Polymorphic expression of glutathione transferases A1, M1, P1 and T1 in epithelial ovarian cancer: a Serbian case-control study

PURPOSE: Since several studies have proposed that epithelial ovarian cancer should not be considered as a single disease entity and that it results from an accumulation of genetic changes, we aimed to assess the polymorphic expression of major cytosolic glutathione S-transferases (GSTM1, T1, A1 and P1) with respect to ovarian cancer susceptibility and aggressiveness. METHODS: This case-control study was conducted on 93 newly diagnosed epithelial ovarian cancer patients and 178 healthy matched controls. The multiplex polymerase chain reaction (PCR) was used to detect homozygous deletions of GSTM1 and GSTT1 genes. Analysis of the single nucleotide polymorphism (SNP) GSTA1 C69T was performed using PCR-restriction fragment length polymorphism (RFLP), while for SNP GSTP1 Ile105Val real-time PCR was used. RESULTS: No significant association to ovarian cancer risk was found for individual GSTM1, GSTA1 and GSTP1 genotypes (p>0.05). However, the carriers of GSTT1-active genotype were at 2-fold higher risk of ovarian cancer development (95%CI: 1.00-4.01, p=0.049), which was even more elevated in the subgroup of patients with positive family history of cancer. Moreover, the frequency of all three GST genotypes that might be associated to ovarian cancer risk (GSTT1-active, GSTA1-active and GSTP1-referent) was significantly higher in patients than in the control group (p=0.042). Even more, patients who were carriers of combination of these three genotypes represented over 64% of the total number of patients within any of the International Federation of Gynecology and Obstetrics (FIGO) stages of ovarian cancer. CONCLUSIONS: This study provides supportive evidence that GSTs might affect both susceptibility and progression of ovarian cance

Adherent but Not Suspension-Cultured Embryoid Bodies Develop into Laminated Retinal Organoids

Human induced pluripotent stem cells (iPSCs) are differentiated into three-dimensional (3D) retinal organoids to study retinogenesis and diseases that would otherwise be impossible. The complexity and low yield in current protocols remain a technical challenge, particularly for inexperienced personnel. Differentiation protocols require labor-intensive and time-consuming dissection of optic vesicles (OVs). Here we compare this method with a suspension method of developing retinal organoids. iPSCs were differentiated with standard protocols but the suspension-grown method omitted the re-plating of embryoid bodies and dissection of OVs. All other media and treatments were identical between developmental methods. Developmental maturation was evaluated with RT-qPCR and immunocytochemistry. Dissection- and suspension-derived retinal organoids displayed temporal biogenesis of retinal cell types. Differences in retinal organoids generated by the two methods of differentiation included temporal developmental and the organization of neural retina layers. Retinal organoids grown in suspension showed delayed development and disorganized retinal layers compared to the dissected retinal organoids. We found that omitting the re-plating of EBs to form OVs resulted in numerous OVs that were easy to identify and matured along a retinal lineage. While more efficient, the suspension method led to retinal organoids with disorganized retinal layers compared to those obtained using conventional dissection protocols

Functional Evaluation of Splicing for Variants of Uncertain Significance in Patients with Inherited Retinal Diseases

Inherited retinal diseases (IRD) comprise a heterogeneous set of clinical and genetic disorders that lead to blindness. Given the emerging opportunities in precision medicine and gene therapy, it has become increasingly important to determine whether DNA variants with uncertain significance (VUS) are responsible for patients’ IRD. This research was performed to assess the functional consequence of six VUS identified in patients with IRD. Clinical assessments included an ophthalmic examination, best-corrected visual acuity, and kinetic perimetry. Imaging was acquired with the Optos ultra-widefield camera and spectral domain optical coherence tomography (SD-OCT). Genetic testing was performed by Molecular Vision Laboratories. VUS that were predicted to alter splicing were analyzed with a minigene assay, which revealed that VUS in the genes OPA1, CNGB1, and CLUAP1 altered spicing mechanisms. Due to emerging gene and cell therapies, these results expand the genotype-phenotype correlations for patients diagnosed with an IRD

Genetic Diagnosis for 64 Patients with Inherited Retinal Disease

The overlapping genetic and clinical spectrum in inherited retinal degeneration (IRD) creates challenges for accurate diagnoses. The goal of this work was to determine the genetic diagnosis and clinical features for patients diagnosed with an IRD. After signing informed consent, peripheral blood or saliva was collected from 64 patients diagnosed with an IRD. Genetic testing was performed on each patient in a Clinical Laboratory Improvement Amendments of 1988 (CLIA) certified laboratory. Mutations were verified with Sanger sequencing and segregation analysis when possible. Visual acuity was measured with a traditional Snellen chart and converted to a logarithm of minimal angle of resolution (logMAR). Fundus images of dilated eyes were acquired with the Optos® camera (Dunfermline, UK). Horizontal line scans were obtained with spectral-domain optical coherence tomography (SDOCT; Spectralis, Heidelberg, Germany). Genetic testing combined with segregation analysis resolved molecular and clinical diagnoses for 75% of patients. Ten novel mutations were found and unique genotype phenotype associations were made for the genes RP2 and CEP83. Collective knowledge is thereby expanded of the genetic basis and phenotypic correlation in IRD

Lincosamide Synthetase—A Unique Condensation System Combining Elements of Nonribosomal Peptide Synthetase and Mycothiol Metabolism

<div><p>In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of <i>S. lincolnensis</i> strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of <i>ccbZ</i> gene adjacent to <i>ccbN</i>, the counterpart of <i>lmbN</i>, suggesting the gene rearrangement, evident also from still active internal translation start in <i>lmbN</i>, and indicating the direction of lincosamide biosynthesis evolution. The <i>in vitro</i> test with LmbN-CP, LmbC and the newly identified <i>S. lincolnensis</i> phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a <i>holo</i>-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.</p></div

Comparative analysis of lincomycin and celesticetin biosynthetic gene clusters.

<p>The genes coding for proteins with already confirmed/published functions are marked (C), the functions of other encoded proteins were assigned either according to sequence analysis combined with the prediction (P) in literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118850#pone.0118850.ref014" target="_blank">14</a>], or based on BLAST analysis only (S). The genes in bold were selected for further testing. For shared homologous genes (below the bold line) the percentage of mutual identity of encoded proteins is shown, for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118850#pone.0118850.s001" target="_blank">S1 Document</a>. C8—octose; LIN—lincomycin; CEL—celesticetin;</p><p><i>*⁾</i>—the relevant function, biosynthetic step assignment and sequence identity value concern only the marked gene; MFS—major facilitator superfamily; salicylate—both biosynthesis and attachment of the salicylate unit; AS—biosynthesis of amino sugar; PPL—biosynthesis of PPL</p><p>Comparative analysis of lincomycin and celesticetin biosynthetic gene clusters.</p

The biological activity assay of <i>S. lincolnensis</i> inactivation mutants.

<p>The inhibition zones correspond to the antibiotic production. WT—wild type strain (positive control); ΔT, ΔN, N-ΔID, N-ΔCP indicate the respective disrupted lincomycin biosynthetic gene or its part; NC—not complemented; PPL or MTL—complemented by the respective intermediate of lincomycin biosynthesis. For each sample, at least two independent cultivations were evaluated in triplicates as described in Material and methods. A typical result is shown.</p