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DNA degradation with ozone.

DNA was ozonized in solution and the reaction was followed with polarimetry and with iodimetry. Polarimetry was used to determine the molar ratio DNA/O(3) when the DNA optical activity vanishes completely. At a molar ratio DNA/O(3)=2.3 the supramolecular structure of DNA collapses completely. Instead, iodimetry shows that the ozonolysis proceeds until all the nucleobases have been destroyed, an event which occurs at a molar ratio DNA/O(3)=1.1. The ozonolysis of DNA was also followed spectrophotometrically. DNA is reactive with ozone also in the solid state, as fixed bed. Clear indication about its oxidation derives from the FT-IR spectra from polarimetric measurements and from thermal analysis performed by thermogravimetric analysis (TGA), differential thermogravimetric analysis (DTG) and from differential thermal analysis (DTA). Particular remarkable is the fact that RNA has been found much less reactive toward ozone in the solid state than DNA.

Animals↗

Multiplexed fluorescence detection of phosphorylation, glycosylation, and total protein in the proteomic analysis of breast cancer refractoriness.

The Multiplexed Proteomics (MP) technology is a new approach that permits quantitative, multicolor fluorescence detection of proteins in one-dimensional or two-dimensional gels. This methodology allows for multiplexed identification and differential analysis of phosphoproteins, glycoproteins, and total proteins within a single gel electrophoresis experiment. Here the MP system was applied to the differential proteomic analysis of pregnancy-induced refractoriness to breast cancer using a rat model system. Differential analyses identified multiple proteins with altered phosphorylation, glycosylation, or protein expression patterns.

Animals↗

Differential gene expression analysis by micro-preparative capillary gel electrophoresis.

Differential display analysis by cDNA fractionation, collection of differentially expressed fractions of interests and their downstream characterization is demonstrated. cDNA pools from two strains of Cochliobolus heterostrophus fungus were generated by specific restriction digestion and selective ligation. Micropreparative separation and isolation of differentially expressed transcript representatives were accomplished by high-performance capillary gel electrophoresis. The collected individual DNA molecules were polymerase chain reaction amplified and sequenced to create expressed sequence tags for the genes of interests. High resolving power and sensitivity of capillary gel electrophoresis enabled fast and automated processing of minute amounts of cDNA samples with high precision.

Base Sequence↗

Differential pathophysiological mechanisms of reduced P300 amplitude in schizophrenia and depression: a single trial analysis.

In order to address basic mechanisms behind a reduced averaged P300 wave in schizophrenia and depression, 17 unmedicated schizophrenic and 11 unmedicated depressive subjects were tested in an 'oddball paradigm' against healthy controls matched for gender and age. The amplitude distributions of single trials' maximum positive deflections after stimulation (P300) for both target and nontarget stimuli were determined, which served as a basis for calculating the discrimination index d'. This index characterizes differences in the electrophysiological responses to target and nontarget stimuli of a subject being engaged in a discrimination task. As a main result d' was significantly lower for schizophrenics than for their controls. Directly compared to depressive subjects, schizophrenics also depicted a statistically significant decrease of the discrimination index, which could not be explained by differences in age. Although the averaged P300 signals did not show any significant differences between the two diagnostic groups, the approach of calculating d' on the basis of single trial analysis differentiated between schizophrenics and depressives. In conclusion, schizophrenic patients revealed different functional features when generating event-related potentials in an 'oddball paradigm' compared to healthy controls and depressives.

Adult↗

MKMC enables reference-free transcriptomic analysis using k-mer representations.

