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Proliferation and apoptosis measurements by color image analysis based on differential absorption.

Cell proliferation and apoptosis indices are important indicators for the prognosis and treatment of a variety of cancers. A method is described using differential absorption color image analysis to measure proliferation and apoptosis in tumor sections using BrdU (5' bromodeoxyuridine) incorporation and immunohistochemistry and terminal deoxytransferase nick end-labeling (TUNEL). Nuclei were labeled with streptavidin-peroxidase-diaminobenzidine (DAB) secondary detection. The differential absorption method uses a computer-controlled microscope equipped with a tunable filter and digital camera to take advantage of the spectral differences of stained objects of interest. Images collected at defined wavelengths are divided and scaled to form ratio images in which the hematoxylin- or DAB-stained nuclei have intensity ranges far above those of surrounding structures. Using brightness thresholding followed by selection based on nuclear size and shape parameters, binary images were formed of the BrdU/apoptotic-positive tumor and all the tumor nuclei for subsequent counting and calculations of proliferation and apoptotic indices.

Animals↗

Differential scanning calorimetric analysis of antifreeze protein activity in the common mealworm, Tenebrio molitor.

Antifreeze proteins (AFP) are able to inhibit the growth of ice-crystals at temperatures below the equilibrium freezing point (Tf) of hemolymph. The analysis of AFP activity has commonly involved the use of direct microscopic observation of a sample following inoculation with ice. The resulting activity, defined as the amount of thermal hysteresis observed between Tf and the subsequent rapid growth of ice, has been reported to range up to 7 degrees C. However, most studies report high level of variation, possibly due to ice-crystal size variability and the presence of non-visible ice nuclei. We describe a new method of analysis of AFP activity using differential scanning calorimetry (DSC). DSC analysis reveals much higher activity, up to 10 degrees C, with less variation observed within a sample, and is not subject to the difficulty of accurate assessment of ice-crystal volume.

Animals↗

SimpleMicrobiome: An integrated web-based platform for streamlined microbiome data analysis and visualization.

Microbiome studies require multiple analytical steps after initial sequence processing. These steps commonly include data harmonization, preprocessing, taxonomic profiling, diversity analysis, differential abundance testing, predictive modeling, network inference, and preparation of publication-ready outputs. Although robust packages are available for many of these tasks, routine use often depends on command-line workflows, repeated data reformatting, and method-specific scripting. These requirements can limit accessibility for experimental researchers and complicate consistent analysis across interdisciplinary teams. We developed SimpleMicrobiome, a web-based R Shiny platform that integrates established microbiome analysis methods into a single interactive downstream workflow. The application accepts standard abundance, taxonomy, and metadata tables, supports interactive preprocessing and sample filtering, and provides modules for taxa profile visualization, alpha and beta diversity analysis, ANCOM-BC2 and MaAsLin2 differential abundance testing, Random Forest modeling with SHAP-based interpretation, microbial association network inference using SparCC and SPIEC-EASI through NetCoMi, correlation heatmaps, and dbRDA/CAP-style association biplots. The platform is implemented as a modular Shiny application so that preprocessing choices are propagated across downstream analyses, results can be exported as figures and tables, and the same application can be run through the public server, source-code installation, or a Docker image. SimpleMicrobiome consolidates major downstream microbiome analysis tasks in an accessible browser-based environment while retaining links to established analytical frameworks. The platform may reduce technical barriers for non-programming users, improve consistency across exploratory and reporting-oriented analyses, and support collaborative microbiome research. The public application is available at https://simplemicrobiome.mglab.org, the source code is available at https://github.com/yjcho2252/SimpleMicrobiome, and a Docker image for local deployment is available at https://hub.docker.com/r/mglab2252/simplemicrobiome.

differential abundance↗

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters the differentiation pattern of human keratinocytes in organotypic culture.

