ABSTRACT
Metasurfaces demonstrate excellent capabilities in manipulating the phase, amplitude and polarization of light. Metalens, as a typical kind of metasurface devices, shows great prospect in simplifying imaging systems. However, like diffractive optical elements, intrinsic dispersion of metasurfaces is high. Thus, significant chromatic aberration is present in common metalenses, deteriorating imaging quality under broadband illumination condition and limiting their applications. To tackle this problem, broadband achromatic metalenses have been proposed and demonstrated in the visible and near-infrared wavelength regions so far. However, broadband achromatic metalens working in the mid-wave and long-wave infrared is still rare. In this paper, thanks to the ingenious design of meta-units that provide the required local phase and phase dispersion, several all-silicon broadband achromatic metalenses working in the mid-wave infrared (3-5â µm) or long-wave infrared (8-14â µm) wavelengths are proposed. Numerical simulation results demonstrate that the designed broadband achromatic metalenses can provide a near-constant focal length with small deviations and an average focusing efficiency of about 70% over the whole operation bandwidths. In addition, these metalenses hold near diffraction-limited focusing capability and polarization-independent focusing features. The achromatic metalenses proposed here are beneficial for improving imaging quality under broadband illumination and increasing detection efficiency of mid-wave and long-wave infrared detection systems.
ABSTRACT
The complexity of the chemistry behind the hydrothermal conversion is enormous. Components interact with their own physical and chemical structure, making it harsh to understand the conversion as a whole. Herein, the six-water recirculation and loading nano SiO2 experiment in a one-pot hydrothermal carbonization procedure was designed to elucidate the mechanism of regulating the functional groups and microporous structure of the hydrochar surface. The hydrochar prepared by the second circulating liquid and loading nano-SiO2 (HBC-R2/Si) was equipped most enriched functional groups (carboxyl = 11.48 µmol/g, phenolic hydroxyl = 52.98 µmol/g, lactone groups = 46.52 µmol/g) and suitable pore size (1.90 nm-1.93 nm) as a sorbent riched in hemicellulose. The sorption kinetics (equilibrium reached ≈ 480 min) are approximately evenly fitted by the pseudo-second-order, Weber and Morris, and Elovich models, indicating that membranes and particles diffusion, pore diffusion, and surface sorption coexisted in the sorption of methylene blue (MB) on the hydrochar materials. Simultaneously, all hydrochar materials achieved over 25% MB removal within 90 min (liquid membrane diffusion) and over 40% for HBC-R2 and HBC-R2/Si, suggesting that liquid membrane diffusion is the predominant rate-limiting step. Pearson's correlation analysis and Mantel's analysis announced that the cation exchange capacity (CEC), pore size, and carboxyl groups on the hemicellulose affect the sorption capacity by limiting the pore diffusion procedure. However, the CEC and the phenolic hydroxyl groups on the cellulose and hemicellulose affect the sorption rate by limiting membrane diffusion. Three consecutive sorption/desorption cycles confirmed the high stability and reusability of HBC-R2/Si composites.
Subject(s)
Carbon , Cellulose , Carbon/chemistry , Kinetics , Methylene Blue/analysis , Surface Properties , AdsorptionABSTRACT
Snow is one of the most critical sources of freshwater, which influences the global water cycle and climate change. However, it is difficult to monitor global snow variations with high spatial-temporal resolution using traditional techniques due to their costly and labor-intensive nature. Nowadays, the Global Positioning System Interferometric Reflectometry (GPS-IR) technique can measure the average snow depth around a GPS antenna using its signal-to-noise ratio (SNR) data. Previous studies focused on the use of GPS data at sites located in flat areas or on very gentle slopes. In this contribution, we propose a strategy called the Tilted Surface Strategy (TSS), which uses the SNR data reflected only from the flat quadrants to estimate the snow depth instead of the conventional strategy, which employs all the SNR data reflected from the whole area around a GPS antenna. Three geodetic GPS sites from the Plate Boundary Observatory (PBO) project were chosen in this experimental study, of which GPS sites p683 and p101 were located on slopes with their gradients up to 18% and the site p025 was located on a flat area. Comparing the snow depths derived with the GPS-IR TSS method with the snow depth results provided with the GPS-PBO, i.e., GPS-IR with the conventional strategy, the Snowpack Telemetry (SNOTEL) network measurements and gridded Snow Data Assimilation System (SNODAS) estimates, it was found that the snow depths derived with the four methods had a good agreement, but the snow depth time series with the GPS-IR TSS method were closer to the SNOTEL measurements and the SNODAS estimates than those with GPS-PBO method. Similar observations were also obtained from the cumulative snowfall time series. Results generally indicated that for those GPS sites located on slopes, the TSS strategy works better.
