Array and distribution of actin filaments in guard cells contribute to the determination of stomatal aperture

Actin filaments in guard cells and their dynamics function in regulating stomatal movement. In this study, the array and distribution of actin filaments in guard cells during stomatal movement were studied with two vital labeling, microinjection of alexa-phalloidin in Vicia faba and expression of GFP-mTn in tobacco. We found that the random array of actin filaments in the most of the closed stomata changed to a ring-like array after stomatal open. And actin filaments, which were throughout the cytoplasm of guard cells of closed stomata (even distribution), were mainly found in the cortical cytoplasm in the case of open stomata (cortical distribution). These results revealed that the random array and even distribution of actin filaments in guard cells may be required for keeping the closed stomata; similarly, the ring-like array and cortical distribution of actin filaments function in sustaining open stomata. Furthermore, we found that actin depolymerization, the trait of moving stomata, facilitates the transformation of actin array and distribution with stomatal movement. So, the depolymerization of actin filaments was favorable for the changes of actin array and distribution in guard cells and thus facilitated stomatal movement.     

Inv21p12q22del21q22 and intellectual disability

Chromosomal rearrangements are common in humans. Pericentric inversions are among the most frequent aberrations (1–2%). Most inversions are balanced and do not cause problems in carriers unless one of the breakpoints disrupts important functional genes, has near submicroscopic copy number variants or hosts “cryptic” complex chromosomal rearrangements. Pericentric inversions can lead to imbalance in offspring. Less than 3% of Down syndrome patients have duplication as a result of parental pericentric inversion of chromosome 21. We report a family with an apparently balanced pericentric inversion of chromosome 21. The proband, a 23-year-old female was referred for prenatal diagnosis at 16 weeks gestation because of increased nuchal translucency. She has a familial history of Down's syndrome and moderate intellectual disability, a personal history of four spontaneous abortions and learning difficulties. Peripheral blood and amniotic fluid samples were collected to perform proband's and fetus' cytogenetic analyses. Additionally, another six family members were evaluated and cytogenetic analysis was performed. Complementary FISH and MLPA studies were carried out. An apparent balanced chromosome 21 pericentric inversion was observed in four family members, two revealed a recombinant chromosome 21 with partial trisomy, and one a full trisomy 21 with an inverted chromosome 21. Array CGH analysis was performed in the mother and the brother's proband. MLPA and aCGH studies identified a deletion of about 1.7 Mb on the long arm of inverted chromosome 21q22.11. We believe the cause of the intellectual disability/learning difficulties observed in the members with the inversion is related to this deletion. The recombinant chromosome 21 has a partial trisomy including the DSCR with no deletion. The risk for carriers of having a child with multiple malformations/intellectual disability is about 30% depending on whether and how this rearrangement interferes with meiosis.     

20 novel point mutations and one large deletion in EXT1 and EXT2 genes: Report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple exostosis

Background

Hereditary multiple exostosis represents the most frequent bone tumor disease in humans. It consists of cartilage deformities affecting the juxta-ephyseal region of long bones. Usually benign, exostosis could degenerate in malignant chondrosarcoma form in less than 5% of the cases. Being caused by mutations in the predicted tumor suppressor genes, EXT1 (chr 8q23-q24) and EXT2 (chr 11p11-p12) genes, HMEs are usually inherited with an autosomal dominant pattern, although “de novo” cases are not infrequent.

Aim

Here we present our genetic diagnostic report on the largest Southern Italy cohort of HME patients consisting of 90 subjects recruited over the last 5 years.

Results

Molecular screening performed by direct sequencing of both EXT1 and EXT2 genes, by MLPA and Array CGH analyses led to the identification of 66 mutations (56 different occurrences) and one large EXT2 deletion out of 90 patients (74.4%). The total of 21 mutations (20 different occurrences, 33.3%) and the EXT2 gene deletion were novel. In agreement with literature data, EXT1 gene mutations were scattered along all the protein sequence, while EXT2 lesions fell in the first part of the protein. Conservation, damaging prediction and 3-D modeling, in-silico, analyses, performed on three novel missense variants, confirmed that at least in two cases the novel aminoacidic changes could alter the structure stability causing a strong protein misfolding.

Conclusions

Here we present 20 novel EXT1/EXT2 mutations and one large EXT2 deletion identified in the largest Southern Italy cohort of patients affected by hereditary multiple exostosis.     

Amino Acid Residues 489–503 of Dihydropyridine Receptor (DHPR) β1a Subunit Are Critical for Structural Communication between the Skeletal Muscle DHPR Complex and Type 1 Ryanodine Receptor

The β_1a subunit is a cytoplasmic component of the dihydropyridine receptor (DHPR) complex that plays an essential role in skeletal muscle excitation-contraction (EC) coupling. Here we investigate the role of the C-terminal end of this auxiliary subunit in the functional and structural communication between the DHPR and the Ca²⁺ release channel (RyR1). Progressive truncation of the β_1a C terminus showed that deletion of amino acid residues Gln⁴⁸⁹ to Trp⁵⁰³ resulted in a loss of depolarization-induced Ca²⁺ release, a severe reduction of L-type Ca²⁺ currents, and a lack of tetrad formation as evaluated by freeze-fracture analysis. However, deletion of this domain did not affect expression/targeting or density (Q_max) of the DHPR-α_1S subunit to the plasma membrane. Within this motif, triple alanine substitution of residues Leu⁴⁹⁶, Leu⁵⁰⁰, and Trp⁵⁰³, which are thought to mediate direct β_1a-RyR1 interactions, weakened EC coupling but did not replicate the truncated phenotype. Therefore, these data demonstrate that an amino acid segment encompassing sequence ⁴⁸⁹QVQVLTSLRRNLSFW⁵⁰³ of β_1a contains critical determinant(s) for the physical link of DHPR and RyR1, further confirming a direct correspondence between DHPR positioning and DHPR/RyR functional interactions. In addition, our data strongly suggest that the motif Leu⁴⁹⁶-Leu⁵⁰⁰-Trp⁵⁰³ within the β_1a C-terminal tail plays a nonessential role in the bidirectional DHPR/RyR1 signaling that supports skeletal-type EC coupling.     

