Integrating imaging and genomic data for the discovery of distinct glioblastoma subtypes: a joint learning approach.

Glioblastoma is a highly heterogeneous disease, with variations observed at both phenotypical and molecular levels. Personalized therapies would be facilitated by non-invasive in vivo approaches for characterizing this heterogeneity. In this study, we developed unsupervised joint machine learning between radiomic and genomic data, thereby identifying distinct glioblastoma subtypes. A retrospective cohort of 571 IDH-wildtype glioblastoma patients were included in the study, and pre-operative multi-parametric MRI scans and targeted next-generation sequencing (NGS) data were collected. L21-norm minimization was used to select a subset of 12 radiomic features from the MRI scans, and 13 key driver genes from the five main signal pathways most affected in glioblastoma were selected from the genomic data. Subtypes were identified using a joint learning approach called Anchor-based Partial Multi-modal Clustering on both radiomic and genomic modalities. Kaplan-Meier analysis identified three distinct glioblastoma subtypes: high-risk, medium-risk, and low-risk, based on overall survival outcome (p < 0.05, log-rank test; Hazard Ratio = 1.64, 95% CI 1.17-2.31, Cox proportional hazard model on high-risk and low-risk subtypes). The three subtypes displayed different phenotypical and molecular characteristics in terms of imaging histogram, co-occurrence of genes, and correlation between the two modalities. Our findings demonstrate the synergistic value of integrated radiomic signatures and molecular characteristics for glioblastoma subtyping. Joint learning on both modalities can aid in better understanding the molecular basis of phenotypical signatures of glioblastoma, and provide insights into the biological underpinnings of tumor formation and progression.

Clinical measures, radiomics, and genomics offer synergistic value in AI-based prediction of overall survival in patients with glioblastoma.

Multi-omic data, i.e., clinical measures, radiomic, and genetic data, capture multi-faceted tumor characteristics, contributing to a comprehensive patient risk assessment. Here, we investigate the additive value and independent reproducibility of integrated diagnostics in prediction of overall survival (OS) in isocitrate dehydrogenase (IDH)-wildtype GBM patients, by combining conventional and deep learning methods. Conventional radiomics and deep learning features were extracted from pre-operative multi-parametric MRI of 516 GBM patients. Support vector machine (SVM) classifiers were trained on the radiomic features in the discovery cohort (n = 404) to categorize patient groups of high-risk (OS < 6 months) vs all, and low-risk (OS ≥ 18 months) vs all. The trained radiomic model was independently tested in the replication cohort (n = 112) and a patient-wise survival prediction index was produced. Multivariate Cox-PH models were generated for the replication cohort, first based on clinical measures solely, and then by layering on radiomics and molecular information. Evaluation of the high-risk and low-risk classifiers in the discovery/replication cohorts revealed area under the ROC curves (AUCs) of 0.78 (95% CI 0.70-0.85)/0.75 (95% CI 0.64-0.79) and 0.75 (95% CI 0.65-0.84)/0.63 (95% CI 0.52-0.71), respectively. Cox-PH modeling showed a concordance index of 0.65 (95% CI 0.6-0.7) for clinical data improving to 0.75 (95% CI 0.72-0.79) for the combination of all omics. This study signifies the value of integrated diagnostics for improved prediction of OS in GBM.

Effects of systemic inflammation on relapse in early breast cancer.

Chronic inflammation has been a proposed mechanism of resistance to aromatase inhibitors in breast cancer. Stratifying by HER2 status, a matched case-control study from the Wellness After Breast Cancer-II cohort was performed to assess whether or not elevated serum inflammatory biomarkers (C-Reactive protein [CRP], interleukin-6 [IL-6], and serum amyloid A [SAA]) and/or the presence of a high-risk IL-6 promoter genotype were associated with recurrence of hormone receptor positive (HR+) early breast cancer. Estrogen levels were also measured and correlated with biomarkers and disease outcomes. CRP and SAA were significantly associated with an increased risk of recurrence in the HR+/HER2- group, but not the HR+/HER2+ group. Mean serum estrogen levels were non-significantly elevated in patients who relapsed vs. non-relapsed patients. Surprisingly, high-risk IL-6 promoter polymorphisms were strongly associated with HER2+ breast cancer relapse, which has potential therapeutic implications, as elevated intracellular IL-6 has been associated with trastuzumab resistance in pre-clinical models.