ABSTRACT
In Global navigation satellite system (GNSS) data processing, integer ambiguity acceptance test is considered as a challenging problem. A number of ambiguity acceptance tests have been proposed from different perspective and then unified into the integer aperture estimation (IA) framework. Among all the IA estimators, the optimal integer aperture (OIA) achieves the highest success rate with the fixed failure rate tolerance. However, the OIA is of less practical appealing due to its high computation complexity. On the other hand, the popular discrimination tests employ only two integer candidates, which are the essential reason for their sub-optimality. In this study, a generalized difference test (GDT) is proposed to exploit the benefit of including three or more integer candidates to improve their performance from theoretical perspective. The simulation results indicate that the third best integer candidates contribute to more than 70% success rate improvement for integer bootstrapping success rate higher than 0.8 case. Therefore, the GDT with three integer candidates (GDT3) achieves a good trade-off between the performance and computation burden. The threshold function is also applied for rapid determination of the fixed failure rate (FF)-threshold for GDT3. The performance improvement of GDT3 is validated with real GNSS data set. The numerical results indicate that GDT3 achieves higher empirical success rate while the empirical failure rate remains comparable. In a 20 km baseline test, the success rate GDT3 increase 7% with almost the same empirical failure rate.
ABSTRACT
PURPOSE: To investigate pan-ethnic SMN1 copy-number and sequence variation by hybridization-based target enrichment coupled with massively parallel sequencing or next-generation sequencing (NGS). METHODS: NGS reads aligned to SMN1 and SMN2 exon 7 were quantified to determine the total combined copy number of SMN1 and SMN2. The ratio of SMN1 to SMN2 was calculated based on a single-nucleotide difference that distinguishes the two genes. SMN1 copy-number results were compared between the NGS and quantitative polymerase chain reaction and/or multiplex ligation-dependent probe amplification. The NGS data set was also queried for the g.27134T>G single-nucleotide polymorphism (SNP) and other SMN1 sequence pathogenic variants. RESULTS: The sensitivity of the test to detect spinal muscular atrophy (SMA) carriers with one copy of SMN1 was 100% (95% confidence interval (CI): 95.9-100%; n = 90) and specificity was 99.6% (95% CI: 99.4-99.7%; n = 6,648). Detection of the g.27134T>G SNP by NGS was 100% concordant with an restriction fragment-length polymorphism method (n = 493). Ten single-nucleotide variants in SMN1 were detectable by NGS and confirmed by gene-specific amplicon-based sequencing. This comprehensive approach yielded SMA carrier detection rates of 90.3-95.0% in five ethnic groups studied. CONCLUSION: We have developed a novel, comprehensive SMN1 copy-number and sequence variant analysis method by NGS that demonstrated improved SMA carrier detection rates across the entire population examined.Genet Med advance online publication 19 January 2017.
