GRAde: a long-read sequencing approach to efficiently identifying the CYP11B1/CYP11B2 chimeric form in patients with glucocorticoid-remediable aldosteronism.

BACKGROUND: Glucocorticoid-remediable aldosteronism (GRA) is a form of heritable hypertension caused by a chimeric fusion resulting from unequal crossing over between 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), which are two genes with similar sequences. Different crossover patterns of the CYP11B1 and CYP11B2 chimeric genes may be associated with a variety of clinical presentations. It is therefore necessary to develop an efficient approach for identifying the differences between the hybrid genes of a patient with GRA. RESULTS: We developed a long-read analysis pipeline named GRAde (GRA deciphering), which utilizes the nonidentical bases in the CYP11B1 and CYP11B2 genomic sequences to identify and visualize the chimeric form. We sequenced the polymerase chain reaction (PCR) products of the CYP11B1/CYP11B2 chimeric gene from 36 patients with GRA using the Nanopore MinION device and analyzed the sequences using GRAde. Crossover events were identified for 30 out of the 36 samples. The crossover sites appeared in the region exhibiting high sequence similarity between CYP11B1 and CYP11B2, and 53.3% of the cases were identified as having a gene conversion in intron 2. More importantly, there were six cases for whom the PCR products indicated a chimeric gene, but the GRAde results revealed no crossover pattern. The crossover regions were further verified by Sanger sequencing analysis. CONCLUSIONS: PCR-based target enrichment followed by long-read sequencing is an efficient and precise approach to dissecting complex genomic regions, such as those involved in GRA mutations, which could be directly applied to clinical diagnosis. The scripts of GRAde are available at https://github.com/hsu-binfo/GRAde .

Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy.

Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.

Water-induced relaxation of a degenerate vibration of guanidinium using 2D IR echo spectroscopy.

The nearly degenerate asymmetric stretch vibrations near 1600 cm(-1) of the guanidinium cation in D-glycerol/D(2)O mixtures having different viscosity were studied by 2D IR photon echo spectroscopy. The polarization-dependent photon echo signal shows two separate frequency distributions in the 2D spectrum in D(2)O, even though only one band is evident from inspection of the linear FTIR spectrum. The split components are more clearly seen at higher viscosity where the distortion of the molecule from 3-fold symmetry is even more evident. The interactions with solvent induce energy transfer between the degenerate component modes on the time scale of 0.5 ps. The energy transfer between modes is directly observed in 2D IR and distinguished by the waiting time dependence of the cross peaks from the transfers between configurations of the distorted ion and solvent. The 2D IR analysis carried out for various polarization conditions gave frequency-frequency auto- and cross-correlation functions for the degenerate components which derive from the solvent induced wagging of the -ND(2) groups of the guanidinium ion.

Ultrafast vibrational spectroscopy of a degenerate mode of guanidinium chloride.

Nearly degenerate asymmetric stretches with perpendicular transition dipole moments of the deuterated guanidinium cation (DGdm(+)) in D(2)O and D-glycerol/D(2)O mixtures at 1600 cm(-1) were investigated by linear FTIR spectroscopy and polarization dependent femtosecond pump-probe spectroscopy. The vibrational coupling of the asymmetric stretches of guanidinium occurs within 0.5 ps and leads to fast decay of the anisotropy to a level of 0.1. A systematic study of the influence of the coherence transfer on pump-probe signals is given. Following this decay, the anisotropy decays with a time constant of 4.1 ps in D(2)O by rotational diffusion about an axis perpendicular to the DGdm(+) mean plane. The presence of aggregation was demonstrated for concentrations higher than 0.2 M.

Characterization of microstructures fabricated by two-photon polymerization using coherent anti-stokes Raman scattering microscopy.

