Regioselective and Enantioselective Nickel-Catalyzed Intermolecular Reductive Coupling of Aliphatic Alkenes with Imines.

Unactivated aliphatic alkenes are particularly desirable as starting materials because they are readily accessible in large quantities, but the enantioselective intermolecular reductive coupling of unactivated alkenes with imines is challenging. In this paper, we report a method for nickel-catalyzed intermolecular reductive coupling reactions between aliphatic alkenes and imines to yield chiral amines with excellent enantioselectivities and good linear selectivities. The reaction conditions are compatible with a broad range of aliphatic alkenes, including those derived from bioactive molecules. The success of this method can be attributed to the use of newly developed monodentate chiral spiro phosphine ligands.

Correlation of distribution characteristics and dynamic changes of gut microbiota with the efficacy of immunotherapy in EGFR-mutated non-small cell lung cancer.

BACKGROUND: The effects of gut microbiota and metabolites on the responses to immune checkpoint inhibitors (ICIs) in advanced epidermal growth factor receptor (EGFR) wild-type non-small cell lung cancer (NSCLC) have been studied. However, their effects on EGFR-mutated (EGFR +) NSCLC remain unknown. METHODS: We prospectively recorded the clinicopathological characteristics of patients with advanced EGFR + NSCLC and assessed potential associations between the use of antibiotics or probiotics and immunotherapy efficacy. Fecal samples were collected at baseline, early on-treatment, response and progression status and were subjected to metagenomic next-generation sequencing and ultra-high-performance liquid chromatography-mass spectrometry analyses to assess the effects of gut microbiota and metabolites on immunotherapy efficacy. RESULTS: The clinical data of 74 advanced EGFR + NSCLC patients were complete and 18 patients' fecal samples were dynamically collected. Patients that used antibiotics had shorter progression-free survival (PFS) (mPFS, 4.8 vs. 6.7 months; P = 0.037); probiotics had no impact on PFS. Two dynamic types of gut microbiota during immunotherapy were identified: one type showed the lowest relative abundance at the response time point, whereas the other type showed the highest abundance at the response time point. Metabolomics revealed significant differences in metabolites distribution between responders and non-responders. Deoxycholic acid, glycerol, and quinolinic acid were enriched in responders, whereas L-citrulline was enriched in non-responders. There was a significant correlation between gut microbiota and metabolites. CONCLUSIONS: The use of antibiotics weakens immunotherapy efficacy in patients with advanced EGFR + NSCLC. The distribution characteristics and dynamic changes of gut microbiota and metabolites may indicate the efficacy of immunotherapy in advanced EGFR + NSCLC.

Heterogeneity in the immune microenvironment of bone metastasis in driver-positive non-small cell lung cancer.

Mutations in epidermal growth factor receptor and anaplastic lymphoma kinase are common driver events in non-small cell lung cancer (NSCLC), which are associated with a high frequency of bone metastases (BMs). While the bone marrow represents a specialized immune microenvironment, the immune repertoire of BMs remains unknown. Considering the higher incidence of BMs in driver gene-positive NSCLCs, and the unique biology of the bone, herein, we assessed the infiltrating immune cells and T cell receptor (TCR) profile of BMs in driver-positive NSCLCs. Immune profile of BMs in driver gene-positive NSCLC were assessed in 10 patients, where 6 had driver gene-positive mutation. TCR and bulk RNA sequencing were performed on malignant bone samples. The diversity and clonality of the TCR repertoire were analyzed. The cellular components were inferred from bulk gene expression profiles computationally by CIBERSORT. Although BMs were generally regarded as immune-cold tumors, immune cell composition analyses showed co-existence of cytotoxic and suppressor immune cells in driver-positive BM samples, as compared to primary lung. Analysis of the TCR repertoire indicated a trend of higher diversity and similar clonality in the driver-positive compared with the driver-negative subsets. In addition, we identified two cases that showed the opposite response to immune checkpoint blockade. A comparison of these two patients' BM samples showed more highly amplified clones, fewer M2 macrophages and more activated natural killer cells in the responder. In summary, BMs in NSCLC are heterogeneous in their immune microenvironment, which might be related to differential clinical outcomes to immune checkpoint blockade.

Effects of sericin on oxidative stress and PI3K/AKT/mTOR signal pathway in cryopreserved mice ovarian tissue.

