complex matrix samples
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Candida haemulonii complex (Candida haemulonii [I], Candida duobushaemulonii [II], and Candida haemulonii var. vulnera [III]) has become relevant in recent times, not so much because of a high incidence in human clinical sample cultures but because of its remarkable antifungal resistance. The objective of this study was to evaluate several methods for the identification of this uncommon species of Candida. Ten isolates of C. haemulonii were identified by biochemical and proteomic methods, and their antifungal susceptibility testing was performed by both commercial and reference methods. MALDI-TOF MS (Vitek MS and Vitek MS PRIME) and Vitek2 correctly identified these genera but API method did not. There was a good correlation between the commercial methods and the reference methods for the AST. In conclusion Vitek MS, Vitek MS PRIME, and Vitek2 systems, but not API32C, are reliable for identification of C. haemulonii complex. Furthermore, MALDI-TOF MS systems could identify to the subspecies level. Commercial methods for antifungal susceptibility testing are valid for the study of this species and confirm amphotericin B and to azole resistance.
Matrix metalloproteinases (MMP)-2 and MMP-9 play important roles in inflammation as well as in pain processes. For this reason, we compared the concentrations of these enzymes in skin and serum of patients with complex regional pain syndrome (CRPS), other pain diseases and healthy subjects. We analyzed ipsi- and contralateral skin biopsies of 18 CRPS patients, as well as in 10 pain controls and 9 healthy subjects. Serum samples were analyzed from 20 CRPS, 17 pain controls and 17 healthy subjects. All samples were analyzed with ELISA. Concentrations were then compared to clinical data as well as to quantitative sensory testing data.MMP-2 was increased in both ipsi- and contralateral skin biopsies of CRPS patients compared to healthy subjects. While low ipsilateral MMP-2 was associated with trophic changes, contralateral MMP-2 inversely correlated with the CRPS severity. MMP-9 was also locally increased in ipsilateral CRPS skin, and higher ipsi- and contralateral MMP-9 levels correlated with CRPS severity. We conclude that MMP-2 and MMP-9 are differently expressed depending on the clinical phenotype in CRPS. PERSPECTIVE: This article describes an upregulation of MMPs in CRPS and pain controls and shows different expression of MMP-2 and -9 depending on clinical phenotype in CRPS. These results provide evidence that MMP-2 and -9 play a key role in CRPS pathophysiology.
- MeSH
- biopsie MeSH
- dospělí MeSH
- komplexní regionální syndromy bolesti metabolismus patofyziologie MeSH
- kůže MeSH
- lidé středního věku MeSH
- lidé MeSH
- matrixová metaloproteinasa 2 metabolismus MeSH
- matrixová metaloproteinasa 9 metabolismus MeSH
- stupeň závažnosti nemoci MeSH
- zánět metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
A method for the fast isolation, propagation, and characterization of very low count bacteriophages active against pathogenic bacterial strains is described in this study. Bacteriophages with a count of 102 phage particles were dynamically adhered from the maximum 10 mL blood plasma sample onto the nanostructured part of the fused silica capillary. One-step propagation of phage particles of genus Kayvirus inside the etched capillary on 104Staphylococcus aureus host cells increased their number to 6 × 104 phage particles. Phage particles were concentrated online and separated by capillary electrophoretic methods. No phage replication occurred when the phage-resistant S. aureus or Escherichia coli cells were used. Two-step phage propagation in the capillary allowed an increase in the total virion count to up to 6 × 105 phage particles and subsequent off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the phage zone collected after capillary electrophoresis. Relative standard deviations of the phage peak area were at most 2.3%. We expect that the method of isolating bacteriophages from blood plasma and their simultaneous identification will facilitate clinical studies of phage preparations and contribute to pharmacokinetics studies during phage therapy. This approach is also suitable for capturing and enriching new phages from the environment when a susceptible indicator strain is available.