Traditional RNA-seq analysis depends heavily on genome alignment and gene annotation, limiting its utility in non-model organisms and introducing biases that can obscure regulatory complexity. We present MKMC (Multi-sample Kmer Counter), a scalable, reference-free toolkit for RNA-seq analysis that leverages k-mer-based statistics to detect biological variation without requiring alignment. MKMC integrates fast k-mer counting, abundance matrix generation, normalization, dimensionality reduction, and differential analysis into a unified workflow. Across diverse datasets, MKMC recapitulates key biological signals-including sex differences in killifish liver-and matches alignment-based pipelines in differential expression analysis and transcriptomic age prediction. Notably, MKMC detects isoform-specific events missed by traditional methods, one of which we validated using in situ hybridization. These results reveal previously hidden isoform-level regulatory events that contribute to sex- and age-associated transcriptional programs. MKMC offers a robust, extensible alternative to alignment-based approaches, enabling transcriptomic discovery across both model and non-model systems. While we focus here on RNA-seq as a primary application, MKMC is broadly applicable to any k-mer-based analysis of next-generation sequencing data.

MKMC↗

Sensitivity analysis of a nonlinear lumped parameter model of HIV infection dynamics.

A formal sensitivity analysis is performed on a delay differential equation model for the viral dynamics of an in vivo HIV infection during protease inhibitor therapy. We present results of both a differential analysis as well as a principle component based analysis and provide evidence that suggests the exact times at which specific parameters have the most influence over the solution. We offer insight into the pairwise mathematical relationships between the productively infected T-cell death rate delta, the viral plasma clearance rate c, and the time delay tau between infection and viral production as they relate to the viral dynamics. The results support the claim that the presence of a nonzero delay has a major impact on the model dynamics. Lastly, we comment upon the inadequacies of an alternative principle component based analysis.

Computer Simulation↗

The effect of pH and triethanolamine on sulfisoxazole complexation with hydroxypropyl-beta-cyclodextrin.

A novel complexation of sulfisoxazole with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was studied. Two systems were used: binary complexes prepared with HP-beta-CD and multicomponent system (HP-beta-CD and the basic compound triethanolamine (TEA)). Inclusion complex formation in aqueous solutions and in solid state were investigated by the solubility method, thermal analysis (differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)), Fourier-transform infrared spectroscopy (FT-IR) and dissolution studies. The solid complexes of sulfisoxazole were prepared by freeze-drying the homogeneous concentrated aqueous solutions in molar ratios of sulfisoxazole:HP-beta-CD 1:1 and 1:2, and sulfisoxazole:TEA:HP-beta-CD 1:1:2. FT-IR and thermal analysis showed differences among sulfisoxazole:HP-beta-CD and sulfisoxazole:TEA:HP-beta-CD and their corresponding physical mixtures and individual components. The HP-beta-CD solubilization of sulfisoxazole could be improved by ionization of the drug molecule through pH adjustments. However, larger improvements of the HP-beta-CD solubilization are obtained when multicomponent systems are used, allowing to reduce the amount of CD necessary to prepare the target formulation.

2-Hydroxypropyl-beta-cyclodextrin↗

Case study: acceleration/deceleration injury with angular kyphosis.

OBJECTIVE: To discuss the case of a patient who received upper cervical chiropractic care after trauma-induced arcual kyphosis in the cervical spine. A practical application of conservative management for posttrauma cervical spine injury in the private office setting is described. CLINICAL FEATURES: A 17-year-old female patient suffered an unstable C3/C4 motor segment after a lateral-impact motor vehicle collision. Additional symptoms on presentation included vertigo, tinnitus, neck and shoulder pain, and confusion. INTERVENTION AND OUTCOME: Conservative management consisted exclusively of upper cervical-specific adjustments guided by radiographic analysis and paraspinal bilateral skin temperature differential analysis of the cervical spine. During 10 weeks of care and 22 office visits, all symptoms subsided and the instability of C3/C4 motor segment appeared to be completely resolved. CONCLUSION: This study provides support for the use of upper cervical chiropractic management in cervical spine trauma cases. The clinical work-up consisted of physical examination, radiographic analysis, computer-administered and scored cognitive function testing, and audiometric examination. After conservative care, these examinations were repeated and demonstrated that the objective findings concurred with the subjective improvements reported by the patient.

Acceleration↗

Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos.