Contrasting spectroscopic response of human hemoglobin in presence of graphene oxides and its reduced form: Comparative approach with carbon quantum dots.

Recently, a considerable amount of research is being directed towards study of graphene oxide (GO) and its reduced form (RGO) since their exposed functional groups make them better candidates in nanobiotechnolgy. In order to assess their biocompatibility, the nature of interactions between Human Hemoglobin (HHb) and GO/RGO are monitored since a comparative spectroscopic approach towards understanding their nature of interactions has not been investigated previously. UV-vis spectroscopy reveals hyperchromicity for HHb-GO system and hypochromicity for HHb-RGO system in the region of absorption of tryptophan/tyrosine residues. Notably, although steady-state fluorescence static quenching of HHb for GO and enhancement of fluorescence for RGO is noticed, but average fluorescence-lifetime is remaining unchanged in presence of GO/RGO. Calorimetric data illustrates three-site and five-site binding model to be the best-fit model for GO and RGO respectively. Also, synchronous fluorescence quenching corresponding to alterations in microenvironment of tryptophan/ tyrosine residues is observed only in presence of GO. Likewise FTIR spectroscopy elucidates involvement of both amide I and amide II bond of HHb backbone through H-bonding interaction only for GO. Furthermore RLS spectra demonstrate an increase and a decrease in signal for GO and RGO respectively. Surprisingly, secondary structure of HHb is maintained upon interaction with both GO/RGO, as revealed by CD spectroscopy, thus supporting their potential application in biological microenvironment. Thus it appears that the spectroscopic properties of HHb upon interaction with GO is altered upon its reduction to RGO. Furthermore the role of HHb as good candidate for bimolecular interaction has been highlighted.

Fluorescence enhancement via aggregation effect due to microenvironmental alterations in human hemoglobin protein in presence of carbon quantum dots (CQD): Comparative spectroscopic approach.

CQDs have emerged with outstanding properties as a star member of carbon nanomaterial family and in order to reveal its wide-range of application in biological microenvironment the interactions between human hemoglobin (HHb) and CQD and also with ethylenediamine-functionalized CQD (NCQD) are assessed using several techniques. Firstly, UV-vis absorption spectra of HHb reveal hyperchromic effect in the region of absorbance of tryptophan and tyrosine residues and also hypochromicity of Soret band in presence of CQD and NCQD. Interestingly, steady-state fluorescence spectroscopy reveal distinct fluorescence enhancement of HHb with significant red shift thereby indicating exposures of tryptophan and tyrosine residues to a more hydrophilic environment. However synchronous fluorescence spectra reveal that the microenvironment of tryptophan and tyrosine residues is altered in opposite manner, i.e. exposure of tryptophan residues to a more hydrophilic environment and the tyrosine residues to a more hydrophobic environment. Moreover the fluorescence enhancement is observed to be accompanied by increase in average fluorescence-lifetime and decrease in steady-state anisotropy thus signifying a decrease in restriction of rotational motion. Furthermore tryptophan residues within HHb appear to interact more with CQD compared to NCQD. Thermodynamic parameters as revealed by Isothermal Titration Calorimetry (ITC) demonstrate that electrostatic, hydrogen bonding and hydrophobic interactions are the predominant modes of interactions in presence of CQD. Whereas hydrophobic and hydrogen bonding interactions are the major interacting forces in presence of NCQD with five-site sequential binding as best-fit model in both the cases. Such interactions also appear to be associated with an increase in aggregation of HHb as evident from the measurements by atomic force microscopy (AFM) and dynamic light scattering (DLS) study. Although FT-IR spectra display alteration of amide I band, but the overall secondary structure of HHb seems to be nearly retained even in presence of CQDs, as evident in the CD spectra. These observations thus highlight the potential biomedical application of CQDs in biological microenvironment of human especially as drug-delivery system. Also bimolecular interaction of HHb as a model protein with other nanoparticles at the nano bio-interface has been outlined.

Molecular diagnosis of somatic overgrowth conditions: A single-center experience.