Ovarian tissue cryopreservation is an effective fertility protective strategy for preadolescent female cancer patients, whose tumor treatment cannot be delayed. In the present study, the effects of sericin, as an antioxidant, on mice ovarian tissue freezing and thawing were investigated. Mice ovarian tissues were cryopreserved and thawed in medium containing 0.5% or 1%sericin (w/v), and 0.1 mM melatonin. Then, the follicular morphology was observed. The levels of antioxidant enzymes were determined, including glutathione (GSH), glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) and catalase (CAT). Moreover, the levels of nitric oxide (NO), malondialdehyde (MDA) and lactate dehydrogenase (LDH) were also tested. Besides, apoptosis-related proteins Bcl-2 and Bax were determined. Our results showed that 1% sericin maintained follicular morphology, inhibited apoptosis, decreased MDA and NO levels, and boosted endogenous antioxidant enzyme levels, while had no significant effect on LDH levels. Furthermore, these effects may be related with the activation of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of Rapamycin (mTOR) signaling pathway, as demonstrated by increased PI3K, p-AKT and mTOR levels. These findings demonstrate that 1% sericin may reduce oxidative stress and protect ovarian tissues during freezing and thawing via PI3K/AKT/mTOR signaling pathway.

Genetic association study of intron variants in the forkhead box protein P3 gene in Chinese patients diagnosed with cervical cancer.

The aim of this study was to investigate the effects of forkhead box protein P3 (FOXP3) intron single nucleotide variants (SNVs) in high-risk human papilloma virus (HR-HPV) infection and cervical cancer (CC) malignant lesions. We performed FOXP3 genotyping in 350 patients with CC and 350 healthy controls using the ImLDR multiple single nucleotide polymorphism genotyping technology. The heterozygous mutation TC in rs2294021 decreased the risk of HR-HPV infection and CC malignant lesions (TC vs. TT: OR = 0.71, 95% CI = 0.51-0.99); the dominant model TC+CC and allele C in rs2294021 decreased the risk of CC malignant lesions (TC+CC vs. TT: OR = 0.69, 95% CI = 0.50-0.95; C vs. T: OR = 0.78, 95% CI = 0.63-0.97). The heterozygous mutation GA, dominant model GA+AA and allele A in rs3761549 also decreased the risk of HR-HPV infection and CC malignant lesions (GA vs. GG: OR = 0.70, 95% CI = 0.51-0.96; GA+AA vs. GG: OR = 0.69, 95% CI = 0.51-0.94; A vs. G: OR = 0.75, 95% CI = 0.58-0.96). Patients with CC and HR-HPV infection carrying rs2294021 TC and rs3761549 GA had lower expression of FOXP3 protein. Haplotype analysis revealed that T-C-A decreased the risk of HR-HPV infection. Furthermore, we found a significant association between immune cells infiltration and prognosis in patients with CC. Our findings demonstrated that rs2294021 and rs3761549 variants may protect against HR-HPV and CC malignant lesions by downregulating FOXP3 and that FOXP3 was associated with immune cells infiltration, which affected the prognosis of CC.

Chronic cough in asthma is associated with increased airway inflammation, more comorbidities, and worse clinical outcomes.

Background: Cough is often the most prominent and intractable symptom reported by patients with asthma, but few studies have explored the characteristics of patients with asthma and with chronic cough (CC) in a real-world setting. Methods: In a prospective cohort study, patients ages ≥ 18 years with stable asthma were consecutively recruited at the West China Hospital, Sichuan University. The patients were classified as having asthma with CC (the CC group) or asthma with non-CC (the non-CC group) after 3 months of optimized asthma therapy according to standard guidelines. Multidimensional assessment was performed at baseline, followed by a 12-month follow-up to assess asthma exacerbations. Results: Of 323 patients with asthma, 127 patients were assigned to the CC group and 196 patients were assigned to the non-CC group. The participants with CC were older and had more airflow obstruction; worse asthma control and quality of life; increased airway inflammation; upper respiratory tract infection as a trigger; and more comorbidities, such as psychological dysfunction, rhinitis, chronic obstructive pulmonary disease, and bronchiectasis. They reported greater work productivity loss and daily activity impairment, and increased moderate-to-severe exacerbations. Conclusion: The participants with asthma and with CC had a significant disease burden, with increased exacerbations, health-care utilization, and impaired work productivity and daily activity. These observations indicated potential clinical implications in patients with asthma and with CC, and call for more attention to this aspect of asthma.