Subject(s)
Genetic Carrier Screening , High-Throughput Nucleotide Sequencing/methods , Muscular Atrophy, Spinal/genetics , Survival of Motor Neuron 1 Protein/genetics , Gene Dosage , Humans , Muscular Atrophy, Spinal/ethnology , Polymorphism, Single Nucleotide , Reproducibility of Results , Sensitivity and Specificity , Survival of Motor Neuron 2 Protein/geneticsABSTRACT
BACKGROUND: Primary immunodeficiency diseases (PIDDs) are inherited disorders of the immune system. The most severe form, severe combined immunodeficiency (SCID), presents with profound deficiencies of T cells, B cells, or both at birth. If not treated promptly, affected patients usually do not live beyond infancy because of infections. Genetic heterogeneity of SCID frequently delays the diagnosis; a specific diagnosis is crucial for life-saving treatment and optimal management. OBJECTIVE: We developed a next-generation sequencing (NGS)-based multigene-targeted panel for SCID and other severe PIDDs requiring rapid therapeutic actions in a clinical laboratory setting. METHODS: The target gene capture/NGS assay provides an average read depth of approximately 1000×. The deep coverage facilitates simultaneous detection of single nucleotide variants and exonic copy number variants in one comprehensive assessment. Exons with insufficient coverage (<20× read depth) or high sequence homology (pseudogenes) are complemented by amplicon-based sequencing with specific primers to ensure 100% coverage of all targeted regions. RESULTS: Analysis of 20 patient samples with low T-cell receptor excision circle numbers on newborn screening or a positive family history or clinical suspicion of SCID or other severe PIDD identified deleterious mutations in 14 of them. Identified pathogenic variants included both single nucleotide variants and exonic copy number variants, such as hemizygous nonsense, frameshift, and missense changes in IL2RG; compound heterozygous changes in ATM, RAG1, and CIITA; homozygous changes in DCLRE1C and IL7R; and a heterozygous nonsense mutation in CHD7. CONCLUSION: High-throughput deep sequencing analysis with complete clinical validation greatly increases the diagnostic yield of severe primary immunodeficiency. Establishing a molecular diagnosis enables early immune reconstitution through prompt therapeutic intervention and guides management for improved long-term quality of life.
Subject(s)
Sequence Analysis, DNA , Severe Combined Immunodeficiency/diagnosis , Severe Combined Immunodeficiency/genetics , Adolescent , Child , Female , Genetic Variation , Humans , Male , Pathology, Molecular/standards , Pathology, Molecular/trendsABSTRACT
PURPOSE: Next-generation sequencing (NGS) has been widely applied to clinical diagnosis. Target-gene capture followed by deep sequencing provides unbiased enrichment of the target sequences, which not only accurately detects single-nucleotide variations (SNVs) and small insertion/deletions (indels) but also provides the opportunity for the identification of exonic copy-number variants (CNVs) and large genomic rearrangements. METHOD: Capture NGS has the ability to easily detect SNVs and small indels. However, genomic changes involving exonic deletions/duplications and chromosomal rearrangements require more careful analysis of captured NGS data. Misaligned raw sequence reads may be more than just bad data. Some mutations that are difficult to detect are filtered by the preset analytical parameters. "Loose" filtering and alignment conditions were used for thorough analysis of the misaligned NGS reads. Additionally, using an in-house algorithm, NGS coverage depth was thoroughly analyzed to detect CNVs. RESULTS: Using real examples, this report underscores the importance of the accessibility to raw sequence data and manual review of suspicious sequence regions to avoid false-negative results in the clinical application of NGS. Assessment of the NGS raw data generated by the use of loose filtering parameters identified several sequence aberrations, including large indels and genomic rearrangements. Furthermore, NGS coverage depth analysis identified homozygous and heterozygous deletions involving single or multiple exons. CONCLUSION: Our results demonstrate the power of deep NGS in the simultaneous detection of point mutations and intragenic exonic deletion in one comprehensive step.Genet Med 18 5, 513-521.
Subject(s)
Genetic Diseases, Inborn/diagnosis , Genetic Diseases, Inborn/genetics , High-Throughput Nucleotide Sequencing/methods , INDEL Mutation/genetics , Algorithms , DNA Copy Number Variations/genetics , Exons , Genetic Diseases, Inborn/pathology , Heterozygote , High-Throughput Nucleotide Sequencing/trends , Homozygote , Humans , Polymorphism, Single Nucleotide/genetics , Sequence Deletion/geneticsABSTRACT
Mutations in the MUT gene, which encodes the mitochondrial enzyme methylmalonyl-CoA mutase, are responsible for the mut form of methylmalonic aciduria (MMA). In this study, a next generation sequencing (NGS) based gene panel was used to analyze 53 patients that had been diagnosed with mut MMA by somatic cell complementation analysis. A total of 54 different mutations in MUT were identified in 48 patients; 16 novel mutations were identified, including 1 initiation site mutation (c.2T>C [p.M1?]), 1 missense mutation (c.566A>T [p.N189I]), 2 nonsense mutations (c.129G>A [p.W43*] and c.1975C>T [p.Q659*]), 2 mutations affecting splice sites (c.753+3A>G and c.754-2A>G), 8 small insertions, deletions, and duplications (c.29dupT [p.L10Ffs*39], c.55dupG [p.V19Gfs*30], c.631_633delGAG [p.E211del], c.795_796insT [p.M266Yfs*7], c.1061delCinsGGA [p.S354Wfs*20], c.1065_1068dupATGG [p.S357Mfs*5], c.1181dupT [p.L394Ffs*30], c.1240delG [p.E414Kfs*17]), a large insertion (c.146_147ins279), and a large deletion involving exon 13. Phenotypic rescue and cDNA analysis were used to confirm that the c.146_147ins279 and c.631_633delGAG mutations were associated with the decreased methylmalonyl-CoA mutase function observed in the patient fibroblasts. In five patients, the NGS panel did not confirm the diagnosis made by complementation analysis. One of these patients was found to carry 2 novel mutations (c.433G > A [p.E145K] and c.511A>C [p.N171H]) in the SUCLG1 gene.