We demonstrate the possibility to image microstructures fabricated by two-photon polymerization (TPP) using coherent anti-Stokes Raman scattering (CARS) microscopy. The imaging contrast based on chemical selectivity attained by CARS microscopy is used to gather qualitative information on TPP. Upon the basis of detailed knowledge of the characteristic signatures of the photoresist Raman spectrum, quantitative relationships between laser writing conditions and polymer cross-linking are demonstrated. The increase in degree of polymer conversion as a function of laser average power follows a sigmoidal profile which is interpreted in terms of a simple model based on the polymerization mechanism of the photoresist.

Association of novel chymotrypsin C gene variations and haplotypes in patients with chronic pancreatitis in Chinese in Taiwan.

BACKGROUND/AIMS: Variations and haplotypes of the chymotrypsin C (CTRC) gene in Chinese patients with chronic pancreatitis (CP) and control subjects with genotype-phenotype correlation were investigated. METHODS: One hundred and twenty-six patients with CP were analyzed. The entire sequence of coding regions of exons 2, 3 and 7 and their neighboring intronic regions in introns 1, 2 and 6 of the CTRC gene were analyzed using PCR sequence-specific primers and direct sequencing. The exonic region of exon 7 and the neighboring intronic region of intron 6 were also analyzed in 90 geographically matched healthy control subjects. RESULTS: In total, 4 novel variations were identified in exons 2, 3 and 7 in 3 CP patients. A total of 2.3% (3/126) of our CP patients carried variations of the CTRC gene. We also first identified six new intronic variations in intron 6 which had not been reported before. The GAGGGG, GAGGAG and GAGTAG haplotypes assembled by six locus intronic variations c.640-41/c.640-40/c.640-39/c.640-37/c.640-36/c.640-35 in intron 6 were associated with a significantly higher susceptibility risk of CP (OR 66.75, 37.00, and 9.37, respectively). CONCLUSION: Novel CTRC gene variations and haplotypes are associated with CP in a Chinese population.

Autoimmune pancreatitis associated with high prevalence of gastric ulcer independent of Helicobacter pylori infection status.

OBJECTIVES: The relationship between Helicobacter pylori status and host tumor necrosis factor alpha (TNF-alpha) promoter susceptibility in ulcers inautoimmune pancreatitis (AIP) is unknown. We sought to study the frequency of peptic ulcer, the association of peptic ulcer with H. pylori and host TNF-alpha promoter haplotype in AIP and nonautoimmune chronic pancreatitis. METHODS: Esophagogastroduodenoscopy (EGD) was performed in 40 patients with AIP and 113 patients with nonautoimmune chronic pancreatitis (CP). The status of H. pylori infection was determined. Genotyping and 5-locus haplotype assembly of the TNF-alpha promoter were performed. The correlation between clinical characteristics, endoscopic findings, Helicobacter pylori infection status, and TNF-alpha promoter polymorphism and haplotype was analyzed. RESULTS: The frequencies of gastric ulcer (GU) was higher in patients with AIP compared with patients with nonautoimmune CP (22.5% vs 4.4%, P = 0.001). Duodenal ulcer (DU) was more prevalent than GU in both patients with AIP and patients with nonautoimmune CP. There was no difference in the positive status of H. pylori and TNF-alpha promoter polymorphism/haplotype. CONCLUSIONS: Our results demonstrated that GU was more prevalent in AIP compared with nonautoimmune CP. Positive H. pylori status and host TNF-alpha promoter susceptibility could not explain the pathogenesis of higher GU prevalence and pathogenesis of AIP in our population.

Lipoprotein lipase mutation S447X associated with pancreatic calcification and steatorrhea in hyperlipidemic pancreatitis.