Efficacy and safety of switching from brand-name to domestic generic levetiracetam in children with epilepsy. / å·¦ä¹™æ‹‰è¥¿å¦çš„å›½äº§ä»¿åˆ¶è¯æ›¿æ¢æ²»ç–—å„¿ç«¥ç™«ç—«çš„ç–—æ•ˆåŠå®‰å ¨æ€§ç ”ç©¶.

OBJECTIVES: To study the efficacy and safety of domestic generic levetiracetam in replacement of brand-name levetiracetam in the treatment of children with epilepsy. METHODS: A retrospective analysis was performed on the medical data of 154 children with epilepsy who received domestic generic levetiracetam in the inpatient or outpatient service of Guangdong Provincial People's Hospital from May 2019 to December 2020. Domestic generic levetiracetam and brand-name levetiracetam were compared in terms of efficacy and safety. RESULTS: For these 154 children, the epilepsy control rate was 77.3% (119/154) at baseline. At 6 months after switching to domestic generic levetiracetam, the epilepsy control rate reached 83.8% (129/154), which showed a significant increase (P<0.05). There was no significant change in the frequency of seizures from baseline to 6 months after switching (P>0.05). The incidence of refractory epilepsy in children with no response after switching treatment was significantly higher than that in children with response (P<0.05). Before switching, only 1 child (0.6%) experienced somnolence, while after switching, 3 children (1.9%) experienced mild adverse drug reactions, including dizziness, somnolence, irritability, and bad temper. CONCLUSIONS: Switching from brand-name to generic levetiracetam is safe and effective and holds promise for clinical application, but more prospective randomized controlled trials are required in future.

Small Airway Dysfunction in Asthma Is Associated with Perceived Respiratory Symptoms, Non-Type 2 Airway Inflammation, and Poor Responses to Therapy.

BACKGROUND: Emerging evidence has indicated that small airway dysfunction (SAD) contributes to the clinical expression of asthma. OBJECTIVES: The aim of the study was to explore the relationships of SAD assessed by forced expiratory flow between 25 and 75% (FEF25-75%), with clinical and inflammatory profile and treatment responsiveness in asthma. METHOD: In study I, dyspnea intensity (Borg scale), chest tightness, wheezing and cough (visual analog scales, VASs), and pre- and post-methacholine challenge testing (MCT) were analyzed in asthma patients with SAD and non-SAD. In study II, asthma subjects with SAD and non-SAD underwent sputum induction, and inflammatory mediators in sputum were detected. Asthma patients with SAD and non-SAD receiving fixed treatments were prospectively followed up for 4 weeks in study III. Spirometry, Asthma Control Questionnaire (ACQ), and Asthma Control Test (ACT) were carried out to define treatment responsiveness. RESULTS: SAD subjects had more elevated ΔVAS for dyspnea (p = 0.027) and chest tightness (p = 0.032) after MCT. Asthma patients with SAD had significantly elevated interferon (IFN)-γ in sputum (p < 0.05), and Spearman partial correlation found FEF25-75% significantly related to IFN-γ and interleukin-8 (both having p < 0.05). Furthermore, multivariable regression analysis indicated SAD was significantly associated with worse treatment responses (decrease in ACQ ≥0.5 and increase in ACT ≥3) (p = 0.022 and p = 0.032). CONCLUSIONS: This study indicates that SAD in asthma predisposes patients to greater dyspnea intensity and chest tightness during bronchoconstriction. SAD patients with asthma are characterized by non-type 2 inflammation that may account for poor responsiveness to therapy.

[Long-term follow-up of efficacy of insulin pump in the treatment of children with type 1 diabetes mellitus].