Subject(s)
Amino Acid Metabolism, Inborn Errors/diagnosis , Amino Acid Metabolism, Inborn Errors/genetics , High-Throughput Nucleotide Sequencing , Methylmalonyl-CoA Mutase/genetics , Adolescent , Adult , Amino Acid Metabolism, Inborn Errors/physiopathology , Cells, Cultured , Child , Child, Preschool , Codon, Nonsense/genetics , Exons/genetics , Female , Fibroblasts/metabolism , Humans , INDEL Mutation/genetics , Infant , Male , Methylmalonic Acid/metabolism , Mitochondria/enzymology , Mutation, Missense/genetics , Phenotype , Sequence Deletion/genetics , Succinate-CoA Ligases/genetics , Young AdultABSTRACT
Next generation sequencing (NGS) based gene panel testing is increasingly available as a molecular diagnostic approach for inborn errors of metabolism. Over the past 40 years patients have been referred to the Vitamin B12 Clinical Research Laboratory at McGill University for diagnosis of inborn errors of cobalamin metabolism by functional studies in cultured fibroblasts. DNA samples from patients in which no diagnosis was made by these studies were tested by a NGS gene panel to determine whether any molecular diagnoses could be made. 131 DNA samples from patients with elevated methylmalonic acid and no diagnosis following functional studies of cobalamin metabolism were analyzed using the 24 gene extended cobalamin metabolism NGS based panel developed by Baylor Miraca Genetics Laboratories. Gene panel testing identified two or more variants in a single gene in 16/131 patients. Eight patients had pathogenic findings, one had a finding of uncertain significance, and seven had benign findings. Of the patients with pathogenic findings, five had mutations in ACSF3, two in SUCLG1 and one in TCN2. Thus, the NGS gene panel allowed for the presumptive diagnosis of 8 additional patients for which a diagnosis was not made by the functional assays.
Subject(s)
Metabolism, Inborn Errors/diagnosis , Metabolism, Inborn Errors/genetics , Methylmalonic Acid/metabolism , Vitamin B 12 Deficiency/diagnosis , Vitamin B 12 Deficiency/genetics , Vitamin B 12/metabolism , Vitamin B Complex/metabolism , Amino Acid Metabolism, Inborn Errors/genetics , Child, Preschool , Coenzyme A Ligases/genetics , Female , Genetic Testing/methods , Genetic Variation , Genotype , Heterozygote , High-Throughput Nucleotide Sequencing/methods , Humans , Infant , Infant, Newborn , Male , Molecular Diagnostic Techniques , Mutation , Phenotype , Succinate-CoA Ligases/geneticsABSTRACT
PURPOSE: We aimed to demonstrate the detection of exonic deletions using target capture and deep sequencing data. METHODS: Sequence data from target gene capture followed by massively parallel sequencing were analyzed for the detection of exonic deletions using the normalized mean coverage of individual exons. We compared the results with those obtained from high-density exon-targeted array comparative genomic hybridization and applied similar analysis to examine samples from patients with pathogenic exonic deletions. RESULTS: Thirty-eight samples, each containing 2,134, 2,833, or 4,688 coding exons from different panels, with a total of 103,863 exons, were analyzed by capture-massively parallel sequencing and array comparative genomic hybridization. Ten deletions detected by array comparative genomic hybridization were all detected by massively parallel sequencing, whereas only two of three duplications were detected. We were able to detect all pathogenic exonic deletions in 11 positive cases. Thirty-one exonic copy number changes from nine perspective clinical samples were also identified. CONCLUSION: Our results demonstrated the feasibility of using the same set of sequence data to detect both point mutations and exonic deletions, thus improving the diagnostic power of massively parallel sequencing-based assays.