BACKGROUND: Somatic overgrowth conditions, including Proteus syndrome, Sturge-Weber syndrome, and PIK3CA-related overgrowth spectrum, are caused by post-zygotic pathogenic variants, result in segmental mosaicism, and give rise to neural, cutaneous and/or lipomatous overgrowth. These variants occur in growth-promoting pathways leading to cellular proliferation and expansion of tissues that arise from the affected cellular lineage. METHODS: We report on 80 serial patients evaluated for somatic overgrowth conditions in a diagnostic laboratory setting, including three prenatal patients. In total, 166 tissues from these 80 patients were subjected to targeted sequencing of an 8-gene panel capturing 10.2 kb of sequence containing known pathogenic variants associated with somatic overgrowth conditions. Deep next-generation sequencing was performed with the IonTorrent PGM platform at an average depth typically >5,000×. RESULTS: Likely pathogenic or pathogenic variants were identified in 36 individuals and variants of unknown significance in four. The overall molecular diagnostic yield was 45% but was highly influenced by both submitted tissue type and phenotype. In the prenatal setting, two patients had pathogenic variants identified in cultured amniocytes but in a third patient, the pathogenic variant was only present in post-natal tissues. Finally, expanding the test to include full gene sequencing of PIK3CA in contrast to targeted sequencing identified likely pathogenic variants in 3 of 7 patients that tested negative on the original panel. CONCLUSION: Next-generation sequencing has enabled sensitive detection of somatic pathogenic variants associated with overgrowth conditions. However, as the pathogenic variant allele frequency varies by tissue type within an individual, submission of affected tissue(s) greatly increases the chances of a molecular diagnosis.

Interactions of Fluorescein Dye with Spherical and Star Shaped Gold Nanoparticles.

UV-vis absorption, FT-IR, steady state fluorescence and fluorescence lifetime measurements were made on Fluorescein dye (Fl dye) molecules in presence of gold nanoparticles of different morphologies: spherical gold nanoparticles (GNP) and star shaped gold nanoparticles (GNS). The experimental observations demonstrate that Fl dye molecules form dimers when adsorbed on nanosurface of spherical gold particles. On the other hand possibly due to lack of adsorption on the surface of GNS the dye molecules were unable to form dimers. The projected tips on the surface of GNS may possibly hinder the dyes to adsorb on the surface of this nanoparticle. From the spectral analysis and measurements of thermodynamic parameters it is inferred that two different types of ground state interactions occur between Fl-dye-GNP and Fl dye-GNS systems. Both the observed negative values of the thermodynamic parameters ΔH and ΔS in the case of the former system predict the possibility of occurrences of hydrogen bonding interactions between two neighboring Fl dye molecules when adsorbed on the nanosurface of GNP. On the other hand in Fl dye-GNS system electrostatic interactions appear to occur, as evidenced from negative ΔH and positive value of ΔS, between the positive charges residing on the tips of the nanoparticles and anionic form of Fl dye. It has been concluded that as the adsorption of organic dyes on solid surfaces is prerequisite for the degradation of dye pollutants, the present experimental observations demonstrate that GNP could be used as a better candidate than GNS in degradation mechanism of the xanthenes dyes.

To reveal the nature of interactions of human hemoglobin with gold nanoparticles having two different morphologies (sphere and star-shaped) by using various spectroscopic techniques.

The nature of interactions between heme protein human hemoglobin (HHb) and gold nanoparticles of two different morphologies that is GNP (spherical) and GNS (star-shaped) have been investigated by using UV-vis absorption, steady state fluorescence, synchronous fluorescence, resonance light scattering (RLS), time resolved fluorescence, FT-IR, and circular dichroism (CD) techniques under physiological condition of pH ~7 at ambient and different temperatures. Analysis of the steady state fluorescence quenching of HHb in aqueous solution in the presence of GNP and GNS suggests that the nature of the quenching is of static type. The static nature of the quenching is also confirmed from time resolved data. The static type of quenching also indicates the possibility of formation of ground state complex for both HHb-GNP and HHb-GNS systems. From the measurements of Stern-Volmer (SV) constants KSV and binding constants, KA and number of binding sites it appears that HHb forms stronger binding with GNP relative to GNS. Analysis of the thermodynamic parameters indicates that the formation of HHb-GNP and HHb-GNS complexes are spontaneous molecular interaction processes (∆G<0). In both cases hydrogen bonding and van der Waals interactions play a dominant role (∆H<0, ∆S<0). Synchronous fluorescence spectroscopy further reveals that the ground state complex formations of HHb-GNP and HHb-GNS preferably occur by binding with the amino acid tyrosine through hydrogen bonding interactions. Moreover the α-helicity contents of the proteins as obtained from the circular dichroism (CD) spectra appears to be marginally reduced by increasing concentrations of GNP and GNS and the α-helical structures of HHb retain its identity as native secondary structure in spite of complex formations with GNP or GNS. These findings demonstrate the efficiency of biomedical applications of GNP and GNS nanoparticles as well as in elucidating their mechanisms of action as drugs or drug delivery systems in human.