- MeSH
- lidé MeSH
- methicilin rezistentní Staphylococcus aureus * MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- stafylokokové bakteriofágy genetika MeSH
- stafylokokové infekce * MeSH
- Staphylococcus aureus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MALDI-TOF MS is currently becoming the method of choice for rapid identification of bacterial species in routine diagnostics. Yet, this method suffers from the inability to differentiate reliably between some closely related bacterial species including those of the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex, namely A. baumannii and Acinetobacter nosocomialis. In the present study, we evaluated a protocol which was different from that used in the Bruker Daltonics identification system (MALDI BioTyper) to improve species identification using a taxonomically precisely defined set of 105 strains representing the four validly named species of the ACB complex. The novel protocol is based on the change in matrix composition from alpha-cyano-4-hydroxycinnamic acid (saturated solution in water:acetonitrile:trifluoroacetic acid, 47.5:50:2.5, v/v) to ferulic acid (12.5mgml(-1) solution in water:acetonitrile:formic acid 50:33:17, v/v), while the other steps of sample processing remain unchanged. Compared to the standard protocol, the novel one extended the range of detected compounds towards higher molecular weight, produced signals with better mass resolution, and allowed the detection of species-specific signals. As a result, differentiation of A. nosocomialis and A. baumannii strains by cluster analysis was improved and 13 A. nosocomialis strains, assigned erroneously or ambiguously by using the standard protocol, were correctly identified.
- MeSH
- Acinetobacter chemie klasifikace izolace a purifikace MeSH
- bakteriologické techniky metody MeSH
- odběr biologického vzorku metody MeSH
- shluková analýza MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro-proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non-contracted tissue (lateral side; n = 13). We used label-free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin-C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
- MeSH
- dítě MeSH
- extracelulární matrix - proteiny metabolismus MeSH
- hmotnostní spektrometrie MeSH
- kalcinóza MeSH
- lidé MeSH
- pes equinovarus etiologie metabolismus MeSH
- předškolní dítě MeSH
- prospektivní studie MeSH
- proteom MeSH
- transformující růstový faktor beta metabolismus MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a complex role in the pathogenesis of atherosclerosis. We compared (1) the histopathological findings in patients with abdominal aortic aneurysms (AAA) and aortoiliac occlusive disease (AOD); (2) the expression of MMP-2/MMP-9 and TIMP-1/TIMP-2 in aortic layers, inflammatory cells and smooth muscle cells (SMCs), aiming to identify the common underlying pathogenic mechanisms of the disease development. Samples were obtained from 30 patients with AAA and 30 with AOD. Aortic histology and immunohistochemistry were performed to evaluate inflammatory changes and MMP and TIMP expression. Thrombosis and ulceration were more frequent in AOD than in AAA. The MMP-9 expression was elevated in all aortic layers of AAA patients and in media/adventitia of AOD patients, mainly followed by lower expression of its inhibitor TIMP-1. Higher MMP-9 expression was also found in SMCs and macrophages of both AAA and AOD specimens, while higher TIMP-1/TIMP-2 were predominantly observed in the lymphocytes and macrophages of the aneurysm. These results showed that both conditions exhibited increased MMP-9 expression; however, the MMP expression pattern differed to some degree between the aneurysms and occlusive disease. The variations in molecular mechanisms underlying dilatative/stenosing disease warrant further investigation.
- MeSH
- aneurysma břišní aorty metabolismus MeSH
- arteriální okluzní nemoci metabolismus MeSH
- imunohistochemie MeSH
- lidé MeSH
- matrixová metaloproteinasa 9 metabolismus MeSH
- matrixové metaloproteinasy metabolismus MeSH
- myocyty hladké svaloviny metabolismus MeSH
- nemoci aorty metabolismus MeSH
- tkáňový inhibitor metaloproteinasy 1 metabolismus MeSH
- tkáňový inhibitor metaloproteinasy 2 metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In this study we present an innovative method for the preparation of fully hydrated samples of microbial biofilms of cultures Staphylococcus epidermidis, Candida parapsilosis and Candida albicans. Cryo-scanning electron microscopy (cryo-SEM) and high-pressure freezing (HPF) rank among cutting edge techniques in the electron microscopy of hydrated samples such as biofilms. However, the combination of these techniques is not always easily applicable. Therefore, we present a method of combining high-pressure freezing using EM PACT2 (Leica Microsystems), which fixes hydrated samples on small sapphire discs, with a high resolution SEM equipped with the widely used cryo-preparation system ALTO 2500 (Gatan). Using a holder developed in house, a freeze-fracturing technique was applied to image and investigate microbial cultures cultivated on the sapphire discs. In our experiments, we focused on the ultrastructure of the extracellular matrix produced during cultivation and the relationships among microbial cells in the biofilm. The main goal of our investigations was the detailed visualization of areas of the biofilm where the microbial cells adhere to the substrate/surface. We show the feasibility of this technique, which is clearly demonstrated in experiments with various freeze-etching times.
