Quantitative analysis
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Lysosomes are the terminal end of catabolic pathways in the cell, as well as signaling centers performing important functions such as the recycling of macromolecules, organelles, and nutrient adaptation. The importance of lysosomes in human health is supported by the fact that the deficiency of most lysosomal genes causes monogenic diseases called as a group Lysosomal Storage Diseases (LSDs). A common phenotypic hallmark of LSDs is the expansion of the lysosomal compartment that can be detected by using conventional imaging methods based on immunofluorescence protocols or overexpression of tagged lysosomal proteins. These methods require the alteration of the cellular architecture (i.e., due to fixation methods), can alter the behavior of cells (i.e., by the overexpression of proteins), and require sample preparation and the accurate selection of compatible fluorescent markers in relation to the type of analysis, therefore limiting the possibility of characterizing cellular status with simplicity. Therefore, a quantitative and label-free methodology, such as Quantitative Phase Imaging through Digital Holographic (QPI-DH), for the microscopic imaging of lysosomes in health and disease conditions may represent an important advance to study and effectively diagnose the presence of lysosomal storage in human disease. Here we proof the effectiveness of the QPI-DH method in accomplishing the detection of the lysosomal compartment using mouse embryonic fibroblasts (MEFs) derived from a Mucopolysaccharidosis type III-A (MSP-IIIA) mouse model, and comparing them with wild-type (WT) MEFs. We found that it is possible to identify label-free biomarkers able to supply a first pre-screening of the two populations, thus showing that QPI-DH can be a suitable candidate to surpass fluorescent drawbacks in the detection of lysosomes dysfunction. An appropriate numerical procedure was developed for detecting and evaluate such cellular substructures from in vitro cells cultures. Results reported in this study are encouraging about the further development of the proposed QPI-DH approach for such type of investigations about LSDs.
- Klíčová slova
- digital holography, intracellular specificity, label‐free imaging, lysosomal storage diseases, lysosomes, quantitative phase imaging,
- MeSH
- fibroblasty metabolismus patologie MeSH
- kvantitativní fázové zobrazování MeSH
- lidé MeSH
- lyzozomální nemoci z ukládání metabolismus patologie genetika diagnóza MeSH
- lyzozomy * metabolismus MeSH
- mukopolysacharidóza III metabolismus patologie genetika MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Haloalkane dehalogenases are microbial enzymes that cleave a carbon-halogen bond in halogenated compounds. The haloalkane dehalogenase LinB, isolated from Sphingomonas paucimobilis UT26, is a broad-specificity enzyme. Fifty-five halogenated aliphatic and cyclic hydrocarbons were tested for dehalogenation with the LinB enzyme. The compounds for testing were systematically selected using a statistical experimental design. Steady-state kinetic constants K(m) and k(cat) were determined for 25 substrates that showed detectable cleavage by the enzyme and low abiotic hydrolysis. Classical quantitative structure-activity relationships (QSARs) were used to correlate the kinetic constants with molecular descriptors and resulted in a model that explained 94% of the experimental data variability. The binding affinity of the tested substrates for this haloalkane dehalogenase correlated with hydrophobicity, molecular surface, dipole moment, and volume:surface ratio. Binding of the substrate molecules in the active site pocket of LinB depends nonlinearly on the size of the molecules. Binding affinity increases with increasing substrate size up to a chain length of six carbon atoms and then decreases. Comparative binding energy (COMBINE) analysis was then used to identify amino acid residues in LinB that modulate its substrate specificity. A model with three statistically significant principal components explained 95% of the experimental data variability. van der Waals interactions between substrate molecules and the enzyme dominated the COMBINE model, in agreement with the importance of substrate size in the classical QSAR model. Only a limited number of protein residues (6-8%) contribute significantly to the explanation of variability in binding affinities. The amino acid residues important for explaining variability in binding affinities are as follows: (i) first-shell residues Asn38, Asp108, Trp109, Glu132, Ile134, Phe143, Phe151, Phe169, Val173, Trp207, Pro208, Ile211, Leu248, and His272, (ii) tunnel residues Pro144, Asp147, Leu177, and Ala247, and (iii) second-shell residues Pro39 and Phe273. The tunnel and the second-shell residues represent the best targets for modulating specificity since their replacement does not lead to loss of functionality by disruption of the active site architecture. The mechanism of molecular adaptation toward a different specificity is discussed on the basis of quantitative comparison of models derived for two protein family members.