A comparison of Illumina and Ion Torrent sequencing platforms in the context of differential gene expression.

BACKGROUND: Though Illumina has largely dominated the RNA-Seq field, the simultaneous availability of Ion Torrent has left scientists wondering which platform is most effective for differential gene expression (DGE) analysis. Previous investigations of this question have typically used reference samples derived from cell lines and brain tissue, and do not involve biological variability. While these comparisons might inform studies of tissue-specific expression, marked by large-scale transcriptional differences, this is not the common use case. RESULTS: Here we employ a standard treatment/control experimental design, which enables us to evaluate these platforms in the context of the expression differences common in differential gene expression experiments. Specifically, we assessed the hepatic inflammatory response of mice by assaying liver RNA from control and IL-1ß treated animals with both the Illumina HiSeq and the Ion Torrent Proton sequencing platforms. We found the greatest difference between the platforms at the level of read alignment, a moderate level of concordance at the level of DGE analysis, and nearly identical results at the level of differentially affected pathways. Interestingly, we also observed a strong interaction between sequencing platform and choice of aligner. By aligning both real and simulated Illumina and Ion Torrent data with the twelve most commonly-cited aligners in the literature, we observed that different aligner and platform combinations were better suited to probing different genomic features; for example, disentangling the source of expression in gene-pseudogene pairs. CONCLUSIONS: Taken together, our results indicate that while Illumina and Ion Torrent have similar capacities to detect changes in biology from a treatment/control experiment, these platforms may be tailored to interrogate different transcriptional phenomena through careful selection of alignment software.

Designing of an artificial light energy converter in the form of short-chain dyad when combined with core-shell gold/silver nanocomposites.

UV-vis absorption, steady state and time resolved fluorescence and absorption spectroscopic investigations demonstrate that the short chain dyad MNTMA when combined with gold-silver core-shell (Au@Ag) nanocomposite , forms elongated conformers in the excited state whereas for the dyad - Ag (spherical) system the majority of dyads remains in a folded conformation. In the dyad-core-shell nanocomposite system, energy wasting charge recombination rate slows down primarily due to elongated conformation and thus it may be anticipated that this hybrid nanocomposite system may serve as a better light energy conversion device.

Use of Spectroscopic Techniques to Reveal the Nature of the Interactions of Two Sialic Acid Specific Lectins with Gold Nanoparticles.

From UV-vis absorption, steady state and time resolved fluorescence measurements coupled with circular dichroism (CD) spectral studies, it was revealed that among the two lectins: Sambucus nigra agglutinin (SNA) and Saraca indica (saracin II), SNA forms stronger binding complex in the ground state with gold nanoparticles (GNPs). From the measurements of Stern-Volmer (SV) constants Ksv, and binding constants K(A) and number of binding sites two important inferences could be drawn. Firstly, the fluorescence quenching is primarily due to static quenching and secondly SNA forms stronger binding with GNPs relative to the other lectin saracin II. Synchronous fluorescence spectral measurements further substantiate this proposition of exhibiting the fully exposed tryptophan residue in case of SNA. It appears that the lectin SNA adopted a relatively looser conformation with the extended polypeptide structures leading to the exposure of the hydrophobic cavities which favoured stronger binding with GNPs. CD measurements demonstrate that gold nanoparticles when interact with the lectins (glycoproteins), no significant distortion in the structural pattern of the later occurs. The unaltered identity in the secondary structural pattern of both SNA and saracin II in presence of gold nanoparticles hints that GNPs may be used as useful drug or drug delivery systems.

Time Resolved Spectroscopic Studies on a Novel Synthesized Photo-Switchable Organic Dyad and Its Nanocomposite Form in Order to Develop Light Energy Conversion Devices.