OBJECTIVE: To study the clinical effect of continuous subcutaneous insulin infusion (CSⅡ) versus multiple daily injection (MDI) on blood glucose control in children with type 1 diabetes mellitus (T1DM). METHODS: A retrospective analysis was performed on the medical data of 91 children with T1DM who were treated with CSⅡ for more than 1 year and 75 children with T1DM who were treated with MDI. The two groups were compared in terms of glycosylated hemoglobin (HbA1C) and the recurrence of diabetic ketoacidosis (DKA) to evaluate the difference in the efficacy during the 3-year follow-up. A survey was conducted for the children in the CSⅡ group and their family members to investigate the degree of satisfaction with insulin pump. RESULTS: There was no significant difference in age, sex, and course of diabetes between the CSⅡ and MDI groups at disease onset and in the first year, the second year, and the third year of follow-up (P > 0.05). There was no significant difference in the HbA1C level between the two groups at disease onset (P > 0.05), but in the first year of follow-up, the CSⅡ group had a significantly lower HbA1C level than the MDI group (P=0.04). There was no significant difference in the HbA1C level between the two groups in the second year and the third year of follow-up (P > 0.05). The CSⅡ group had a higher proportion of children with HbA1C < 7.5% than the MDI group in the first year, the second year, and the third year of follow-up (P > 0.05). Within the 3 years of follow-up, 2 children in the CSⅡ group and 8 in the MDI group experienced the recurrence of DKA. In the third year of follow-up, there was no significant difference in blood pressure and blood lipids between the CSⅡ and MDI groups (P > 0.05). Most children and their family members (87%) were satisfied with CSⅡ treatment. CONCLUSIONS: Children with T1DM treated with CSⅡ have a better control of blood glucose than those treated with MDI, and children and their family members are satisfied with CSⅡ treatment. Therefore, it holds promise for clinical application.

Antioxidative effect of melatonin on cryopreserved ovarian tissue in mice.

Cryopreservation of ovarian tissues (OTs) has become the most effective way to preserve the fertility of female cancer patients. However, cryopreservation of OTs is still relatively at an experimental stage. The aim of study is to examine the effect of melatonin (MTL) on cryopreserved-thawed OTs. Fragments of OTs were cryopreserved in medium containing different concentrations (0 mM, 0.001 mM, 0.01 mM, 0.1 mM and 1 mM) of MLT. The endogenous enzymes (GSH-PX, GSH, SOD, CAT and T-AOC), MDA and ROS levels were all evaluated after cryopreservation. Our results showed that the 0.1 mM of MLT significantly improved the survival and diameter of follicles (P < 0.001). Meanwhile, the antioxidant enzymes activities (including GSH-PX, GSH, SOD, CAT and T-AOC) were enhanced and MDA content were significantly decreased in 0.1 mM of MLT group compared to other groups (P < 0.001). Additionally, compared to the control group, MTL of 0.1 mM resulted in a significantly lower ROS level. In conclusion, MLT protects the quality of cryopreserved OTs by decreasing oxidative stress level and the optimal concentration is 0.1 mM.

Clinical and Inflammatory Features of Exacerbation-Prone Asthma: A Cross-Sectional Study Using Multidimensional Assessment.

BACKGROUND: Reducing asthma exacerbations is a major target of current clinical guidelines, but identifying features of exacerbation-prone asthma (EPA) using multidimensional assessment (MDA) is lacking. OBJECTIVE: To systemically explore the clinical and inflammatory features of adults with EPA in a Chinese population. METHODS: We designed a cross-sectional study using the Severe Asthma Web-based Database from the Australasian Severe Asthma Network (ASAN). Eligible Chinese adults with asthma (n = 546) were assessed using MDA. We stratified patients based on exacerbation frequency: none, few (1 or 2), and exacerbation prone (≥3). Univariate and multivariable negative binomial regression analyses were performed to investigate features associated with the frequency of exacerbations. RESULTS: Of 546 participants, 61.9% had no exacerbations (n = 338), 29.6% had few exacerbations (n = 162), and 8.4% were exacerbation prone (n = 46) within the preceding year. EPA patients were characterized by elevated blood and sputum eosinophils but less atopy, with more controller therapies but worse asthma control and quality of life (all p < 0.05). In multivariable models, blood and sputum eosinophils (adjusted rate ratio = 2.23, 95% confidence interval = [1.26, 3.84] and 1.67 [1.27, 2.21], respectively), FEV1 (0.90 [0.84, 0.96]), bronchodilator responsiveness (1.16 [1.05, 1.27]), COPD (2.22 [1.41, 3.51]), bronchiectasis (2.87 [1.69, 4.89]), anxiety (2.56 [1.10, 5.95]), and depression (1.94 [1.20, 3.13]) were found. Further, upper respiratory tract infection (1.83 [1.32, 2.54]) and food allergy (1.67 [1.23, 2.25]) were at high risk of asthma symptom triggers. CONCLUSION: EPA is a clinically recognizable phenotype associated with several recognizable traits that could be addressed by targeted treatment.