- MeSH
- alkany chemie metabolismus MeSH
- chemické modely MeSH
- halogenované uhlovodíky chemie metabolismus MeSH
- hydrolasy chemie metabolismus MeSH
- katalýza MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- kvantitativní vztahy mezi strukturou a aktivitou * MeSH
- kvantová teorie MeSH
- molekulární modely MeSH
- multivariační analýza MeSH
- Sphingomonas enzymologie MeSH
- statistické modely MeSH
- substrátová specifita MeSH
- termodynamika MeSH
- vazebná místa MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- validační studie MeSH
- Názvy látek
- alkany MeSH
- haloalkane dehalogenase MeSH Prohlížeč
- halogenované uhlovodíky MeSH
- hydrolasy MeSH
This paper summarizes the effects of irregular shape on the results of a quantitative X-ray fluorescence (XRF) micro-analysis. These effects become relevant when an XRF analysis is performed directly on an investigated material. A typical example is XRF analyses of valuable and historical objects whose measurements should be performed non-destructively and non-invasively, without taking samples. Several measurements and computer simulations were performed for selected metallic materials and shapes to evaluate the accuracy and precision of XRF. The described experiments and the corresponding Monte Carlo simulations were related to the XRF device designed and utilized at the Czech Technical University. It was found that the relative uncertainty was typically about 5-10% or even higher in quantitative analyses of minor elements due to irregular shapes of surfaces. This must be considered in cases of the interpretation of XRF results, especially in the cultural heritage sciences. The conclusions also contain several recommendations on how to measure objects under hard-to-define geometric conditions with respect to reduction in the surface effect in quantitative or semi-quantitative XRF analyses.
- Klíčová slova
- Monte Carlo method, X-ray fluorescence, metal, quantitative analysis,
- Publikační typ
- časopisecké články MeSH
Hypokinetic dysarthria (HD) and freezing of gait (FOG) are both axial symptoms that occur in patients with Parkinson's disease (PD). It is assumed they have some common pathophysiological mechanisms and therefore that speech disorders in PD can predict FOG deficits within the horizon of some years. The aim of this study is to employ a complex quantitative analysis of the phonation, articulation and prosody in PD patients in order to identify the relationship between HD and FOG, and establish a mathematical model that would predict FOG deficits using acoustic analysis at baseline. We enrolled 75 PD patients who were assessed by 6 clinical scales including the Freezing of Gait Questionnaire (FOG-Q). We subsequently extracted 19 acoustic measures quantifying speech disorders in the fields of phonation, articulation and prosody. To identify the relationship between HD and FOG, we performed a partial correlation analysis. Finally, based on the selected acoustic measures, we trained regression models to predict the change in FOG during a 2-year follow-up. We identified significant correlations between FOG-Q scores and the acoustic measures based on formant frequencies (quantifying the movement of the tongue and jaw) and speech rate. Using the regression models, we were able to predict a change in particular FOG-Q scores with an error of between 7.4 and 17.0 %. This study is suggesting that FOG in patients with PD is mainly linked to improper articulation, a disturbed speech rate and to intelligibility. We have also proved that the acoustic analysis of HD at the baseline can be used as a predictor of the FOG deficit during 2 years of follow-up. This knowledge enables researchers to introduce new cognitive systems that predict gait difficulties in PD patients.