Embryonic lethality of thrombomodulin-deficient mice has indicated an essential role for this regulator of blood coagulation in murine development. Here, the embryonic expression pattern of thrombomodulin was defined by surveying beta-galactosidase activity in a mouse strain in which the reporter gene was placed under the regulatory control of the endogenous thrombomodulin promoter via homologous recombination in embryonic stem cells. The murine trophoblast was identified as a previously unrecognized anatomical site where TM expression is conserved between humans and mice and may exert a critical function during postimplantation development. Targeted reporter gene expression in mesodermal precursors of the endothelial cell lineage defined thrombomodulin as an early marker of vascular differentiation. Analysis of the thrombomodulin promoter in differentiating ES cells and in transgenic mice provided evidence for a disparate and cell type-specific gene regulatory control mechanism in the parietal yolk sac. The thrombomodulin promoter as defined in this study will allow the targeting of gene expression to the parietal yolk sac of transgenic mice and the initiation of investigations into the role of parietal endoderm in placental function.

Animals↗

Expression of cell cycle regulatory genes during primordial-primary follicle transition in the mouse ovary.

OBJECTIVE: To elucidate the genes involved in the ovarian primordial-primary follicle transition. DESIGN: Experimental animal study. SETTING: Research unit of university. ANIMAL(S): Day-1 and day-5 female ICR strain mice. INTERVENTION(S): We conducted forward and reverse suppression subtractive hybridization using ovarian mRNAs from day-1 and day-5 mice. MAIN OUTCOME MEASURE(S): After subtractive hybridization, the differential expression of certain genes between day-1 and day-5 ovaries was confirmed by Northern blot analysis. Differential expression of genes between primordial and primary follicles that are involved in the cell-cycle regulation was confirmed by laser capture microdissection (LCM) of follicles followed by real-time polymerase chain reaction (PCR). RESULT(S): In toto, 347 clones were sequenced and analyzed with the BLAST and RIKEN programs. Sequences of 312 clones significantly matched database entries, and 35 clones were novel. Among them, differential expression of five genes between day-1 and day-5 ovaries was confirmed by Northern blot analysis. In addition, differential expression between primordial and primary follicles of 11 more genes, particularly related to the cell-cycle regulation, was confirmed using LCM followed by real-time PCR. CONCLUSION(S): We reported the ovarian expression of many cell-cycle regulating genes during the primordial-primary follicle transition process. Results of the present study may provide insight for further study of the molecular mechanism(s) involved in primordial-primary follicle transition.

Animals↗

The gene expression profile in refractory periodontitis patients.

BACKGROUND: There are no specific bacterial profiles or diagnostic tests capable of identifying refractory periodontitis patients before a treatment regimen is initiated. Therefore, in this high-risk cohort of patients who do not respond appropriately, host factors that might be partly under genetic control may play a crucial role in their susceptibility. Specifically, we tested the hypothesis that patients with refractory periodontitis have multiple upregulated and/or downregulated genes that might be important in influencing clinical risk. METHODS: Oral subepithelial connective tissues were harvested aseptically from seven refractory periodontitis and seven periodontally well-maintained patients. An RNA isolation kit was used to isolate total RNA from tissue samples that had been stabilized in the RNA stabilizing reagent. The isolated total RNA was then subjected to gene expression profiling using the microarray to measure gene expression levels. The retrieved data were analyzed with a computer program for the differential analysis of gene expression microarray experiments. In addition, real-time polymerase chain reaction (PCR) analysis was performed on selected samples to confirm the microarray data's gene expression patterns. RESULTS: A total of 68 upregulated and six downregulated genes were identified that were differentially expressed at least two-fold out of 22,283 genes we analyzed. The selected model provided a 93% intrinsic validation along with a 93% extrinsic validation. To validate the microarray data, five upregulated genes (lactotransferrin [LTF], matrix metalloproteinase-1 [MMP-1], MMP-3, interferon induced-15 [IFI-15], and Homo sapiens hypothetical protein MGC5566) and two downregulated genes (keratin 2A [KRT2A] and desmocollin-1 [DSC-1]) were randomly selected for further analysis by real-time PCR. The relative RNA expression level of these genes measured by real-time PCR was similar to those measured by microarrays. CONCLUSION: The combined use of microarray technology with the computer program for the differential analysis of gene expression microarray experiments provided a set of candidate genes that may serve as novel therapeutic intervention points and improved diagnostic and screening procedures for high-risk individuals.