- MeSH
- biofilmy MeSH
- Candida albicans ultrastruktura MeSH
- Candida parapsilosis ultrastruktura MeSH
- elektronová kryomikroskopie metody MeSH
- extracelulární matrix ultrastruktura MeSH
- mikroskopie elektronová rastrovací metody MeSH
- mrazové lámání metody MeSH
- Staphylococcus epidermidis ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Direct analysis of complex samples is demonstrated by the at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to capillary electrophoresis (CE). The hyphenation of the preparative and the analytical technique is achieved through a 3D-printed microextraction device with an HF located in a sample vial of a commercial CE instrument. The internal geometry of the device guides the CE separation capillary into the HF and the CE injection of the HF-LPME extract is performed directly from the HF lumen. The 3D-printing process ensures uniform dimensions of the devices, their constant position inside the sample vial, and excellent repeatability of the HF-LPME as well as the CE injection. The devices are cheap (∼0.01 €) and disposable, thus eliminating any possible sample-carryover, moreover, the at-line CE analysis of the extract is performed fully autonomously with no need for operator's intervention. The developed HF-LPME/CE-UV method is applied to the determination of acidic drugs in dried blood spot and wastewater samples and is characterized by excellent repeatability (RSD, 0.6-9.6%), linearity (r2, 0.9991-0.9999), enrichment (EF, 29-97), sensitivity (LOD, 0.2-3.4 μg/L), and sample throughput (7 samples/h). A further improvement of selected characteristics of the analytical method is achieved by the at-line coupling of HF-LPME to capillary isotachophoresis (ITP) with electrospray ionization-mass spectrometry (ESI-MS). The HF-LPME/ITP-ESI-MS system facilitates enhanced selectivity, matrix-free analytical signals, and up to 34-fold better sensitivity due to the use of ESI-MS detection and additional on-capillary ITP preconcentration of the HF-LPME extracts.
Monitoring the selectivity of supported liquid membranes (SLMs) is of paramount importance since the amount and type of compounds that are transferred across a SLM directly influence the transfer efficiency, reproducibility and accuracy. To apply a correct SLM in particular sample pretreatment, rapid determination of the transfer of analytes and matrix compounds across the SLM is necessary, which requires the use of an analytical method with universal detection technique. Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C(4)D) has proven to be a useful tool for the determination of SLM selectivity. Background electrolyte solution consisting of 1M acetic acid (pH 2.4) was used for simultaneous separation and detection of three basic drugs (nortriptyline, haloperidol and loperamide) and major matrix components (inorganic cations, proteins, amino acids, etc.) after electromembrane extraction (EME) of standard solutions and complex samples. The CE-C(4)D method has evidenced for the first time that large proteins, such as human serum albumin, are efficiently retained on all examined SLMs and that transfer of other matrix components and the analytes is strongly SLM dependent. Excellent transfer of the analytes was achieved across SLMs impregnated with 2-nitrophenyl octyl ether (NPOE) or 1-ethyl-2-nitrobenzene, however, an increased co-extraction of interfering matrix components, which disabled quantitative determination of haloperidol with the current CE-C(4)D setup, was observed for the latter. After addition of a commonly used ion carrier (bis(2-ethylhexyl)phosphate) to NPOE, a wide range of matrix components were transferred across the SLM with no measurable transfer of the analytes. Best selectivity regarding transfer of the basic drugs and elimination of matrix components was obtained using SLM impregnated with NPOE. An optimized EME-CE-C(4)D method was used to determine the basic drugs in various samples and satisfactory analytical parameters were obtained.
- MeSH
- adsorpce MeSH
- aminokyseliny analýza izolace a purifikace MeSH
- elektroforéza kapilární přístrojové vybavení metody MeSH
- extrakce na pevné fázi přístrojové vybavení metody MeSH
- léčivé přípravky analýza izolace a purifikace MeSH
- lidé MeSH
- membrány umělé MeSH
- proteiny analýza izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cellular processes such as tissue regeneration, inflammation, and migration require the proteolysis of the extracellular matrix and the proteolytic activation of signaling molecules. A widely used and accessible technique for studying these processes is gelatin zymography, particularly for investigating matrix metalloproteinases (MMPs), though it is not limited to them. This method is favored for its simplicity, low cost, and robustness. Despite certain limitations, it remains a preferred approach for the initial investigation of complex samples.Here, we present a protocol applicable to various sample sources, including proteases from human cell lines and bacteria isolated from chronic wounds. We also explore changes in protease activity within exudates from human chronic wounds, a challenging analysis for more complex techniques. Additionally, we emphasize the potential to extend the basic protocol to study the conditions under which proteases are active.