Bioluminescence reporter for monitoring G2-phase cell cycle arrest in vivo.

The bioengineered luciferase reporter has been widely used for monitoring of a variety of molecular events in living cells because of their ability to provide highly sensitive quantitation with broad linearity. In the present study, we made a cyclin A2-luciferase (CYCA-Luc) fusion protein and examined the utility of this optical reporter for monitoring G2-phase cell cycle arrest in living animals. In vitro luciferase assay and in vivo bioluminescence imaging assay showed that the lithium chloride (LiCl), G2-phase-specific drug, induced G2-phase arrest of cell cycle and increased the activity of this reporter under in vitro or in vivo conditions, and this reporter can also be potentially used in high-throughput screening efforts aimed at discovering novel anti-cancer drugs that will cause cell cycle arrest at the G2-phase in cultivated cell lines and animal models.

ESRP1 Induces Cervical Cancer Cell G1-Phase Arrest Via Regulating Cyclin A2 mRNA Stability.

Accumulating evidence indicates that epithelial splicing regulatory protein 1 (ESRP1) can inhibit the epithelial-to-mesenchymal transition (EMT), thus playing a central role in regulating the metastatic progression of tumors. However, it is still not clear whether ESRP1 directly influences the cell cycle, or what the possible underlying molecular mechanisms are. In this study, we showed that ESRP1 protein levels were significantly correlated with the Ki-67 proliferative index (r = -0.521; p < 0.01), and that ESRP1 overexpression can significantly inhibit cervical carcinoma cell proliferation and induced G1-phase arrest by downregulating cyclin A2 expression. Importantly, ESRP1 can bind to GGUGGU sequence in the 3'UTR of the cyclin A2 mRNA, and ESRP1 overexpression significantly decreases the stability of the cyclin A2 mRNA. In addition, our experimental results confirm that ESRP1 overexpression results in enhanced CDC20 expression, which is known to be responsible for cyclin A2 degradation. This study provides the first evidence that ESRP1 overexpression induces G1-phase cell cycle arrest via reducing the stability of the cyclin A2 mRNA, and inhibits cervical carcinoma cell proliferation. The findings suggest that the ESRP1/cyclin A2 regulatory axis may be essential as a regulator of cell proliferation, and may thus represent an attractive target for cervical cancer prevention and treatment.

Solvent diffusion in polymer-embedded hollow nanoparticles studied by in situ small angle X-ray scattering.

In situ time-resolved small-angle X-ray scattering is introduced as a method to monitor the diffusion of a solvent in ceramic hollow nanoparticles (HNPs) supported by a polymer gel scaffold. Changes in the form factor were matched to discrete scattering models. A consecutive reaction kinetic model is used to analyze different stages of solvent diffusion. Rate constants and diffusion coefficients are extracted. By taking the diffusion of low molecular poly(ethylene glycol) in poly(ethylene oxide)-embedded HNPs as a model case, it was found that it took about 0.7 s for the solvent to diffuse through the 6 nm thick shell of HNPs and another 1.2 s to fill the inner cavity, while the diffusion coefficient was of the order of 1018 m2 s-1. The results demonstrate that the method can simultaneously measure solvent penetration into the polymer gel and into embedded sub-100 nm HNPs.

Distribution and prognosis of uncommon metastases from non-small cell lung cancer.

BACKGROUND: According to the literature and our experience, the most common sites of non-small cell lung cancer (NSCLC) metastases include the brain, bone, liver, adrenal glands, contralateral lung and distant lymph nodes. Metastases to other organs are relatively rare. There have been numerous case reports and a few small case series of uncommon metastases derived from NSCLC. METHODS: We defined all organs except the common metastatic sites mentioned above as uncommon sites of metastasis. Patients with uncommon metastases among 2,872 consecutive NSCLC patients with stage IV disease at the Guangdong Lung Cancer Institute (GLCI) from 2006 to 2012 were included in this study. The diagnosis of uncommon metastases was based on pathology or imaging studies. RESULTS: Uncommon metastases were diagnosed in 193 cases at anatomical sites such as the soft tissue, kidney, pancreas, spleen, peritoneum, intestine, bone marrow, eye, ovary, thyroid, heart, breast, tonsil and nasal cavity. Uncommon metastases were identified as independent poor prognostic factors through a multivariate analysis with a HR (hazard ratio) of 1.29 [95% confidence interval (CI) 1.09-1.52, P < 0.01]. Those patients who received systemic therapy plus local treatment had a better survival rate than did those who received systemic therapy only (P < 0.01); all patients received best supportive care. CONCLUSIONS: Metastases to the above mentioned sites are infrequent. The presentation of uncommon metastases tends to indicate a poor outcome, and selected patients may benefit from local treatment.