Aged↗

Identification of potential biomarkers and mechanisms for keloid disorder based on comprehensive bioinformatics analysis and machine learning algorithms.

BACKGROUND: Keloid disorder (KD) encompasses a spectrum of fibroproliferative dermal conditions, the pathogenesis remains complex and incompletely understood. This study sought to identify biomarkers and potential therapeutic targets for KD through an integrative bioinformatics approach and machine learning analysis of RNA sequencing data. METHODS: RNA sequencing was performed on skin tissue samples from 13 patients with KD and 14 healthy controls. Using weighted gene co-expression network analysis and differential expression analysis revealed differentially expressed key module genes, and the CytoHubba plugin identified candidate genes. Subsequently analyzed using least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE) methods to pinpoint feature genes associated with KD. Following this, biomarkers were determined through expression level validation, enrichment analysis, and immune infiltration analysis. RESULTS: A total of 420 differentially expressed key module genes were identified, and the top 10 genes with DMNC values were selected as candidate genes. Five feature genes were selected through LASSO and SVM-RFE, with NID2, MFAP2, COL8A1, and P4HA3 showing significant expression differences between KD and control samples, along with consistent expression patterns across datasets, identified as potential biomarkers. These four biomarkers were proved to possess high diagnostic potential, and they were found to exhibit significant positive correlations with one another. Functional enrichment analysis indicated that the primary KEGG pathways associated with these biomarkers included "steroid hormone biosynthesis" and "cytokine-cytokine receptor interaction." Moreover, immune infiltration analysis revealed that the four biomarkers were negatively correlated with type 17 T helper cells and positively correlated with 15 immune cell types, including activated B cells and central memory CD4 T cells. CONCLUSION: In conclusion, NID2, MFAP2, COL8A1, and P4HA3 were identified as key biomarkers for KD, offering new avenues for more targeted and effective diagnostic and therapeutic strategies for managing this condition.

Humans↗

Genes differentially expressed in human lung fibroblast cells transformed by glycidyl methacrylate.

OBJECTIVE: To define the differences in gene expression patterns between glycidyl methacrylate (GMA)-transformed human lung fibroblast cells (2BS cells) and controls. METHODS: The mRNA differential display polymerase chain reaction (DD-PCR) technique was used. cDNAs were synthesized by reverse transcription and amplified by PCR using 30 primer combinations. After being screened by dot blot analysis, differentially expressed cDNAs were cloned, sequenced and confirmed by Northern blot analysis. RESULTS: Eighteen differentially expressed cDNAs were cloned and sequenced, of which 17 were highly homologous to known genes (homology = 89%-100%) and one was an unknown gene. Northern blot analysis confirmed that eight genes encoding human zinc finger protein 217 (ZNF217), mixed-lineage kinase 3 (MLK-3), ribosomal protein (RP) L15, RPL41, RPS 16, TBX3, stanniocalcin 2 (STC2) and mouse ubiquitin conjugating enzyme (UBC), respectively, were up-regulated, and three genes including human transforming growth factor beta inducible gene (Betaig-h3), alpha-1,2-mannosidase 1A2 (MAN 1A2) gene and an unknown gene were down-regulated in the GMA-transformed cells. CONCLUSION: Analysis of the potential function of these genes suggest that they may be possibly linked to a variety of cellular processes such as transcription, signal transduction, protein synthesis and growth, and that their differential expression could contribute to the GMA-induced neoplastic transformation.

Air Pollutants, Occupational↗

Comparative profiling of the mammalian mitochondrial proteome: multiple aconitase-2 isoforms including N-formylkynurenine modifications as part of a protein biomarker signature for reactive oxidative species.