Subject(s)
Exons , Genetic Testing , High-Throughput Nucleotide Sequencing , Sequence Deletion , Comparative Genomic Hybridization , DNA Copy Number Variations , Exome , Female , Genes, Recessive , Genetic Diseases, Inborn/diagnosis , Genetic Diseases, Inborn/genetics , Genetic Testing/methods , Genotype , Humans , INDEL Mutation , Male , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Cell-free fetal DNA (cffDNA) screening is a valuable tool in clinical practice for detecting chromosomal abnormalities and autosomal dominant (AD) conditions. This study introduces a novel proof-of-concept assay designed for autosomal recessive (AR) cffDNA screening, focusing on cases involving the NPC1 gene. We aim to illustrate the significant benefits of AR cffDNA screening in managing high-risk pregnancies, specifically where biallelic pathogenic variants in NPC1 cause Niemann-Pick disease, type C1 (NPC), a disorder marked by progressive neurodegeneration. Three participants for this study were recruited and gave consent to a hospital in Saudi Arabia. These participants were either carriers of NPC or had a first- or second-degree relative affected by the disorder. No specific criteria were set for the age of the participants. All were between 15 and 18 weeks of gestation. Using amplicon-based next-generation sequencing (NGS), we analyzed the zygosity and variants in cffDNA extracted from maternal peripheral blood. After amplicon NGS, analysis was completed by a custom data analysis pipeline that included in-house-built data processing scripts and commonly used software packages. Importantly, the results were not disclosed to the patients. Our findings showed that in all three cases, AR cffDNA screening results were consistent with standard invasive diagnostic testing. This screening method offers several advantages: it provides critical information to families earlier in the pregnancy compared to invasive diagnostic tests, and it helps to alleviate parental anxiety. Moreover, this non-invasive method can determine pregnancy status in the first trimester for known familial variants. Future research may extend this approach to screen for known disease-causing variants in common AR conditions.
ABSTRACT
OBJECTIVE: To analyze the centrosome abnormalities in the malignant transformation of human bronchial epithelial cells (BEAS-2B) induced by coal tar pitch smoke extracts and to investigate the role and action mechanism of centrosome in the lung cancer induced by coal tar pitch. METHODS: Medium-temperature coal tar pitch smoke extracts were used to treat immortalized human bronchial epithelial cells (BEAS-2B) and establish a malignant transformation model. The treated BEAS-2B cells were used as exposure group, and solvent control group and normal control group were also set for passage culture. The changes of centrosome in BEAS-2B cells seeded on coverslips were evaluated by indirect immunofluorescence assay. The mRNA expression of p53, p21, and cyclin E in BEAS-2B cells was measured by real-time quantitative RT-PCR, and their protein levels in BEAS-2B cells seeded on coverslips were measured by semiquantitative immunohistochemical analysis. RESULTS: The overall rate of centrosome abnormalities in BEAS-2B cells at passage 20 was 6.56±1.01% in the exposure group, significantly higher than those in the normal control group (3.40±0.86%) and solvent control group (3.14±0.59%) (P < 0.05). In addition, the exposure group had a significantly higher overall rate of centrosome abnormalities in BEAS-2B cells at passage 30 compared with the normal control group and solvent control group (22.39±9.5% vs 4.34±1.04%, P < 0.05; 22.39±9.5% vs 4.33±1.20%, P < 0.05). Compared with the normal control group and solvent control group, the exposure group had significantly decreased mRNA and protein expression of p53 and significantly increased mRNA and protein expression of cyclin E in BEAS-2B cells at passages 20 and 30 (P < 0.05). CONCLUSION: Centrosome abnormalities occur before the malignant transformation in BEAS-2B cells treated with coal tar pitch smoke extracts, and they may be mediated by the p53/p21/cyclin E signaling pathway.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Centrosome/metabolism , Coal Tar , Epithelial Cells/metabolism , Cell Line , Cell Transformation, Neoplastic/pathology , Centrosome/pathology , Cyclin E/metabolism , Epithelial Cells/cytology , Humans , Signal Transduction , Smoke , Tumor Suppressor Protein p53/metabolismABSTRACT