UV-vis absorption, steady state and time resolved spectroscopic investigations in pico and nanosecond time domain were made in the different environments on a novel synthesized dyad, 3-(2-methoxynaphthalen-1-yl)-1-(4-methoxyphenyl)prop-2-en-1-one (MNTMA) in its pristine form and when combined with gold (Au) nanoparticles i.e., in its nanocomposite structure. Both steady state and time resolved measurements coupled with the DFT calculations performed by using Gaussian 03 suit of software operated in the linux operating system show that though the dyad exhibits mainly the folded conformation in the ground state but on photoexcitation the nanocomposite form of dyad prefers to be in elongated structure in the excited state indicating its photoswitchable nature. Due to the predominancy of elongated isomeric form of the dyad in the excited state in presence of Au Nps, it appears that the dyad MNTMA may behave as a good light energy converter specially in its nanocomposite form. As larger charge separation rate (kcs ~ 4 x 10(8) s-1) is found relative to the rate associated with the energy wasting charge recombination processes (kcR ~ 3 x 10(5) s-1) in the nanocomposite form of the dyad, it demonstrates the suitability of constructing the efficient light energy conversion devices with Au-dyad hybrid nanomaterials.

Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells & Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA.

Diagnostic and prognostic indicators are key components to achieve the goal of personalized cancer therapy. Two distinct approaches to this goal include predicting response by genetic analysis and direct testing of possible therapies using cultures derived from biopsy specimens. Optimally, the latter method requires a rapid assessment, but growing xenograft tumors or developing patient-derived cell lines can involve a great deal of time and expense. Furthermore, tumor cells have much different responses when grown in 2D versus 3D tissue environments. Using a modification of existing methods, we show that it is possible to make tumor-fragment (TF) spheroids in only 2-3 days. TF spheroids appear to closely model characteristics of the original tumor and may be used to assess critical therapy-modulating features of the microenvironment such as hypoxia. A similar method allows the reproducible development of spheroids from mixed tumor cells and fibroblasts (mixed-cell spheroids). Prior literature reports have shown highly variable development and properties of mixed-cell spheroids and this has hampered the detailed study of how individual tumor-cell components interact. In this study, we illustrate this approach and describe similarities and differences using two tumor models (U87 glioma and SQ20B squamous-cell carcinoma) with supporting data from additional cell lines. We show that U87 and SQ20B spheroids predict a key microenvironmental factor in tumors (hypoxia) and that SQ20B cells and spheroids generate similar numbers of microvesicles. We also present pilot data for miRNA expression under conditions of cells, tumors, and TF spheroids.

Evaluation of Mutational Testing of Preneoplastic Barrett's Mucosa by Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Endoscopic Samples for Detection of Concurrent Dysplasia and Adenocarcinoma in Barrett's Esophagus.

Barrett's intestinal metaplasia (BIM) may harbor genomic mutations before the histologic appearance of dysplasia and cancer and requires frequent surveillance. We explored next-generation sequencing to detect mutations with the analytical sensitivity required to predict concurrent high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus by testing nonneoplastic BIM. Formalin-fixed, paraffin-embedded (FFPE) routine biopsy or endoscopic mucosal resection samples from 32 patients were tested: nonprogressors to HGD or EAC (BIM-NP) with BIM, who never had a diagnosis of dysplasia or EAC (N = 13); progressors to HGD or EAC (BIM-P) with BIM and a worse diagnosis of HGD or EAC (N = 15); and four BIM-negative samples. No mutations were detected in the BIM-NP (0 of 13) or BIM-negative samples, whereas the BIM-P samples had mutations in 6 (75%) of 8 cases in TP53, APC, and CDKN2A (P = 0.0005), detected in samples with as low as 20% BIM. We found that next-generation sequencing from routine FFPE nonneoplastic Barrett's esophagus samples can detect multiple mutations in minute areas of BIM with high analytical sensitivity. Next-generation sequencing panels for detection of TP53 and possibly combined mutations in other genes, such as APC and CDKN2A, may be useful in the clinical setting to improve dysplasia and cancer surveillance in patients with Barrett's esophagus.

Chromosome 3 status combined with BAP1 and EIF1AX mutation profiles are associated with metastasis in uveal melanoma.