The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.

Aconitate Hydratase↗

Protein kinase C isoform expression during the differentiation of 3T3-L1 preadipocytes: loss of protein kinase C-alpha isoform correlates with loss of phorbol 12-myristate 13-acetate activation of nuclear factor kappaB and acquisition of the adipocyte phenotype.

The regulated expression of protein kinase C (PKC) isoforms was examined during the differentiation program of 3T3-L1 preadipocytes. In a parallel analysis, differentiation was blocked by treatment of the cells with tumor necrosis factor-alpha (TNF) to determine differentiation-specific changes in isoform expression from growth or treatment-induced effects. This analysis revealed that the expression of the conventional PKC-alpha isoform was reduced by 85% as cells attained the adipocyte phenotype. PKC-beta expression was measurable only during the early stages of the differentiation process and was not detectable in fully differentiated cells. An upregulation of PKC-theta, a novel PKC isoform, occurred during the latter stage of differentiation. Expression of PKC-zeta an atypical PKC isoform suggested to participate in TNF signal transduction, occurred throughout the time course with similar levels of expression in both preadipocytes and adipocytes. Nuclear run-on analysis demonstrated an approximately 85% reduction in the transcription of the PKC-alpha gene during differentiation. The reduced expression of this isoform corresponded with the decreased ability to activate nuclear factor kapppaB (NF-kappaB) in response to phorbol 12-myristate 13-acetate (PMA) treatment in the adipocytes. These data suggest that PMA responsiveness in 3T3-L1 adipocytes is markedly diminished.

Adipocytes↗

[Analysis of sister chromatid differentiation in myelodysplastic syndrome and its clinical application].

OBJECTIVE: To verify the value of sister chromatid differentiation (SCD) analysis of bone marrow cells in establishing the diagnosis and predicting prognosis of myelodysplastic syndrome (MDS). METHODS: S-bromodeoxyuridine-SCD and karyotyping studies were performed in 50 cases with clinically suspected MDS. RESULTS: Prolonged cell cycle time (CT) was found in 33 of the 50 cases (66.0%). In 13 cases with acute myeloid leukemia (AML), all had prolonged CT. However, only 2 (10.5%) of the 19 cases with nonmalignant hematological disorders had prolonged CT. There were significant differences of CT between the cases with MDS and those with AML, aplastic anemia (AA) and benign hypercellular anemias as well as healthy individuals. However, there were no significant differences between healthy individuals and cases with AA and, with benign hepercellular anemia. CT gradually prolonged during the evolution of MDS. 24 of the 50 patients (48.0%) were found to have clonal chromosomal abnormalities. In seven cases not compatible with MDS according to FAB criteria, six were found to have clonal chromosomal abnormalities. The mortality rates were 11.8 and 50.0% in the groups of normal SCD and of prolonged SCD, respectively (P < 0.05). The rates of leukemia transformation in both the groups were 5.9% and 16.7% (P > 0.05). The median survival of both the groups was 12 months and 8 months (P = 0.051). CONCLUSION: SCD analysis is valuable for establishing the diagnosis and predicting the prognosis of MDS.

Adolescent↗

[Poorly differentiated laryngeal tumors. An electron microscopy analysis].

Poorly differentiated laryngeal carcinomas, which light microscopically are made up of anisomorphic nuclei, undifferentiated cytoplasm and hardly visible cell membranes, show electronmicroscopically a remarquable polymorphism. The tumor cells, which form solid or trabecular groups of different sizes often with poorly defined borderlines to the stroma, are subdivided according to their ultrastructural features in spinosa, secretory and stemcell types. Lymphocytes, plasmacells and reticular cells may show distinct focal proliferation or may be completely absent. In some cases, there was a transition between tumour tissue and metaplastic ciliated and glandular epithelium with concomitant reduction of normal glands. By comparison, besides hyperplastic and metaplastic squamous and ciliated epithelium, normal mucosal biopsies reveal invaginations of pseudostratified ciliated epithelium.

Actin Cytoskeleton↗