Human exposure to the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a severe skin pathology known as chloracne. In these studies we employed a three-dimensional, organotypic model system to study the effects of TCDD on human skin. This model uses the spontaneously immortalized human keratinocyte cell line NIKS and recapitulates both the three-dimensional microenvironment and epithelial-mesenchymal interactions found in intact human skin. Treatment of the organotypic cultures with TCDD causes alterations in the pattern of keratinocyte terminal differentiation. Analysis at both the light and electron microscope levels reveals a fully differentiated cornified layer in TCDD-treated organotypic cultures at earlier time points than observed in vehicle (dimethyl sulfoxide)-treated controls. Furthermore, TCDD-treated organotypic cultures exhibit aberrant distribution of several differentiation-specific protein markers. Basal cells in TCDD- and DMSO-treated organotypic cultures show no differences in proliferation as measured by quantification of 5-bromo-2'-deoxyuridine (BrdU)-positive nuclei. No aberrant BrdU uptake was detected outside of the basal layer. Neither TUNEL labeling nor immunohistochemical staining with an antibody to active caspase-3 revealed increased apoptosis in TCDD-treated organotypic cultures relative to controls. These data clearly indicate that TCDD modulates homeostasis in a model of human stratifying epithelium independent of changes in proliferation and apoptosis, exclusively by impacting keratinocyte terminal differentiation. This TCDD-induced effect on differentiation-specific proteins results in profound changes in the tissue architecture.

Antimetabolites↗

Preparation and characterization of biodegradable PLA polymeric blends.

The purpose of this study was to fine-tune the mechanical properties of high molecular-weight poly-L-lactic acid (PLLA), especially to increase its toughness without sacrificing too much of its original strength. Besides of its long degradation time, PLLA is usually hard and brittle, which hinders its usage in medical applications, i.e., orthopedic and dental surgery. Some modifications, such as the addition of plasticizers or surfactants/compatibilizers, are usually required to improve its original properties. PDLLA can degrade quickly due to its amorphous structure, thus shortening the degradation time of PLLA/PDLLA blends. Blends of biodegradable poly-L-lactic acid (PLLA) and poly-DL-lactic acid (PDLLA) or polycaprolactone (PCL), in addition to a third component, the surfactant-a copolymer of ethylene oxide and propylene oxide, were prepared by blending these three polymers at various ratios using dichloromethane as a solvent. The weight percentages of PLLA/PDLLA (or PCL) blends were 100%/0%, 80%/20%, 60%/40%, 50%/50%, 40%/60%, 20%/80% and 0%/100%, respectively. Physical properties such as the crystalline melting point, glass transition point (T(g)), phase behavior, degradation behavior, and other mechanical properties were characterized by thermogravimetric analysis, differential scanning calorimetry (DSC), infrared spectroscopy, gel permeation chromatography, and dynamic mechanical analysis (DMA). DSC data indicate that PLLA/PDLLA blends without the surfactant had two T(g)'s. With the addition of the surfactant, there was a linear shift of the single T(g) as a function of composition, with lower percentages of PLLA producing lower glass transition temperatures indicating that better miscibility had been achieved. DMA data show that the 40/60 PLLA/PDLLA blends without the surfactant had high elastic modulus and elongation, and similar results were observed after adding 2% surfactant into the blends. The 50/50 PLLA/PDLLA/2% surfactant blend had the highest elastic modulus, yield strength, and break strength compared with other ratios of PLLA/PDLLA/2% surfactant blends. The elongation at break of 50/50 PLLA/PDLLA was similar to that of PLLA. Again, the elongation at break of 50/50 PLLA/PDLLA/2% surfactant was almost 1.2-1.9 times higher than that of 50/50 PLLA/PDLLA and PLLA. Elongation of PLLA increased with the addition of PCL, but the strength decreased at the same time. In conclusions, adding PDLLA and surfactant to PLLA via solution-blending may be an effective way to make PLLA tougher and more suitable to use in orthopedic or dental applications.

Biocompatible Materials↗

Crystal structure determination of thymoquinone by high-resolution X-ray powder diffraction.