BACKGROUND: The factors that whether and how genes involving lipid metabolism including lipoprotein lipase (LPL) and apolipoprotein CII (apo CII) influence occurrence of acute attack of pancreatitis and chronic pancreatitis is not clear. GOALS: The aim of this study was to determine the association of LPL and apo CII genes with acute attack of pancreatitis and chronic pancreatitis in patients with hyperlipidemic pancreatitis (HLP) and hypertriglyceridemia (HTG). STUDY: We performed genetic analysis of 134 patients in Taiwan with HTG (53 with HLP and 81 without HLP). The entire coding and intronic regions of the LPL and apo CII genes were identified with heteroduplex analytical techniques or high resolution melting analysis. All mutations were confirmed by sequencing analysis. Correlation of phenotype and genotype was also analyzed. RESULTS: The frequency of LPL gene mutation rates in HLP patients (17.0%, 9 of 53) was significantly higher than that without HLP attack (4.9%, 4 of 81) (P<0.0001). A total of 10.4% (14 of 134) of our HTG patients carried LPL or apo CII mutation. The most common LPL gene mutation was S447X. There is a high prevalence (77.8%) of HLP attack in HTG patients carrying S447X mutation. Multivariate analysis in HLP patients indicated that the presence of LPL mutation and episode of acute attack were independent risks for pancreatic calcification and steatorrhea. CONCLUSIONS: This is the first complete genetic study analyzing the association of LPL and apo CII mutation in a HLP population. LPL S447X mutation is associated with a higher risk of pancreatic calcification and steatorrhea than those previously known factors in HLP patients.

Association of cystic fibrosis transmembrane conductance regulator (CFTR) mutation/variant/haplotype and tumor necrosis factor (TNF) promoter polymorphism in hyperlipidemic pancreatitis.

BACKGROUND: The mechanism by which hypertriglyceridemia (HTG) leads to pancreatitis is not clear. We sought to determine whether the genes involved in pancreatic ductal or acinar cell injury, including the cationic trypsinogen gene [protease, serine, 1 (trypsin 1) (PRSS1)], the pancreatic secretory trypsin inhibitor gene [serine peptidase inhibitor, Kazal type 1 (SPINK1)], the cystic fibrosis transmembrane conductance regulator gene [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette subfamily C, member 7) (CFTR)], and inflammation genes such as tumor necrosis factor [tumor necrosis factor, TNF superfamily, member 2 (TNF)] are associated with hyperlipidemic pancreatitis (HLP) in patients with HTG. METHODS: We performed genetic analysis of 126 HTG patients in Taiwan (46 with HLP and 80 without HLP). The entire coding and intronic regions of the PRSS1, SPINK1, and CFTR genes were identified by heteroduplex analysis techniques and were confirmed by sequencing analysis. The presence of 125G/C, 1001 + 11C>T, 1540A>G (Met470Val), 2694T>G, and 4521G>A in CFTR, the presence of 272C>T in SPINK1, and TNF promoter polymorphisms (nucleotide positions 1031, 863, 857, 308, and 308) were measured by direct sequencing. RESULTS: Of the 126 HTG patients, 13 (10.3%) carried a CFTR mutation. No PRSS1 or SPINK1 mutations were detected in our patients or in HTG controls. The CFTR gene mutation rates in HTG with and without HLP were 26.1% (12 of 46) and 1.3% (1 of 80), respectively (P <0.0001). The CFTR gene mutations were all Ile556Val. A multivariate analysis of HTG patients indicated that triglycerides, CFTR 470Val, and TNF promoter 863A were independent risk markers for HLP. CONCLUSIONS: This genetic study is the first one to address the association of HLP with the CFTR mutation/variant/haplotype and TNF promoter polymorphism in a Chinese HTG population. The results suggest that the occurrence of HLP is multifactorial and polygenic.

Correlation of the vibrations of the aqueous azide ion with the O-H modes of bound water molecules.

Dual frequency two-dimensional infrared spectroscopy (2D-IR) has been used to investigate the dynamics of the azide-water solvation shell. The memory of the azide transition frequencies is detected in the echo emitted by the OH stretching mode of the ion-bound water molecules. There is a significant positive correlation of the two frequency distributions that decays on a 140 fs time scale. The result confirms that the O-H bond of water molecules in the solvent shell have frequency fluctuations that are considerably slowed from those that are known in bulk water. The positive correlation is attributed to cooperative interactions of coordinated water molecules with an azide ion.