- Klíčová slova
- Acoustic analysis, Freezing of gait, Hypokinetic dysarthria, Parkinson’s disease, Quantitative analysis,
- Publikační typ
- časopisecké články MeSH
The scientific accuracy of ethnobotanical study has significantly grown in the past decades due to the adoption of quantitative methods, mainly represented by indices. These quantitative approaches can provide data amenable to hypothesis testing, statistical validation, and comparative analysis. Plenty of indices are applied nowadays in ethnobotany. However, none of the previously developed indices have argued for comparing general ethnobotanical knowledge between two or more human groups. Hence, this study seeks to cover this methodological gap and proposes a novel index that will provide ethnobotanists with a tangible number representing the general ethnobotanical knowledge of a specific human group. The proposed index will enable researchers in the field to compare ethnobotanical knowledge of two or more ethnic/ religious/ cultural groups; it will also be possible to conduct a comparison within the same group, such as comparing two distanced time periods, genders, and/or age groups. The index complexly employs several factors that can be critical when assessing ethnobotanical knowledge (e.g. total number of species reported by all participants in a particular group, mean number of species reported per participant in a particular group, and mean number of citations per species in a particular group). The index is designed to be mainly used in ethnobotany; however, it is also usable in ethnobiology and may be applicable in other studies related to traditional knowledge assessment.
- Klíčová slova
- BEI, Cross-cultural comparison, Ethnobiology, Ethnobotany, Intra-group comparison, Quantitative analysis, Traditional knowledge,
- MeSH
- etnobotanika * metody MeSH
- lidé MeSH
- srovnání kultur * MeSH
- zdraví - znalosti, postoje, praxe MeSH
- znalosti MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.
- Klíčová slova
- BCR-ABL1, CML, LD-PCR, NGS, pyrosequencing, quantitative analysis of mutant subclones,
- MeSH
- bcr-abl fúzní proteiny chemie genetika MeSH
- chronická myeloidní leukemie genetika patologie MeSH
- DNA analýza genetika metabolismus MeSH
- lidé MeSH
- polymerázová řetězová reakce * MeSH
- sekvenční analýza DNA * MeSH
- srovnávací genomová hybridizace MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- bcr-abl fúzní proteiny MeSH
- DNA MeSH
OBJECTIVE: Surgery is the only curative treatment for primary hyperparathyroidism. Parathyroid scintigraphy is one method used to preoperatively localize the lesion. We examined time-related changes in radiopharmaceutical uptake in parathyroid adenomas (PTAs) and thyroid gland by quantitative single-photon-emission computed tomography (SPECT) imaging to assess differences between rapid and delayed washout patterns. PATIENTS AND METHODS: The study group consisted of 35 histologically verified PTAs after radio-guided surgery extirpation in 33 patients with primary hyperparathyroidism. Patients underwent a three-phase SPECT/CT study of the neck and upper thorax post 99mTc-methoxyisobutylisonitrile (MIBI) injection. Images were reconstructed using a proprietary ordered-subset-conjugate-gradient-maximization algorithm (Siemens xSPECT Quant). PTAs were divided into those with a rapid (group A) and those with a slow (group B) washout pattern. SUVmax values of PTAs and thyroid gland tissue at 10, 90 and 180 min post 99mTc-MIBI injection were recorded and statistically assessed. Retention indexes related to the early examination were calculated for PTA and thyroid gland (RI-PTA and RI-TG). RESULTS: There were 11 PTAs in group A and 24 in group B. Significant between-group differences in PTA SUVmax and PTA/thyroid gland ratios were observed only at 180 min postinjection (P = 0.0297, P = 0.0222, respectively). RI-PTAs differed significantly at 90 and 180 min postinjection (P = 0.0298, P = 0.0431). No differences in PTA volumes, thyroid gland SUVmax values or RI-TG were observed between the groups. CONCLUSION: PTAs with rapid and slow washout patterns have different characteristics on quantitative analysis in later phases. No significant differences in directly measurable quantitative values (SUVmax, PTA/thyroid gland ratio) at the early stages of multi-phase examination were observed.