Cuprous oxide (Cu2O) and cupric oxide (CuO) are widely available and low cost raw materials. Their applications as precursors for wet chemical synthesis of metallic Cu materials are greatly limited due to their insoluble in water and most organic solvents. In this work, copper superfine particles (Cu SPs) are synthesized using Cu2O and CuO as precursors via a heating process in monoethanoamine (MEA). Due to the strong coordinating character, Cu2O and CuO can be partially dissolved in MEA. The dissolved copper source is reduced by MEA at elevated temperature with the drastically releasing of NH3. As the dissolved copper source is reduced, more oxide will be dissolved and finally leads to the full reduction of Cu2O and CuO to produce the Cu SPs. The advantage of this synthesis method is that MEA acts as both the solvent and the reducing agent. The antimicrobial properties are investigated to find that the obtained Cu SPs depress the growth of Escherichia coli (E. coli) and Staphylococcus aureus (St. aureus) efficiently. More interesting, the composites produced via curing Cu2O and CuO with a small amount of MEA also exhibit excellent antimicrobial activity, indicating the MEA curing method is high-efficiency. The synthesis is low cost, high-efficiency, high atom-economy and up-scale synthesizing easily, which will benefit the wide applications of Cu SPs.
Subject(s)
Anti-Infective Agents , Copper , Anti-Infective Agents/pharmacology , Copper/pharmacology , Escherichia coli , Ethanolamine , OxidesABSTRACT
The newly described F-BAR (Fer/CIP4 and Bin, amphiphysin, Rvs) family of proteins includes Cdc42-interacting protein-4 (CIP4), formin-binding protein-17 (FBP-17) and transactivator of cytoskeletal assembly-1 (Toca-1), and drives membrane deformation and invagination. Membrane remodeling affects endocytosis, vesicle budding, and cargo selection. The F-BAR family presents a novel family of proteins, which little is known about their in vivo function. We investigated the physiological role of CIP4, by creating Cip4-null mice through homologous recombination. Compared with their wild-type littermates, the Cip4-null mice displayed lower early post-prandial glucose levels. Adipocytes isolated from Cip4-null mice exhibited increased [(14)C]2-deoxyglucose uptake compared with cells from wild-type mice. The enhanced insulin sensitivity was not due to higher levels of insulin or phospho-Akt, a critical player in insulin signaling. However, higher glucose transporter 4 (GLUT4) levels were detected in muscle membrane fractions in Cip4-null mice under insulin stimulation. Mouse embryonic fibroblasts from Cip4-null mice demonstrated decreased transferrin uptake, fluorescein isothiocyanate-dextran, and horseradish peroxidase uptake, indicating that CIP4 affects multiple modes of endocytosis. These studies demonstrate a physiological role for CIP4 in endocytosis leading to a whole animal phenotype.
Subject(s)
Endocytosis/physiology , Microtubule-Associated Proteins/metabolism , Adipocytes/metabolism , Animals , Biological Transport/genetics , Biological Transport/physiology , Blotting, Southern , Blotting, Western , Cattle , Cells, Cultured , Endocytosis/genetics , Female , Genotype , Glucose/metabolism , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , Male , Mice , Mice, Inbred ICR , Mice, Knockout , Microscopy, Fluorescence , Microtubule-Associated Proteins/genetics , Minor Histocompatibility Antigens , Reverse Transcriptase Polymerase Chain Reaction , Transferrin/metabolismABSTRACT
A novel strategy to systematically tune the pK(a) values of rhodamines is described. This strategy was applied to rationally develop compound 1e with a pK(a) of 6.5, the highest among rhodamine amide derivatives, and it could be employed to detect acidic pH variations in living cells with a turn-on signal.