The crystal structure of 2-isopropyl-5-methyl-1,4-benzoquinone (thymoquinone) and its thermal behavior--as necessary physical and chemical properties--were determined in order to enhance the current understanding of thymoquinone chemical action by using high resolution x-ray powder diffraction, Fourier transform infrared spectroscopy (FTIR), and 3 thermo-analytical techniques thermogravimetric analysis (TGA), differential thermal analysis (DTA), and differential scanning calorimetry (DSC). The findings obtained with high-resolution x-ray powder diffraction and molecular location methods based on a simulated annealing algorithm after Rietveld refinement showed that the triclinic unit cell was a = 6.73728(8) A, b = 6.91560(8) A, c = 10.4988(2) A, alpha = 88.864(2) degrees, beta = 82.449(1) degrees, gamma = 77.0299(9) degrees; cell volume = 472.52(1) A3, Z = 2, and space group P1. In addition, FTIR spectrum revealed absorption bands corresponding to the carbonyl and C-H stretching of aliphatic and vinylic groups characteristically observed in such p-benzoquinones. Also, a chemical decomposition process starting at 65 degrees C and ending at 213 degrees C was noted when TGA was used. DSC allowed for the determination of onset at 43.55 degrees C and a melting enthalpy value of DeltaH(m) = 110.6 J/g. The low value obtained for the fusion point displayed a van der Waals pattern for molecular binding, and the thermograms performed evidence that thymoquinone can only be found in crystalline triclinic form, as determined by DRX methods.

Benzoquinones↗

[The use of discriminant analysis for the differential diagnosis of periodontal diseases].

Basing on rheo- and photoplethysmography combined with mathematical methods and computers the authors have developed a method for the differential diagnosis of periodontal diseases (gingivitis, periodontitis). Diagnostic studies carried out in 200 patients have shown a high probability (at least 92 percent) of the method for the differential diagnosis of periodontal diseases.

Adolescent↗

Whole blood transcriptome profile identifies motor neurone disease RNA biomarker signatures.

Blood-based biomarkers for motor neuron disease are needed for better diagnosis, progression prediction, and clinical trial monitoring. We used whole blood-derived total RNA and performed whole transcriptome analysis to compare the gene expression profiles in (motor neurone disease) MND patients to the control subjects. We compared 42 MND patients to 42 aged and sex-matched healthy controls and described the whole transcriptome profile characteristic for MND. In addition to the formal differential analysis, we performed functional annotation of the genomics data and identified the molecular pathways that are differentially regulated in MND patients. We identified 12,972 genes differentially expressed in the blood of MND patients compared to age and sex-matched controls. Functional genomic annotation identified activation of the pathways related to neurodegeneration, RNA transcription, RNA splicing and extracellular matrix reorganisation. Blood-based whole transcriptomic analysis can reliably differentiate MND patients from controls and can provide useful information for the clinical management of the disease and clinical trials.

Humans↗

Social networks and adult social identities: profiles and correlates of support and rejection.

The growth of interest in social networks has underscored the need for integrative analyses of network dimensions. A three-pronged strategy to achieve this objective was pursued in a study of 261 married women nurses. First, network variables related to important adult social identities were differentiated. Factor analyses revealed four network interaction factors: work support, work rejection, general support, and general rejection. An additional factor analysis differentiated work associates from nonwork network members. Second, cluster analysis of these factor scores identified five different network profiles. The profiles differed in the importance of the woman's work to her nonwork ties as well as the frequency and pattern of different positive and negative interactions. Third, individuals with these contrasting network profiles differed on levels of life satisfaction and psychological symptomatology. The discussion emphasizes social network differences within the broader context of the ecology of human development.

Adaptation, Psychological↗

Midline nasal tissue influences nestin expression in nasal-placode-derived luteinizing hormone-releasing hormone neurons during development.

