NOTCH1 mutation associates with impaired immune response and decreased relapse-free survival in patients with resected T1-2N0 laryngeal cancer.

Background: Patients with early-stage laryngeal cancer, even stage T1-2N0, are at considerable risk of recurrence and death. The genetic and immunologic characteristics of recurrent laryngeal cancer remain unclear. Methods: A total of 52 T1-2N0 laryngeal cancer patients were enrolled. Of these, 42 tissue samples were performed by targeted DNA sequencing, and 21 cases were performed by NanoString immuno-oncology targeted RNA sequencing to identify the distinct molecular bases and immunologic features associated with relapse in patients with early laryngeal cancer, respectively. Results: To the best to our knowledge, we present for the first time an overview of the genomic mutation spectrum of early-stage laryngeal cancers. A total of 469 genomic alterations were detected in 211 distinct cancer-relevant genes, and the genes found to be mutated in more than five patients (>10%) included tumor protein p53 (TP53, 78.5%), FAT atypical cadherin 1 (FAT1, 26%), LDL receptor related protein 1B (LRP1B, 19%), cyclin dependent kinase inhibitor 2A (CDKN2A, 17%), tet methylcytosine dioxygenase 2 (TET2, 17%), notch receptor 1 (NOTCH1, 12%) and neuregulin 1 (NRG1, 12%). Recurrent laryngeal cancer demonstrated a higher tumor mutation burden (TMB), as well as higher LRP1B mutation and NOTCH1 mutation rates. Univariate and multivariate analyses revealed that high TMB (TMB-H) and NOTCH1 mutation are independent genetic factors that are significantly associated with shorter relapse-free survival (RFS). Simultaneously, the results of the transcriptome analysis presented recurrent tumors with NOTCH1 mutation displayed upregulation of the cell cycle pathway, along with decreased B cells score, T cells score, immune signature score and tumor-infiltrating lymphocytes (TILs) score. The Cancer Genome Atlas (TCGA)-laryngeal cancer dataset also revealed weakened immune response and impaired adhesion functions in NOTCH1-mutant patients. Conclusions: Genomic instability and impaired immune response are key features of the immunosurveillance escape and recurrence of early laryngeal cancer after surgery. These findings revealed immunophenotypic attenuation in recurrent tumors and provided valuable information for improving the management of these high-risk patients. Due to the small number of patients in this study, these differences need to be further validated in a larger cohort.

Genomic landscape and tumor mutation burden analysis of Chinese patients with sarcomatoid carcinoma of the head and neck.

BACKGROUND: Sarcomatoid carcinoma (SC) of the head and neck (HN) is a rare disease that has both sarcomatoid and cancerous components. The genetic background and mechanisms of tumorigenesis remain largely unrevealed, and the progress of precision therapy has been limited. METHODS: Targeted DNA-based next-generation sequencing (NGS) was performed by a 539 genes panel of pan-cancer in 12 patients with SC of the HN to identify their genetic alterations and investigate clinically actionable mutations for use in precision treatment. RESULTS: TP53 was identified as the most frequently mutated gene. Genes related to the cell cycling, chromatin remodeling and histone modification were found to be frequently mutated in patients with SC of the HN. Alterations in receptor tyrosine kinases (RTKs) were also found in six patients. In addition, four patients had mutations in members of the downstream RAS and PI3-kinase pathways, PIK3CA was identified as the most frequently mutated gene in this pathway. The tumor mutation burden (TMB) value ranged from 0.71 to 14.71 per megabase, with a median of 4.34. The TMB value of PIK3CA mutation patients was significantly higher than that of PIK3CA wild-type patients. CONCLUSIONS: This was the first study to investigate genomic alterations specifically in Chinese patients with SC of the HN. Our research results showed that 10 out of 12 patients can match the targeted therapies or immunotherapy currently available in clinical practice or active clinical trials, suggesting precision therapy has the potential utility to improve the long-term prognosis for patients with the rare disease. Due to the small number of patients in this study, the findings need to be validated in a larger cohort.

Determination of the enantiomers of alpha-hydroxy- and alpha-amino acids in capillary electrophoresis with contactless conductivity detection.

The enantiomers of the anions of five alpha-hydroxy acids, namely lactic acid, alpha-hydroxybutyric acid, 2-hydroxycaproic acid, 2-hydroxyoctanoic acid and 2-hydroxydecanoic acid, as well as the two alpha-amino acids aspartic acid and glutamic acid, were baseline separated and detected by CE with contactless conductivity detection. Vancomycin was employed as chiral selector and could be used with conductivity detection without having to resort to a partial filling protocol as needed when this reagent is used with UV absorbance measurements. The procedure was successfully applied to the determination of the lactic acid enantiomers in samples of milk and yogurt. Linearity was achieved in the concentration range of 10-500 micromol/L with good correlation coefficients (0.9993 and 0.9990 for L- and D-lactic acid, respectively). The LODs (3 S/N) for L- and D-lactic acid were determined as 2.8 and 2.4 micromol/L, respectively.