阵列生物传感器研究进展

简要介绍了阵列生物传感器的基本原理和分类。根据换能器的不同,评述了光学、电化学、质量型、磁致阻抗等阵列生物传感器的研究进展,并对目前阵列生物传感器研究中存在的问题进行了分析。     

Whole serum BSA antibody screening using a label-free biophotonic nanoparticle array

Bovine serum albumin antibodies (aBSA) have been screened from whole leporine anti serum on a biophotonic array. The array was initially printed with seed gold nanoparticles into a 96-spot configuration, and 130-nm gold nanoparticles were synthesised in situ on the surface of each spot. The gold nanoparticle surface was then functionalized with the proteins bovine serum albumin (BSA), fibrinogen, and immunoglobulin G (IgG) and with the amino acid glycine. The concentration of aBSA in the whole serum was determined using a kinetic analysis of the time-dependent light scattering from the nanoparticles. The aBSA-BSA kinetic parameters derived from the array are k_a = (1.3 ± 0.3) × 10⁵ M⁻¹ s⁻¹, k_d = (4 ± 2) × 10⁻⁴ s⁻¹, and K_D = 3 nM, which compare favorably with those from continuous gold surfaces. The ultimate sensitivity of the array reader to the bulk refractive index (RI) is 1 × 10⁻⁴ refractive index units (RIU), corresponding to 1 μg ml⁻¹ for aBSA. The nanoparticles appear to be more sensitive than the continuous gold surface to the aBSA binding event from whole serum, and this is interpreted in terms of the difference in RI contrast in the plasmon fields.     

Nonparametric testing for DNA copy number induced differential mRNA gene expression

Summary .  The central dogma of molecular biology relates DNA with mRNA. Array CGH measures DNA copy number and gene expression microarrays measure the amount of mRNA. Methods that integrate data from these two platforms may uncover meaningful biological relationships that further our understanding of cancer. We develop nonparametric tests for the detection of copy number induced differential gene expression. The tests incorporate the uncertainty of the calling of genomic aberrations. The test is preceded by a "tuning algorithm" that discards certain genes to improve the overall power of the false discovery rate selection procedure. Moreover, the test statistics are "shrunken" to borrow information across neighboring genes that share the same array CGH signature. For each gene we also estimate its effect, its amount of differential expression due to copy number changes, and calculate the coefficient of determination. The method is illustrated on breast cancer data, in which it confirms previously reported findings, now with a more profound statistical underpinning.     

A Latent Class Model with Hidden Markov Dependence for Array CGH Data

Summary Array CGH is a high‐throughput technique designed to detect genomic alterations linked to the development and progression of cancer. The technique yields fluorescence ratios that characterize DNA copy number change in tumor versus healthy cells. Classification of tumors based on aCGH profiles is of scientific interest but the analysis of these data is complicated by the large number of highly correlated measures. In this article, we develop a supervised Bayesian latent class approach for classification that relies on a hidden Markov model to account for the dependence in the intensity ratios. Supervision means that classification is guided by a clinical endpoint. Posterior inferences are made about class‐specific copy number gains and losses. We demonstrate our technique on a study of brain tumors, for which our approach is capable of identifying subsets of tumors with different genomic profiles, and differentiates classes by survival much better than unsupervised methods.     

Physicochemical features of ultra-high viscosity alginates

The physicochemical characteristics of the ultra-high viscosity and highly biocompatible alginates extracted from Lessonia nigrescens (UHV_N) and Lessonia trabeculata (UHV_T) were analyzed. Fluorescence and ¹H NMR spectroscopies, viscometry, and multi-angle light scattering (MALS) were used for elucidation of the chemical structure, molar mass, and coil size. The sequential structures from NMR spectroscopy showed high guluronate content for UHV_T, but low for UHV_N. Intrinsic viscosity [η] measurements exhibited unusual high values (up to 2750 mL/g), whereas [η] of a commercial alginate was only about 970 mL/g. MALS batch measurements of the UHV-alginates yielded ultra-high values of the weight average molar mass (M_w up to 1.1 × 10⁶ g/mol) and of the z-average gyration radius (〈R_G〉_z up to191 nm). The M_w and 〈R_G〉_z distributions of UHV-alginates and of ultrasonically degraded fractions were determined using size exclusion chromatography combined with MALS and asymmetrical flow-field-flow fractionation. The M_w dependency of [η] and 〈R_G〉_z could be described by [η] = 0.059 ×  and 〈R_G〉_z = 0.103 × . (UHV_N: x = 0.52; UHV_T: x = 0.53) indicating that the monomer composition has no effect on coil expansion. Therefore, the equations can be used to calculate M_w and 〈R_G〉_z values of UHV_T- and UHV_N-alginate mixtures as used in immunoisolation. Furthermore, the simple and inexpensive capillary viscometry can be used for real-time validation of the extraction and purification process of the UHV-alginates.