Neurons differentiating into the luteinizing hormone-releasing hormone (LHRH) neuroendocrine phenotype are derived from the nasal placode. Cells within the vomeronasal organ anlage that turn on LHRH gene and peptide expression subsequently migrate into the forebrain where they influence reproductive function. The molecular and cellular cues regulating differentiation and migration of these cells are unknown. Discovery of developmental markers can indicate proteins directing or associated with differentiation. Analysis of such markers after manipulation of external cues can elucidate important extracellular differentiation signals. Embryonic LHRH neurons were examined in vivo for Mash-1 and nestin, two factors that delineate precursor populations in PNS and forebrain CNS cells. Nestin, but not Mash-1, was detected in early expressing LHRH cells in the vomeronasal organ anlage. These results were duplicated in LHRH neurons maintained in vitro in nasal explants. Such LHRH cells expressed nestin mRNA but not Mash-1 mRNA and were also negative for three other olfactory epithelial developmental transcription factors, Math4A, Math4C/neurogenin1, and NeuroD mRNA. Experimental manipulation of nasal explants revealed dual expression of nestin protein and LHRH in cells proximal to the vomeronasal organ anlage that was dependent upon midline cartilaginous/mesenchymal tissues. Prolonged nestin expression in LHRH cells after midline removal is consistent with nasal midline tissues modulating differentiation of LHRH neurons from the nasal placode.

Animals↗

Weight change in depression.

This report describes the weight changes of 109 outpatients during the course of a depressive illness and relates these changes to several potential predictors: age, gender, diagnosis, and scores on the Hamilton Rating Scale for Depression (HRSD), the Beck Depression Inventory, and the three factors on the Eating Questionnaire. Weight changes ranged from -33 to +50 pounds, with 40% of the patients reporting weight gain, 30% weight loss, and 30% no change in weight. Weight loss occurred more rapidly than did weight gain. The disinhibition factor of the Eating Questionnaire was significantly correlated with weight change during depression and, on a stepwise discriminant function analysis, differentiated weight-gaining from weight-losing patients at a high level of statistical significance. Severity of depression also differentiated weight-gaining from weight-losing patients in the discriminant function analysis, but only on the HRSD and at a level of more modest statistical significance.

Adult↗

Applications of tissue microdissection in cancer genetics.

A major obstacle to apply molecular biologic techniques effectively to the somatic genetic analysis of neoplastic tissue is the presence of abundant nonneoplastic elements in the analyzed specimen. These nonneoplastic elements, including reactive fibrous cells, vascular cells, and a variety of infiltrating white blood cells, may mask genetic alterations that otherwise would be easily detectable if the neoplastic cells were procured selectively. We have developed a microdissection technique which allows to selectively procure and genetically analyze small populations of neoplastic cells from the glass slide. In this report, we describe the microdissection technique and genetic analysis of archival frozen and paraffin-embedded tissue in general. Then, we review some implications of this approach of genetic analysis for mapping and cloning of new genes, identification of premalignant lesions, and differential analysis of different histologic components within the same tumor.

Barrett Esophagus↗

[Determination of lead in urine by differential potentiometric stripping analysis].

In the determination of lead in urine by differential potentiometric stripping analysis (DPSA) urine samples are acidified with hydrochloric acid to a total concentration equal to 0.2 mol/L without prior digestion with mineral acids. The sample is pre-electrolyzed at -1.2V (Vs SCE) for 80 seconds by glassy-carbon electrode coated with mercury. The lead concentrations are evaluated by means of standard addition. Detection limit of the method is 2ppb. The linear relationship occurs in the range of 0-150ppb. The relative standard deviations are 1.6% and the recovery of lead in urine samples is 89.5-110.0%. Compared with dithizone method, this method shows no significant difference in the results obtained.

Humans↗

[Clinico-pharmacologic analysis of the neurotropic action of tranquilizers].

Clinical aspects of the neurotropic effect of tranquillizers are considered. The systemic differentiated analysis of the influence of these psychopharmacological agents on the functional characteristics of the neurological status and psychophysiological condition of patients with neurotic disturbances is carried out. Two types of the neurotropic effect of tranquillizers and their selective vegetotropic effect are discussed. The presented results indicate that the neurotropic action of the psychopharmacological agents is of induced medical character.