[Effect of Ilepcimide Combined Western Drugs on Serum Level of Neuron Specific Enolase in Treating Epilepsy Children Patients].

Objective To observe changes of serum neuron specific enolase (NSE) level in children patients with epilepsy by additional use of ilepcimide (piperine derivative). Methods Totally 107 epilepsy children patients were assigned to the test group (77 cases) and the control group (30 cases) ac- cording to random digit table. Children patients in the control group received anti-epileptic Western drugs only. Those in the test group additionally took ilepcimide, 5 mg/kg per day as initial dose, taken in two times. The dose was gradually added to those without control of epilepsy attack. Added dose within a week should not exceed 10 mg/kg per day. The therapeutic course for all was one year. Electoencephalo- gram (EEG) was performed before treatment, half a year after treatment, and one year after treatment, respectively. Serum NSE level was detected using electrochemiluminescence. Efficacy was assessed after 1-year treatment. Results The total effective rate was 65. 0% (50177) in the test group, with statistical difference as compared with that in the control group [30. 0% (9/30), P <0. 01 ]. Compared with before treatment, serum NES-level obviously decreased in the test group after 0. 5-year treatment and 1- year treatment respectively (P <0. 05, P <0. 01). Besides, serum NES level was lower after 1-year treatment than after 0. 5-year treatment (P <0. 05, P <0. 01). There was no statistical difference in serum NES level between the test group and the control group at each time point (P >0. 05). Results of EEG were obviously superior in the test group (3 with normal range EEG, 5 critically abnormal EEG, 69 abnormal EEG) to the control group (2 with normal range EEG and 75 abnormal EEG) after 1-year treatment, with statistical difference (Z= -2. 33, P <0. 05). There was no statistical difference in EEG results of the control group between before treatment (all abnormal EEG) and after 1-year treatment (3 critically abnormal EEG and 27 abnormal EEG) (Z = -1. 732, P > 0. 05). Conclusion Adding ilepcimide (piperine derivative) for epilepsy children patients could lower serum NSE level and the frequency of seizures, and improve results of EEG.

[Optimization of Extraction Technology for Sericin from Silkworm Cocoon with Orthogonal Design].

OBJECTIVE: To optimize the appropriate extracting technology for sericin from Silkworm cocoon. METHODS: Using sericin extraction rates and sericin content as the indices. The single and orthogonal experiments were used to determine the best conditions. RESULTS: The optimal extraction technology for sericin from Silkworm cocoon was as follows: 1: 30 for the ratio of solid to liquid, 3 h reflux for 2 times of extraction and water temperature at 100 degrees C. The extraction rate of sericin from Silkworm cocoon was 27.1%. CONCLUSION: The optimal extraction technology is stable, feasible, and can provide reference for further pharmacological study on cocoon sericin.

Detecting the spectrum of multigene mutations in non-small cell lung cancer by Snapshot assay.

As molecular targets continue to be identified and more targeted inhibitors are developed for personalized treatment of non-small cell lung cancer (NSCLC), multigene mutation determination will be needed for routine oncology practice and for clinical trials. In this study, we evaluated the sensitivity and specificity of multigene mutation testing by using the Snapshot assay in NSCLC. We retrospectively reviewed a cohort of 110 consecutive NSCLC specimens for which epidermal growth factor receptor (EGFR) mutation testing was performed between November 2011 and December 2011 using Sanger sequencing. Using the Snapshot assay, mutation statuses were detected for EGFR, Kirsten rate sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase catalytic alpha polypeptide (PIK3CA), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), v-ras neuroblastoma viral oncogene homolog (NRAS), dual specificity mitogen activated protein kinase kinase 1 (MEK1), phosphatase and tensin homolog (PTEN), and human epidermal growth factor receptor 2 (HER2) in patient specimens and cell line DNA. Snapshot data were compared to Sanger sequencing data. Of the 110 samples, 51 (46.4%) harbored at least one mutation. The mutation frequency in adenocarcinoma specimens was 55.6%, and the frequencies of EGFR, KRAS, PIK3CA, PTEN, and MEK1 mutations were 35.5%, 9.1%, 3.6%, 0.9%, and 0.9%, respectively. No mutation was found in the HER2, NRAS, or BRAF genes. Three of the 51 mutant samples harbored double mutations: two PIK3CA mutations coexisted with KRAS or EGFR mutations, and another KRAS mutation coexisted with a PTEN mutation. Among the 110 samples, 47 were surgical specimens, 60 were biopsy specimens, and 3 were cytological specimens; the corresponding mutation frequencies were 51.1%, 41.7%, and 66.7%, respectively (P = 0.532). Compared to Sanger sequencing, Snapshot specificity was 98.4% and sensitivity was 100% (positive predictive value, 97.9%; negative predictive value, 100%). The Snapshot assay is a sensitive and easily customized assay for multigene mutation testing in clinical practice.