Protective effect of spleen-yin-nourishing recipe on amyloid beta-peptide-induced damage of primarily cultured rat hippocampal neurons and its mechanism.

OBJECTIVE: To observe the relationship among amyloid beta-peptide (Abeta)-induced neurotoxicity, serum-inducible kinase (SNK)-spine-associated Rap guanosine triphosphatase activating protein (SPAR) pathway and N-methyl-D-aspartate receptor (NMDAR), and to explore the mechanism of the protective effect of spleen-yin nourishing recipe (Zibu Piyin Recipe, ZBPYR) in hippocampal neurons against Abeta-induced neurotoxicity. METHODS: The Abeta(1-40) powder was dissolved in 1 x PBS and incubated at 37 degrees centigrade, and then aggregated fibrillar Abeta(1-40) was obtained 72 h later. We used rat primary hippocampal neurons as cell model. ZBPYR-containing serum was gained by the method of serum pharmacology. ZBPYR-containing serum was added to the culture 1 h before Abeta(1-40) (5 micromol/L) exposure. Cells were harvested 2 h after Abeta(1-40) exposure for total RNA extracting. Then the mRNA expression levels of SNK, SPAR and NMDAR subunits NR1, NR2A and NR2B were detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: After 2-hour Abeta(1-40) exposure, we found that the expression level of SNK mRNA was up-regulated and the expression levels of SPAR, NR1, NR2A and NR2B mRNAs were down-regulated in hippocampal neurons as compared with control group (P < 0.01, P < 0.05). While with ZBPYR-containing serum pretreatment, the expression level of SNK mRNA was down-regulated and the levels of SPAR, NR1, NR2A and NR2B were up-regulated as compared with Abeta(1-40) exposure, and 2% ZBPYR-containing serum showed the best effect (P < 0.05). CONCLUSION: Abeta-induced neurotoxicity was related to SNK-SPAR pathway and NMDAR; ZBPYR-containing serum can protect neurons from Abeta-induced neurotoxicity, and this protective effect may be performed by regulating the expression of NMDAR and blocking of the SNK-SPAR pathway.

Determination of gamma-hydroxybutyric acid in clinical samples using capillary electrophoresis with contactless conductivity detection.

A method for the determination of gamma-hydroxybutyric acid (GHB) in urine and serum samples with capillary electrophoresis using capacitively coupled contactless conductivity detection (CE-C(4)D) was developed. The optimized separation buffer consisting of 20 mM of arginine, 10 mM of maleic acid and 30 microM of cetyltrimethylammonium bromide (CTAB) contained 5 mM vancomycin to facilitate the separation of gamma-hydroxybutyric acid from beta-hydroxybutyric acid (BHB), which is also present in clinical samples. The detection limits in the clinical samples were found to be about 2 microg/ml, which is well below the required sensitivity for the recognition of overdosage and adequate for the determination of endogenous concentrations in urine. The determination of GHB in both types of samples was carried out directly after a fourfold dilution without requiring any derivatization or extraction procedures.

Ion-pair liquid-liquid-liquid microextraction of nerve agent degradation products followed by capillary electrophoresis with contactless conductivity detection.

The four nerve agent degradation products methylphosphonic acid (MPA), ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA) have been successfully extracted from aqueous sample solution by ion-pair liquid-liquid-liquid microextraction. In this procedure, the target analytes in the sample solution were converted into their ion-pair complexes with tri-n-butyl amine and then extracted by an organic solvent (1-octanol) layer on top of the sample solution. Simultaneously, the analytes were back-extracted into a drop of an aqueous acceptor solution which was suspended in the organic phase at a microsyringe needle tip. The factors influential to extraction: type of organic solvent, type of ion-pair reagent and its concentration, pH values of sample solution and acceptor aqueous phase, stirring rate and extraction time were investigated in detail. After extraction, the drop of the acceptor solution was withdrawn into the syringe and injected into a capillary electrophoresis system for analysis. Using contactless conductivity detection, direct quantification of these compounds is possible. Moreover, large-volume sample injection was employed for further preconcentration. Improvements in the limits of detection between 2.5 and 4 orders of magnitude could be achieved and concentrations at the ng/mL level can be determined. This newly established approach was successfully applied to a spiked river water sample.

Enantiomeric separation of 1-phenylethylamine and 1-cyclohexylethylamine in capillary electrophoresis with contactless conductivity detection.

