Automated Design of Probes for rRNA-Targeted Fluorescence In Situ Hybridization Reveals the Advantages of Using Dual Probes for Accurate Identification

Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu).     

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

The ability of a GroEL-based bio-layer interferometry (BLI) assay to detect structurally altered and/or aggregated species of pharmaceutically relevant proteins is demonstrated. Assay development included optimizing biotinylated-GroEL immobilization to streptavidin biosensors, combined with biophysical and activity measurements showing native and biotinylated GroEL are both stable and active. First, acidic fibroblast growth factor (FGF-1) was incubated under conditions known to promote (40°C) and inhibit (heparin addition) molten globule formation. Heat exposed (40°C) FGF-1 exhibited binding to GroEL-biosensors, which was significantly diminished in the presence of heparin. Second, a polyclonal human IgG solution containing 6–8% non-native dimer showed an increase in higher molecular weight aggregates upon heating by size exclusion chromatography (SEC). The poly IgG solution displayed binding to GroEL-biosensors initially with progressively increased binding upon heating. Enriched preparations of the IgG dimers or monomers showed significant binding to GroEL-biosensors. Finally, a thermally treated IgG1 monoclonal antibody (mAb) solution also demonstrated increased GroEL-biosensor binding, but with different kinetics. The bound complexes could be partially to fully dissociated after ATP addition (i.e., specific GroEL binding) depending on the protein, environmental stress, and the assay’s experimental conditions. Transmission electron microscopy (TEM) images of GroEL-mAb complexes, released from the biosensor, also confirmed interaction of bound complexes at the GroEL binding site with heat-stressed mAb. Results indicate that the GroEL-biosensor-BLI method can detect conformationally altered and/or early aggregation states of proteins, and may potentially be useful as a rapid, stability-indicating biosensor assay for monitoring the structural integrity and physical stability of therapeutic protein candidates.     

Assessment of ER Stress and Autophagy Induced by Ionizing Radiation in Both Radiotherapy Patients and Ex Vivo Irradiated Samples

Acute radiation leads to several toxic clinical states and triggers some molecular pathways. To shed light on molecular mechanisms triggered by ionizing radiation (IR), we examined the expression profiles of endoplasmic reticulum (ER) stress and autophagy‐related genes in individuals who were exposed to IR. Blood samples were collected from 50 cancer patients before radiotherapy and on the 5th, 15th, and 25th days of the treatment. Peripheral blood samples from 10 healthy volunteers were also obtained for ex vivo irradiation, divided into five and irradiated at a rate of 373 kGy/h to 0, 0.1, 0.5, 1, and 3Gy γ‐rays using a constant gamma source. GRP78, ATG5, LC3, ATF4, XBP1, and GADD153 genes were analyzed by quantitative real‐time polymerase chain reaction (QRT‐PCR) using beta 2 microglobulin (B2M) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) as references. In both groups, expressions of the selected genes have increased. It can be concluded that IR induces ER stress and related authophagy pathway in the peripheral lymphocyte cells proportionally by dose.     

A Monte Carlo evaluation for effects of probable dimensional uncertainties of low dose rate brachytherapy seeds on dose

The aim of this study is to determine effects of size deviations of brachytherapy seeds on two dimensional dose distributions around the seed. Although many uncertainties are well known, the uncertainties which stem from geometric features of radiation sources are weakly considered and predicted. Neither TG-43 report which is not completely in common consensus, nor individual scientific MC and experimental studies include sufficient data for geometric uncertainties. Sizes of seed and its components can vary in a manufacturing deviation. This causes geometrical uncertainties, too. In this study, three seeds which have different geometrical properties were modeled using EGSnrc-Code Packages. Seeds were designed with all their details using the geometry package. 5% deviations of seed sizes were assumed. Modified seeds were derived from original seed by changing sizes by 5%. Normalizations of doses which were calculated from three kinds of brachytherapy seed and their derivations were found to be about 3%–20%. It was shown that manufacturing differences of brachytherapy seed cause considerable changes in dose distribution.     

Making all parts of the 16S rRNA of Escherichia coli accessible in situ to single DNA oligonucleotides

rRNA accessibility is a major sensitivity issue limiting the design of working probes for fluorescence in situ hybridization (FISH). Previous studies empirically highlighted the accessibility of target sites on rRNA maps by grouping probes into six classes according to their brightness levels. In this study, a recently proposed mechanistic model of FISH, based on the thermodynamics of secondary nucleic acid interactions, was used to evaluate the accessibility of the 16S rRNA of Escherichia coli to fluorescein-labeled oligonucleotides when thermodynamic and kinetic barriers were eliminated. To cover the entire 16S rRNA, 109 probes were designed with an average thermodynamic affinity (DeltaGo (overall)) of -13.5 kcal/mol. Fluorescence intensity was measured by flow cytometry, and a brightness threshold between classes 3 and 4 was used as the requirement for proof of accessibility. While 46% of the probes were above this threshold with conventional 3-h hybridizations, extending the incubation period to 96 h dramatically increased the fraction of bright probes to 86%. Insufficient thermodynamic affinity and/or fluorophore quenching was demonstrated to cause the low fluorescence intensity of the remaining 14% of the probes. In the end, it was proven that every nucleotide in the 16S rRNA of E. coli could be targeted with a bright probe and, therefore, that there were no truly inaccessible target regions in the 16S rRNA. Based on our findings and mechanistic modeling, a rational design strategy involving DeltaGo(overall), hybridization kinetics, and fluorophore quenching is recommended for the development of bright probes.     

