour site and kill cancer cells by destroying mitochondria under a biosafety MPE to 808 nm laser. The photostability and antitumor results are obviously superior to that of a Stokes process. This work provides a design criterion for FUCL dyes to realize effective PDT upon a biosafety optical density, possibly bringing more clinical benefits than conventional MPA materials.

Effects of Subchronic Copper Poisoning on Cecal Histology and Its Microflora in Chickens.

Copper (Cu) is an important trace element with a two-sided effect on the growth performance of animals, which depends on the timing and dosage of Cu addition, etc. The purpose of this study was to determine the effects of oral copper sulfate (CuSO4, 350 ppm) on growth performance, cecal morphology, and its microflora of chickens (n = 60) after 30, 60, and 90 days. The results showed that after 90 days of copper exposure, the chickens lost weight, the cecum mucosa was detached, and vacuolation and inflammatory infiltration occurred at the base of the lamina propria. In addition, using the 16S rDNA sequencing method, we observed that copper exposure changed the richness and diversity of intestinal microorganisms. At the phylum level, Proteobacteria and Actinobacteria both significantly increased, while Bacteroidetes significantly decreased in the Cu group compared with control check (CK) group. At the genus level, the relative abundance of Rikenellaceae_RC9_gut_group decreased significantly, while Ruminococcaceae_UCG-014, Lachnoclostridium, and [Eubacterium]_coprostanoligenes_group increased significantly after copper exposure, and the change in microflora was most significant at 90 days. Moreover, the relevance of genus-level bacteria was altered. PICRUST analysis revealed potential metabolic changes associated with copper exposure, such as Staphylococcus aureus infection and metabolic disorders of nutrients. To sum up, these data show that subchronic copper exposure not only affects the growth and development of chickens but also causes the imbalance of intestinal microflora, which may further induce metabolic disorders in chickens.

YAP1-MAML2 Fusion as a Diagnostic Biomarker for Metaplastic Thymoma.

BACKGROUND: Metaplastic thymoma is a very rare tumor with only a few case reports documented in literature. Hence, its molecular features have not been well explored. MATERIAL AND METHODS: Seventeen specimens of metaplastic thymoma were sequenced and retrospectively analyzed by fluorescence in situ hybridization (FISH) and immunohistochemistry in the study. In addition, seven cases of micronodular thymoma with lymphoid stroma and nine cases of type A thymoma were also investigated. RESULTS: Among these metaplastic thymomas, fifteen cases showed classical histological features, and two cases displayed characteristic micronodular-like growth patterns. DNA and RNA based next-generation sequencing identified and confirmed highly recurrent Yes Associated Protein 1 (YAP1) - Mastermind Like Transcriptional Coactivator 2 (MAML2) translocation (13/17, 76.5%) in metaplastic thymoma but not in micronodular thymoma with lymphoid stroma (0/7, 0%) and type A thymoma (0/9, 0%). In addition, six nonsense mutations were also detected in the metaplastic thymoma. FISH in microdissection specimens indicated that both epithelioid and spindle cell components harbored YAP1-MAML2 gene rearrangements. CONCLUSIONS: Our study explored the genetic alterations in epithelioid and spindle cell components in metaplastic thymoma. Furthermore, YAP1-MAML2 gene rearrangements emerged as a potential diagnostic biomarker helpful for distinguishing metaplastic thymoma from type A and micronodular thymoma with lymphoid stroma.

Effects of N-acetyl-l-cysteine on heat stress-induced oxidative stress and inflammation in the hypothalamus of hens.

The purpose of this study was to discuss the effects of N-acetyl-l-cysteine (NAC) on heat stress-induced oxidative stress and inflammation in the hypothalamus of hens in different periods. A total of 120 Hy-Line variety brown laying hens (12 weeks old) were randomly assigned to 4 groups with 6 replicates. The control group (C group) (22 ± 1 °C) received a basal diet, the NAC-treated group (N group) (22 ± 1 °C) received a basal diet with 1000 mg/kg NAC, and 2 heat-stressed groups (36 ± 1 °C for 10 h per day and 22 ± 1 °C for the remaining time) were fed a basal diet (HS group) or a basal diet with 1000 mg/kg NAC (HS + N group) for 21 consecutive days. The influence of NAC on histologic changes, oxidative stress and proinflammatory cytokine production was measured and analysed in hens with heat stress-induced hypothalamic changes. NAC effectively alleviated the hypothalamic morphological changes induced by heat stress. In addition, NAC attenuated the activity of the Nf-κB pathway activated by heat stress and decreased the expression of the proinflammatory cytokines IL-6, IL-18, TNF-α, IKK, and IFN-γ. In addition, NAC treatment regulated the expression of HO-1, GSH, SOD2 and PRDX3 by regulating the activity of Nrf2 at different time points to resist oxidative stress caused by heat exposure. In summary, dietary NAC may be an effective candidate for the treatment and prevention of heat stress-induced hypothalamus injury by preventing Nf-κB activation and controlling the Nrf2 pathway.