Contactless conductivity detection was employed for the detection of the enantiomers of 1-phenylethylamine and 1-cyclohexylethylamine which were separated in capillary electrophoresis with unprecedented high resolutions R(s) of 2.3 and 3.3, respectively, by using a combination of dimethyl-beta-cyclodextrin and the chiral crown ether 18C6H4 as chiral selectors in a citric acid buffer of pH 2.4. The conductivity measurement enabled the direct detection, i.e. without having to derivatize or resort to indirect methods, of all species including the non-UV-absorbing enantiomers of cyclohexylamine. Detection limits of 0.5 microM were achieved and the determination of enantiomeric ratios of up to 99:1 was found possible.

Separation of enantiomers in capillary electrophoresis with contactless conductivity detection.

Contactless conductivity detection is successfully demonstrated for the enantiomeric separation of basic drugs and amino acids in capillary electrophoresis (CE). Derivatization of the compounds or the addition of a visualization agent as for indirect optical detection schemes were not needed. Non-charged chiral selectors were employed, hydroxypropylated cyclodextrin (CD) for the more lipophilic basic drugs and 18-crown-6-tetracarboxylic acid (18C6H4) for the amino acids. Acidic buffer solutions based on lactic or citric acid were used. The detection limits were determined as 0.3 microM for pseudoephedrine as an example of a basic drug and were in the range from 2.5 to 20 microM for the amino acids.

Decreased expression of spine-associated Rap guanosine triphosphatase-activating protein (SPAR) in glutamate-treated primary hippocampal neurons.

Spine-associated Rap guanosine triphosphatase-activating protein (SPAR) is an important regulator of activity-dependent remodeling of synapses. It is also critically involved in both mature dendritic spine formation and the maintenance of spine maturity. Glutamate is a major neurotransmitter of the brain, and is involved in all aspects of cognitive function, as it is the primary transmitter utilized by the cortical and hippocampal pyramidal neurons. Glutamate has also been associated with neuronal dendritic spine damage. The precise molecular mechanisms underlying dendritic spine damage following glutamate-induced neurotoxicity remain unknown. In the current study, we measured mRNA and protein expression levels of SPAR and serum-inducible kinase (SNK) in primary hippocampal neurons following glutamate treatment. Expression of SPAR and SNK was altered by glutamate treatment, indicating that the SPAR and SNK signaling pathways may be involved in the damage to dendritic spines in hippocampal neurons following excitotoxicity induced by glutamate.

Separating stereoisomers of di-, tri-, and tetrapeptides using capillary electrophoresis with contactless conductivity detection.

The separation and detection of small oligopeptides in CE with contactless conductivity detection were demonstrated. A strongly acidic separation buffer (0.5 M acetic acid) was employed in order to render the species cationic. Separation of the stereoisomers was achieved in typically 10-15 min by using either dimethyl-beta-CD (DM-beta-CD), (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C(6)H(4)), a combination of the two substances, or of histidine, as buffer additives. Calibration curves were determined for isomers of Gly-Asp and H-Pro-Asp-NH(2), in the range of 0.05-0.5 mM and 0.1-1 mM, respectively, and were found to be linear. LODs were determined to be in the order of 1.0 microM. The determination of isomeric impurities down to about 1% was found possible. Species showing good separation could also be successfully determined on an electrophoretic lab-on-chip device, with analysis times of a few minutes.

Determination of different classes of amines with capillary zone electrophoresis and contactless conductivity detection.

The use of contactless conductivity detection for the determination of different organic amines in CE was successfully demonstrated. Aliphatic non UV-absorbing species could be determined along absorbing compounds by measuring the conductivity of their protonated forms. The species tested included short-chained aliphatic primary, secondary and tertiary amines, branched aliphatic amines, diamines, hydroxyl-substituted amines as well as species incorporating aromatic and non-aromatic cyclic moieties. Highest sensitivity was obtained with BGE solutions containing solely acetic acid. A concentration of 0.5 M at a pH value of 2.5 was used. Detection limits were in the order of 1 microM. Complete separation of cis- and trans-1,2-diaminocyclohexane could be achieved by adding 18-crown-6 as modifier to the electrolyte solution.

Enantiomeric separation of underivatized small amines in conventional and on-chip capillary electrophoresis with contactless conductivity detection.

The determination of the enantiomers of small non-UV-absorbing amines which otherwise can only be achieved with difficulty was possible by using a combination of the chiral crown ether (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) and dimethyl-beta-CD as selectors in CE and contactless conductivity measurement for detection. Alkylamines without any other functional group, amino alcohols, species with ether or ester groups and with a cyclic moiety were investigated. The detection limits were found to be about 1.0 microM and the determination is possible up to at least 1.0 mM. The determination of enantiomeric ratios of up to 99.5:0.5 was also found feasible.