Changes in peroxidase activities and soluble proteins in strawberry varieties under salt-stress

Effects of salt stress on the activity of peroxidase (PRX) isozyme and leaf proteins were studied in the three strawberry varieties, Camarosa, Tioga and Chandler. Plants were watered with modified 1/3 Hoagland nutrient solution containing 0 (control), 8.5, 17.0 and 34.0 mM NaCl for 30 days fallowing 20 days acclimation. PRX activity was assayed in leaf extracts and PRX profiles of control and salt treated plants were performed by native polyacrylamide gel electrophoresis (PAGE). During the salt stress Tioga and Camarosa tolerated the cellular damage with less electrolyte leakage while Chandler exhibited a severe cellular damage with the highest (87.5 % in 34.0 mM NaCl treatment) electrolyte leakage. Total soluble protein content was decreased by salinity in Tioga (from 11.35 to 5.86 mg g⁻¹ DW) and Chandler (from 9.35 to 3.90 mg g⁻¹ DW) while it was almost unchanged in Camarosa. Salt stress increased total and specific PRX activity as compared with the control in all the varieties. In native PAGE, one basic isoperoxidase band (Rf=0.27) was observed commonly with different band intensity in all the treatments. Therefore, it might be associated with lignification and recovery of cell membrane damage in strawberry plants under salt stress.     

Effects of intraperitoneally administered ubiquinone on the level of total lipid and fatty acids in rat liver

The purpose of the present study was to evaluate the effect of ubiquinone (coenzyme Q-10) on total lipid and fatty acid composition of liver tissues in rats. Twenty male wistar rats were randomly divided into two groups. The first group was used as a control. The second group received ubiquinone (8 mg/every other day) intraperitoneally. This administration was done for a period of 38 days. Body weight increases in animals fed diets for 38 days were on average 35 g in control group (C), and only 11 g in the ubiquinone group. Total lipid content of liver tissues in the ubiquinone group (UB) decreased significantly (p < 0.0001) compared to the control group (C). The ratio of 22:6 and total omega3 fatty acid in the UB increased (p < 0.01) compared to C. While the level of oleic acid (18:1), palmitoleic acid (16:1) and total monounsaturated fatty acid (MUFA) in UB significantly decreased (p < 0.01, p < 0.001, p < 0.001, respectively), the level of stearic acid (18:0) in liver tissue increased (p < 0.05) in the same group when compared to C. Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme catalyzing the synthesis of monounsaturated fatty acid mainly oleate (18:1). We speculate that ubiquinone inhibits SCD activity. SCD is an important metabolic control point in body weight regulation. Our results indicate that ubiquinone supplementation may have an inhibitory effect on obesity and it seems that the level of 22:6 in liver increased due to ubiquinone.     

GPSM2 mutations cause the brain malformations and hearing loss in Chudley-McCullough syndrome

Autosomal-recessive inheritance, severe to profound sensorineural hearing loss, and partial agenesis of the corpus callosum are hallmarks of the clinically well-established Chudley-McCullough syndrome (CMS). Although not always reported in the literature, frontal polymicrogyria and gray matter heterotopia are uniformly present, whereas cerebellar dysplasia, ventriculomegaly, and arachnoid cysts are nearly invariant. Despite these striking brain malformations, individuals with CMS generally do not present with significant neurodevelopmental abnormalities, except for hearing loss. Homozygosity mapping and whole-exome sequencing of DNA from affected individuals in eight families (including the family in the first report of CMS) revealed four molecular variations (two single-base deletions, a nonsense mutation, and a canonical splice-site mutation) in the G protein-signaling modulator 2 gene, GPSM2, that underlie CMS. Mutations in GPSM2 have been previously identified in people with profound congenital nonsyndromic hearing loss (NSHL). Subsequent brain imaging of these individuals revealed frontal polymicrogyria, abnormal corpus callosum, and gray matter heterotopia, consistent with a CMS diagnosis, but no ventriculomegaly. The gene product, GPSM2, is required for orienting the mitotic spindle during cell division in multiple tissues, suggesting that the sensorineural hearing loss and characteristic brain malformations of CMS are due to defects in asymmetric cell divisions during development.     

MASP1 mutations in patients with facial, umbilical, coccygeal, and auditory findings of Carnevale, Malpuech, OSA, and Michels syndromes

Distinctive facial features consisting of hypertelorism, telecanthus, blepharophimosis, blepharoptosis, epicanthus inversus, periumbilical defects, and skeletal anomalies are seen in autosomal-recessive Carnevale, Malpuech, Michels, and oculo-skeletal-abdominal (OSA) syndromes. The gene or genes responsible for these syndromes were heretofore unknown. We report on three individuals from two consanguineous Turkish families with findings characteristic of these syndromes, including facial dysmorphism, periumbilical depression, mixed hearing loss, radioulnar synostosis, and coccygeal appendage. Homozygosity mapping yielded an autozygous region on chromosome 3q27 in both families. In one family, whole exome sequencing revealed a missense mutation, MASP1 c.2059G>A (p.G687R), that cosegregated with the phenotype. In the second family, Sanger sequencing of MASP1 revealed a nonsense mutation, MASP1 c.870G>A (p.W290X), that also cosegregated with the phenotype. Neither mutation was found in 192 Turkish controls or 1200 controls of various other ancestries. MASP1 encodes mannan-binding lectin serine protease 1. The two mutations occur in a MASP1 isoform that has been reported to process IGFBP-5, thereby playing a critical role in insulin growth factor availability during craniofacial and muscle development. These results implicate mutations of MASP1 as the cause of a human malformation syndrome and demonstrate the involvement of MASP1 in facial, umbilical, and ear development during the embryonic period.