- MeSH
- adenom diagnostické zobrazování chirurgie MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádory příštítného tělíska * diagnostické zobrazování chirurgie MeSH
- počítačové zpracování obrazu MeSH
- senioři MeSH
- SPECT/CT MeSH
- technecium 99mTc sestamibi MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- technecium 99mTc sestamibi MeSH
A set of 4-benzylsulfanyl derivatives of pyridine-2-carbonitriles and pyridine-2-carbothioamides, previously tested for their antimycobacterial activity, were analysed by quantitative structure-activity relationship (QSAR) techniques, using some physicochemical and quantum-chemical parameters. The resulting QSAR revealed that the activity increases with electron withdrawing substituents in the benzyl moiety of studied compounds. HOMO orbitals can play an important role in the description of the mechanism of interactions at the molecular level. Additionally, the results of multiple linear regression indicate the differences between Mycobacterium tuberculosis and M. avium. The hydrophobicity of studied compounds is important for activity against M. avium.
A computer-aided quantitative method for a complex analysis of gel electrophoretograms is presented. The analysis consists of several steps: (i) determination of the background image by methods of mathematical morphology and its subtraction from the gel image, (ii) selection of an appropriate part of the gel lane including curved lanes and lanes with a nonuniform width, (iii) computation of the lane densitogram by averaging several lane-parallel scans, (iv) decomposition of the lane densitogram into component bands using a data selecting algorithm and Marquardt's minimizer. Several different functions for component bands are utilized. It is shown that the densitogram can be decomposed into component bands with reasonable accuracy only if an appropriate model function is chosen. The algorithms are tested on several different gel electrophoretograms which show typical features as a nonuniform background, curved lanes, an asymmetrical band shape and a superposition of small bands on the shoulders of big ones. It is shown that overlapped bands are best approximated by an asymmetrical Gausian curve and an asymmetrical Gauss-Cauchy function. Linear response to the serial dilution of the protein sample is tested.
- MeSH
- denzitometrie MeSH
- DNA bakterií analýza MeSH
- elektroforéza v agarovém gelu metody MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- Escherichia coli genetika MeSH
- matematika MeSH
- metoda nejmenších čtverců * MeSH
- molekulová hmotnost MeSH
- plazmidy MeSH
- počítačové zpracování obrazu * MeSH
- proteiny chemie MeSH
- ribozomální proteiny analýza MeSH
- Streptomyces aureofaciens chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA bakterií MeSH
- proteiny MeSH
- ribozomální proteiny MeSH
Microbial colony growth can serve as a useful readout in assays for studying complex genetic interactions or the effects of chemical compounds. Although computational tools for acquiring quantitative measurements of microbial colonies have been developed, their utility can be compromised by inflexible input image requirements, non-trivial installation procedures, or complicated operation. Here, we present the Spotsizer software tool for automated colony size measurements in images of robotically arrayed microbial colonies. Spotsizer features a convenient graphical user interface (GUI), has both single-image and batch-processing capabilities, and works with multiple input image formats and different colony grid types. We demonstrate how Spotsizer can be used for high-throughput quantitative analysis of fission yeast growth. The user-friendly Spotsizer tool provides rapid, accurate, and robust quantitative analyses of microbial growth in a high-throughput format. Spotsizer is freely available at https://data.csiro.au/dap/landingpage?pid=csiro:15330 under a proprietary CSIRO license.
- Klíčová slova
- bacteria, colony array, large-scale screen, microorganism, synthetic genetic array, yeast,
- MeSH
- algoritmy MeSH
- počítačové zpracování obrazu metody MeSH
- rychlé screeningové testy metody MeSH
- Schizosaccharomyces růst a vývoj MeSH
- software * MeSH
- Publikační typ
- časopisecké články MeSH