Subject(s)
Fluorescent Dyes/chemistry , Rhodamines/chemistry , Cell Survival , Fluorescent Dyes/analysis , HeLa Cells , Humans , Hydrogen-Ion Concentration , Molecular Structure , Rhodamines/analysisABSTRACT
OBJECTIVE: To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer. METHODS: First, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM. RESULTS: It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (P<0.05). The apoptosis rate of A549/Lv-shCyPA (5.01% +/- 0.5%) was higher than control groups (0.35% +/- 0.17%) (P<0.05). Visible tumors were only detectable at 6th day after inoculated by A549/Lv-shCyPA. The xenograft tumors of A549/Lv-shCyPA remarkably delayed tumor growth and remained at a similarly small average size at 38th days after inoculation compared with the control group (P < 0.05). CONCLUSION: Lentiviral-vector-mediated siRNA technique effectively inhibits the expression of CyPA, induces the NSCLC cell apoptosis, inhibits the tumor growth. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for non-small cell lung cancer.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Cyclophilin A/genetics , Lung Neoplasms/genetics , Animals , Cell Line, Tumor , Gene Silencing , Genetic Vectors , Humans , Lentivirus/genetics , Mice , RNA, Small InterferingABSTRACT
Compound 1 was designed and synthesized as a new fluorescent thiol probe. Probe 1 was constructed on the basis of the conjugate 1,4-addition of thiols to alpha,beta-unsaturated ketones. Notably, probe 1 has suitable water solubility, which allows the sensing assay to be performed in water. Probe 1 is highly sensitive for thiols with a 211-fold fluorescence dynamic range and a low detection limit of 9.25x10(-7) M. The major features of probe 1 also include a high selectivity for thiols over other relevant biological species, excitation and emission in the visible region, rapid functioning at pH 7.4, and a good linear relationship between the fluorescence signal and the thiol concentration. Accordingly, these desirable characteristics may render probe 1 as potentially useful for biological applications.
Subject(s)
Fluorescent Dyes/chemical synthesis , Ketones/chemistry , Sulfhydryl Compounds/analysis , Sulfhydryl Compounds/chemistry , Fluorescent Dyes/chemistry , Hydrogen-Ion Concentration , Molecular Structure , Spectrometry, Fluorescence , Stereoisomerism , WaterABSTRACT
The authors have proposed and experimentally demonstrated all-optical logic gates using a single SOA and delay interference filtering that enable simultaneous logic functions of OR and NOR at 40 Gbits/s. The proposed scheme, which utilizes the combinative filtering profile of a delay interferometer and an optical bandpass filter, has great merits for use in generating logic outputs with high quality in terms of pulse shape, extinction ratio, and eye diagram. Reconfiguration between the two gates is achieved by adjusting the tunable filter and the delay interferometer.
ABSTRACT
BACKGROUND: Although mosaic variation has been known to cause disease for decades, high-throughput sequencing technologies with the analytical sensitivity to consistently detect variants at reduced allelic fractions have only recently emerged as routine clinical diagnostic tests. To date, few systematic analyses of mosaic variants detected by diagnostic exome sequencing for diverse clinical indications have been performed. METHODS: To investigate the frequency, type, allelic fraction, and phenotypic consequences of clinically relevant somatic mosaic single nucleotide variants (SNVs) and characteristics of the corresponding genes, we retrospectively queried reported mosaic variants from a cohort of ~ 12,000 samples submitted for clinical exome sequencing (ES) at Baylor Genetics. RESULTS: We found 120 mosaic variants involving 107 genes, including 80 mosaic SNVs in proband samples and 40 in parental/grandparental samples. Average mosaic alternate allele fraction (AAF) detected in autosomes and in X-linked disease genes in females was 18.2% compared with 34.8% in X-linked disease genes in males. Of these mosaic variants, 74 variants (61.7%) were classified as pathogenic or likely pathogenic and 46 (38.3%) as variants of uncertain significance. Mosaic variants occurred in disease genes associated with autosomal dominant (AD) or AD/autosomal recessive (AR) (67/120, 55.8%), X-linked (33/120, 27.5%), AD/somatic (10/120, 8.3%), and AR (8/120, 6.7%) inheritance. Of note, 1.7% (2/120) of variants were found in genes in which only somatic events have been described. Nine genes had recurrent mosaic events in unrelated individuals which accounted for 18.3% (22/120) of all detected mosaic variants in this study. The proband group was enriched for mosaicism affecting Ras signaling pathway genes. CONCLUSIONS: In sum, an estimated 1.5% of all molecular diagnoses made in this cohort could be attributed to a mosaic variant detected in the proband, while parental mosaicism was identified in 0.3% of families analyzed. As ES design favors breadth over depth of coverage, this estimate of the prevalence of mosaic variants likely represents an underestimate of the total number of clinically relevant mosaic variants in our cohort.