Autonomic Nervous System↗

Mutational analysis of morphologic differentiation in Saccharomyces cerevisiae.

A genetic analysis was undertaken to investigate the mechanisms controlling cellular morphogenesis in Saccharomyces cerevisiae. Sixty mutant strains exhibiting abnormally elongated cell morphology were isolated. The cell elongation phenotype in at least 26 of the strains resulted from a single recessive mutation. These mutations, designated generically elm (elongated morphology), defined 14 genes; two of these corresponded to the previously described genes GRR1 and CDC12. Genetic interactions between mutant alleles suggest that several ELM genes play roles in the same physiological process. The cell and colony morphology and growth properties of many elm mutant strains are similar to those of wild-type yeast strains after differentiation in response to nitrogen limitation into the pseudohyphal form. Each elm mutation resulted in multiple characteristics of pseudohyphal cells, including elongated cell shape, delay in cell separation, simultaneous budding of mother and daughter cells, a unipolar budding pattern, and/or the ability to grow invasively beneath the agar surface. Mutations in 11 of the 14 ELM gene loci potentiated pseudohyphal differentiation in nitrogen-limited medium. Thus, a subset of the ELM genes are likely to affect control or execution of a defined morphologic differentiation pathway in S. cerevisiae.

Carrier Proteins↗

Discriminant analysis in the differential diagnosis of hypercalcaemia.

Linear discriminant analysis, a multivariate statistical procedure, applied to serum calcium, phosphate, alkaline phosphatase, bicarbonate, chloride, creatinine and tubular reabsorption of phosphate, proved to be effective in distinguishing patients with Primary Hyperparathyroidism from other hypercalcaemic patients in eithy-four retrospective cases. The application of the model to thirty-four prospective cases enabled us to separate correctly, hyperparathyroid patients from non-parathyroid hypercalcaemic patients.

Diagnosis, Differential↗

Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains.

We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six M. paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 M. paratuberculosis isolates representing different multiplex PCR for IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. paratuberculosis isolates into 20 distinct MLSSR types, consistent with geographic and epidemiologic correlates and with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. paratuberculosis isolates for molecular epidemiologic and population genetic analyses.

Animals↗

Glycoprotein (gp) 96 expression: induced during differentiation of intestinal macrophages but impaired in Crohn's disease.

BACKGROUND: The glycoprotein (gp) 96 links the adaptive with the innate immune system. It is a chaperone with a binding domain for peptides generated by proteasomal degradation. During cellular stress, peptide loaded gp96 can be released and presented to T cells by antigen presenting cells (APCs). METHODS: mRNAs from in vitro differentiated macrophages (iv mac) and normal intestinal macrophages (IMACs) were compared by subtractive hybridisation and Affymetrix GeneChip analysis. Differentiation induced expression of gp96 was investigated in the multicellular spheroid (MCS) model. In vivo gp96 protein expression was detected by double labelling immunohistochemistry of human colon and in the CD4+ CD62L+ T cell transfer mouse model. RESULTS: Five of 76 clones obtained by subtractive hybridisation revealed >99% sequence homology to gp96. Affymetrix GeneChip analysis confirmed induction of gp96 in IMACs. Gp96 mRNA was detected in IMACs from normal and intestinal bowel disease mucosa. Induction of gp96 protein was observed after seven days in the MCS model of IMAC differentiation. Immunohistochemistry confirmed the presence of gp96 protein in IMACs in normal mucosa as well as in mucosa from patients with ulcerative colitis and diverticulitis. In mucosa from Crohn's disease (CD) patients, gp96 protein was not detectable. In the CD4+ CD62L+ T cell transfer mouse model, gp96 was verifiable in non-activated IMACs. CONCLUSION: Gp96 is induced during differentiation of normal IMACs but is not detected in IMACs in CD mucosa. As gp96 has been described as having a role in tolerance induction, this may be relevant for loss of tolerance against luminal bacteria found in CD patients.

Animals↗