CABP4 mutation in mice shows alteration in protein expression level and neuron discharge frequency.

Background: The calcium-binding protein 4 (CABP4) gene is a newly identified epilepsy-related gene that might be associated with a rare type of genetic focal epilepsy; that is, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In vitro, mutant CABP4 causes an increased inward flow voltage of calcium ions and a significant increase in the electrical signal discharge in hippocampus neurons; however, the role of CABP4 in epilepsy has not yet been specifically described, and there is not yet a CABP4 mutant animal model recapitulating the epilepsy phenotype. Methods: We introduced a human CABP4 missense mutation into the C57BL/6J mouse genome and generated a knock-in strain carrying a glycine-to-aspartic acid mutation in the gene. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were performed to evaluate the CABP4 expression level. Slice patch-clamp recording was carried out on pyramidal cells of prefrontal cortex layers II and III. Results: The CABP4G155D/+ mutant mice were viable and born at an expected Mendelian ratio. Surprisingly, the heterozygous (HE) mice did not display either an abnormal appearance or an overt seizure phenotype, and there was no statistically significant difference between the HE and wild-type (WT) mice in terms of overall messenger RNA (mRNA) and protein expression. However, the HE mutant mice showed an imbalance in the amount of protein expressed in the brain regions. Additionally, the patch-clamp recordings from the HE mouse layer II/III cortical pyramidal cells revealed an increase in the frequency of micro-excitatory post-synaptic currents (mEPSCs) but no change in the amplitude was observed. Conclusions: The findings of this study suggest that the CABP4 p.G155D mutation might be one of the mechanisms underlying seizure onset.

Identification of a risk score model based on tertiary lymphoid structure-related genes for predicting immunotherapy efficacy in non-small cell lung cancer.

BACKGROUND: Tertiary lymphoid structures (TLSs) affect the prognosis and efficacy of immunotherapy in patients with non-small cell lung cancer (NSCLC), but the underlying mechanisms are not well understood. METHODS: TLSs were identified and categorized online from the Cancer Digital Slide Archive (CDSA). Overall survival (OS) and disease-free survival (DFS) were analyzed. GSE111414 and GSE136961 datasets were downloaded from the GEO database. GSVA, GO and KEGG were used to explore the signaling pathways. Immune cell infiltration was analyzed by xCell, ssGSEA and MCP-counter. The analysis of WGCNA, Lasso and multivariate cox regression were conducted to develop a gene risk score model based on the SU2C-MARK cohort. RESULTS: TLS-positive was a protective factor for OS according to multivariate cox regression analysis (p = 0.029). Both the TLS-positive and TLS-mature groups exhibited genes enrichment in immune activation pathways. The TLS-mature group showed more activated dendritic cell infiltration than the TLS-immature group. We screened TLS-related genes using WGCNA. Lasso and multivariate cox regression analysis were used to construct a five-genes (RGS8, RUF4, HLA-DQB2, THEMIS, and TRBV12-5) risk score model, the progression free survival (PFS) and OS of patients in the low-risk group were markedly superior to those in the high-risk group (p < 0.0001; p = 0.0015, respectively). Calibration and ROC curves indicated that the combined model with gene risk score and clinical features could predict the PFS of patients who have received immunotherapy more accurately than a single clinical factor. CONCLUSIONS: Our data suggested a pivotal role of TLSs formation in survival outcome and immunotherapy response of NSCLC patients. Tumors with mature TLS formation showed more activated immune microenvironment. In addition, the model constructed by TLS-related genes could predict the response to immunotherapy and is meaningful for clinical decision-making.