PURPOSE: Somatic mutations in GNAQ, GNA11, SF3B1, EIF1AX, and BAP1 have been identified in uveal melanoma (UM). The aim of this study was to determine whether mutations in these genes in primary tumors were associated with metastases in individuals diagnosed with UM. METHODS: A total of 63 UM cases who developed a metastasis within 48 months of primary treatment and 53 UM controls who were metastasis-free over a similar time period were selected for the study. Primary UM cases were screened for mutations in GNAQ, GNA11, SF3B1, EIF1AX, and BAP1. The association of these mutations with tumor characteristics, chromosome 3 copy number, and metastatic status was analyzed by logistic regression to estimate the odds of developing metastasis within 48 months. RESULTS: As expected, tumor diameter, thickness, cilio-choroidal location, and chromosome 3 monosomy were all significantly (P < 0.02) associated with the presence of metastasis. In univariate analysis, GNA11 (odds ratio [OR] 2.5, 95% confidence interval [CI] 1.1-5.5) and BAP1 (OR 6.3, 95% CI 2.7-14.4) mutations were positively associated and EIF1AX mutation (OR 0.13, 95% CI 0.034-0.47) was inversely associated with metastatic status at 48 months after UM treatment. After adjustment for covariates, a chromosome 3 monosomy/BAP1-mutation/EIF1AX-wild-type (WT) mutation profile was strongly associated (OR 37.5, 95% CI 4.3-414) with the presence of metastasis compared with a chromosome 3 disomy/BAP1-WT/EIF1AX mutation profile. CONCLUSIONS: The results suggest that knowledge of mutations in BAP1 and EIF1AX can enhance prognostication of UM beyond that determined by chromosome 3 and tumor characteristics. Tumors with chromosome 3 disomy/BAP1-WT/EIF1AX-WT have a 10-fold increased risk of metastasis at 48 months compared with disomy-3/BAP1-WT/EIF1AX mutant tumors.

Absence of WNT4 gene mutation in a patient with MURCS association.

MURCS (Mullerian duct aplasia, Renal anomalies, and Cervicothoracic Somite dysplasia) association is a group of congenital genito-urinary and skeletal malformations. We report an adolescent girl with the cardinal features of MURCS association, obesity, and clinical findings of hyperandrogenism who did not show any exonic mutation of the WNT4 gene. Our finding excludes WNT4 gene as a candidate for MURCS association and suggests the need for further genetic studies.

Filaggrin-2 variation is associated with more persistent atopic dermatitis in African American subjects.

BACKGROUND: Atopic dermatitis (AD) is a common skin disease characterized by recurrent episodes of itching. Genetic variation associated with the persistence of AD has not been described for African American subjects. OBJECTIVE: We sought to evaluate genetic variation of filaggrin-2 (FLG2) in African American subjects with AD. METHODS: We evaluated a multiyear prospective cohort study of African American children with AD with respect to FLG2 variation based on whole-exome sequencing, followed by a targeted analysis. We ultimately evaluated the association of rs12568784 and rs16833974 with respect to the persistence of AD symptoms over time. RESULTS: Whole-exome analysis was conducted on 60 subjects, revealing a premature stop codon in exon 3 at S2377X (rs12568784) and X2392S (rs150529054) and a large exon 3 deletion mutation, Q2053del224. On the basis of a priori criteria, we then studied rs12568784, rs16833974 (H1249R), and Q2053del224. We noted that patients with S2377X (odds ratio [OR], 0.44; 95% CI, 0.25-0.46) and H1249R (OR, 0.23; 05% CI, 0.12-0.46) were significantly less likely to be free of symptoms of AD, and Q2053del224 (OR, 0.54; 95% CI, 0.16-1.80) trended toward this outcome. S2377X and H1249R were in high linkage disequilibrium (D' = 0.95). CONCLUSIONS: In an African American cohort with AD, FLG2 mutations were associated with more persistent AD. This is the first finding of genetic variation of a skin barrier protein in subjects of African ancestry with AD.

Enhanced sensitivity for detection of low-level germline mosaic RB1 mutations in sporadic retinoblastoma cases using deep semiconductor sequencing.

Sporadic retinoblastoma (RB) is caused by de novo mutations in the RB1 gene. Often, these mutations are present as mosaic mutations that cannot be detected by Sanger sequencing. Next-generation deep sequencing allows unambiguous detection of the mosaic mutations in lymphocyte DNA. Deep sequencing of the RB1 gene on lymphocyte DNA from 20 bilateral and 70 unilateral RB cases was performed, where Sanger sequencing excluded the presence of mutations. The individual exons of the RB1 gene from each sample were amplified, pooled, ligated to barcoded adapters, and sequenced using semiconductor sequencing on an Ion Torrent Personal Genome Machine. Six low-level mosaic mutations were identified in bilateral RB and four in unilateral RB cases. The incidence of low-level mosaic mutation was estimated to be 30% and 6%, respectively, in sporadic bilateral and unilateral RB cases, previously classified as mutation negative. The frequency of point mutations detectable in lymphocyte DNA increased from 96% to 97% for bilateral RB and from 13% to 18% for unilateral RB. The use of deep sequencing technology increased the sensitivity of the detection of low-level germline mosaic mutations in the RB1 gene. This finding has significant implications for improved clinical diagnosis, genetic counseling, surveillance, and management of RB.

Synthesis, characterization, photophysical properties of a novel organic photoswitchable dyad in its pristine and hybrid nanocomposite forms.

In the present paper the method of synthesis and characterization of a novel organic dyad, 3-(1-Methoxy-3,4-dihydro-naphthalyn-2-yl-)-1-p-chlorophenyl propenone, have been reported. In this paper our main thrust is to fabricate new hybrid nanocomposites by combining the organic dyad with different noble metals, semiconductor nanoparticle and noble metal-semiconductor core/shell nanocomposites. In this organic dyad, donor part is 1-Methoxy-3, 4-dihydro-naphthalen-2-carboxaldehyde with the acceptor p-chloroacetophenone. We have carried out steady state and time-resolved spectroscopic measurements on the dyad and its hybrid nanocomposite systems. Some quantum chemical calculations have also been done using Gaussian 03 software to support the experimental findings by theoretical point of view. Both from the theoretical predictions and NMR studies it reveals that in the ground state only extended (E-type or trans-type) conformation of the dyad exists whereas on photoexcitation these elongated conformers are converted into folded forms (Z- or cis-type) of the dyad, showing its photoswitchable character. Time resolved fluorescence spectroscopic (fluorescence lifetime by TCSPC method) measurements demonstrate that in chloroform medium all the organic-inorganic hybrid nanocomposites, studied in the present investigation, possess larger amount of extended conformers relative to folded ones, even in the excited singlet state. This indicates the possibility of slower energy destructive charge recombination rates relative to the rate processes associate with charge-separation within the dyad. It was found that in CHCl3 medium, the computed charge separation rate was found to be approximately 10(8) s(-1) for the dyad alone and other hybrid nanocomposite systems. The rate is found to be faster than the energy wasting charge recombination rate approximately 10(2)-10(1) s(-1), as observed from the transient absorption measurements for the corresponding hybrid systems. It indicates the conformational geometry has a great effect on the charge-separation and recombination rate processes. The suitability for the construction of efficient light energy conversion devices especially with Ag-Dyad nanocomposite of all the systems studied here is hinted from the observed long ion-pair lifetime.

The rates of charge separation and energy destructive charge recombination processes within an organic dyad in presence of metal-semiconductor core shell nanocomposites.

Steady state and time resolved spectroscopic measurements were made at the ambient temperature on an organic dyad, 1-(4-Chloro-phenyl)-3-(4-methoxy-naphthalen-1-yl)-propenone (MNCA), where the donor 1-methoxynaphthalene (1 MNT) is connected with the acceptor p-chloroacetophenone (PCA) by an unsaturated olefinic bond, in presence of Ag@TiO2 nanoparticles. Time resolved fluorescence and absorption measurements reveal that the rate parameters associated with charge separation, k(CS), within the dyad increases whereas charge recombination rate k(CR) reduces significantly when the surrounding medium is changed from only chloroform to mixture of chloroform and Ag@TiO2 (noble metal-semiconductor) nanocomposites. The observed results indicate that the dyad being combined with core-shell nanocomposites may form organic-inorganic nanocomposite system useful for developing light energy conversion devices. Use of metal-semiconductor nanoparticles may provide thus new ways to modulate charge recombination processes in light energy conversion devices. From comparison with the results obtained in our earlier investigations with only TiO2 nanoparticles, it is inferred that much improved version of light energy conversion device, where charge-separated species could be protected for longer period of time of the order of millisecond, could be designed by using metal-semiconductor core-shell nanocomposites rather than semiconductor nanoparticles only.