There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

• MeSH
• Point Mutation genetics   physiology   MeSH
• Databases, Genetic MeSH
• Lyases chemistry   genetics   metabolism   MeSH
• Models, Molecular MeSH
• Computer Simulation MeSH
• Protein Engineering methods   MeSH
• Enzyme Stability genetics   MeSH
• Temperature MeSH
• Computational Biology methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Lurcher mutant mice of the C3H strain provide a model of both cerebellar and retinal degeneration. Therefore, they enable the study of the behavior of cerebellar mutants under disabled visual orientation conditions. We aimed to examine cerebellar Lurcher mutants and wild type mice with intact cerebella with and without retinal degeneration employing the rotarod and Morris water maze tests. The positions of the hidden platform and the starting point in the water maze test were stable so as to enable the use of both idiothetic navigation and visual inputs. The Lurcher mice evinced approximately 90 % shorter fall latencies on the rotarod than did the wild type mice. Retinal degeneration exerted no impact on motor performance. Only the wild type mice with normal retina were able to find the water maze platform efficiently. The wild type mice with retinal degeneration developed immobility (almost 25 % of the time) as a sign of behavioral despair. The Lurchers maintained high swimming activity as a potential manifestation of stress-induced behavioral disinhibition and their spatial performance was related to motor skills and swim speed. We demonstrated that both motor deficit and pathological behavior have the potential to contribute to abnormal performance in spatial tasks. Thus, spatial disability in cerebellar mutants is most likely a complex consequence of multiple disturbances related to cerebellar dysfunction.

• MeSH
• Maze Learning physiology   MeSH
• Retinal Degeneration genetics   pathology   MeSH
• Motor Skills physiology   MeSH
• Cerebellum pathology   MeSH
• Mice, Neurologic Mutants MeSH
• Mice, Inbred C3H MeSH
• Mice MeSH
• Neurodegenerative Diseases genetics   pathology   MeSH
• Blindness genetics   pathology   MeSH
• Space Perception physiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• MeSH
• Fusion Proteins, bcr-abl chemistry   genetics   MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics   pathology   MeSH
• DNA analysis   genetics   metabolism   MeSH
• Humans MeSH
• Polymerase Chain Reaction * MeSH
• Sequence Analysis, DNA * MeSH
• Comparative Genomic Hybridization MeSH
• High-Throughput Nucleotide Sequencing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot.

• MeSH
• Bacteria chemistry   enzymology   MeSH
• Databases, Protein MeSH
• Hydrolases chemistry   genetics   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Internet MeSH
• Protein Conformation, alpha-Helical MeSH
• Protein Conformation, beta-Strand MeSH
• Humans MeSH
• Models, Molecular MeSH
• Mutation * MeSH
• Protein Engineering methods   MeSH
• Protein Stability MeSH
• Thermodynamics MeSH
• User-Computer Interface * MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

"Genetics and Genomics in Medicine is a new textbook written for undergraduate and graduate students, as well as medical researchers, which explains the science behind the uses of genetics and genomics in medicine today. It is not just about rare inherited and chromosomal disorders, but how genetics affects the whole spectrum of human health and disease. DNA technologies are explained, with emphasis on the modern techniques that have revolutionized the use of genetic information in medicine and are indicating the role of genetics in common complex diseases. The detailed, integrative coverage of genetic approaches to treatment and prevention includes pharmacogenomics and the prospects for personalized medicine. Cancers are essentially genetic diseases and are given a dedicated chapter that includes new insights from cancer genome sequencing. Clinical disorders are covered throughout and there are extensive end-of-chapter questions and problems"--Provided by publisher.

The regulation of the human apolipoprotein (apo) B gene that plays a crucial role in lipid metabolism is apparently very complex, with multiple cis- and trans-acting regulatory factors. One of these factors is an enhancer region in the second intron. In this region a point mutation at position + 722 has been found that is detectable by the restriction enzyme StyI. The report of Levy-Wilson et al. (1991) could suggest that the mutant allele (abolished StyI site) is associated with hypocholesterolemia. To investigate further the possible effect of this mutation on plasma cholesterol levels, we have compared the frequency of the mutant allele between 206 hypercholesterolemic Norwegian or Czech subjects on one hand, and 165 hypocholesterolemic Norwegian or Czech subjects on the other hand. No significant difference in frequency was found between the hypercholesterolemic and the hypocholesterolemic groups. This finding indicates either that the mutation at position + 722 does not affect the enhancer activity or that this in vitro enhancer activity is of little or no clinical significance. One of the Norwegian hypercholesterolemic subjects who was of Czech descent possessed the apoB 3500 mutation that leads to defective binding of low density lipoprotein (LDL) to the LDL receptors. Haplotype analysis of the apoB gene in her family showed that the mutation-bearing allele was identical to that reported in other countries, indicating a common gene source.

• MeSH
• Apolipoproteins B * genetics   MeSH
• Point Mutation MeSH
• Cholesterol * deficiency   MeSH
• Child MeSH
• Adult MeSH
• Gene Frequency MeSH
• Genetic Markers MeSH
• Haplotypes MeSH
• Hypercholesterolemia * genetics   MeSH
• Introns * genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Chromosomes, Human, Pair 2 MeSH
• Molecular Sequence Data MeSH
• Polymerase Chain Reaction MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Deoxyribonucleases, Type II Site-Specific MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Aged MeSH
• Enhancer Elements, Genetic * genetics   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Geographicals
• Czechoslovakia MeSH
• Norway MeSH

The regulation of the human apolipoprotein (apo) B gene that plays a crucial role in lipid metabolism is apparently very complex, with multiple cis- and trans-acting regulatory factors. One of these factors is an enhancer region in the second intron. In this region a point mutation at position + 722 has been found that is detectable by the restriction enzyme StyI. The report of Levy-Wilson et al. (1991) could suggest that the mutant allele (abolished StyI site) is associated with hypocholesterolemia. To investigate further the possible effect of this mutation on plasma cholesterol levels, we have compared the frequency of the mutant allele between 206 hypercholesterolemic Norwegian or Czech subjects on one hand, and 165 hypocholesterolemic Norwegian or Czech subjects on the other hand. No significant difference in frequency was found between the hypercholesterolemic and the hypocholesterolemic groups. This finding indicates either that the mutation at position + 722 does not affect the enhancer activity or that this in vitro enhancer activity is of little or no clinical significance. One of the Norwegian hypercholesterolemic subjects who was of Czech descent possessed the apoB 3500 mutation that leads to defective binding of low density lipoprotein (LDL) to the LDL receptors. Haplotype analysis of the apoB gene in her family showed that the mutation-bearing allele was identical to that reported in other countries, indicating a common gene source.

• MeSH
• Apolipoproteins B genetics   MeSH
• Point Mutation MeSH
• Cholesterol deficiency   MeSH
• Child MeSH
• Adult MeSH
• Gene Frequency MeSH
• Genetic Markers MeSH
• Haplotypes MeSH
• Hypercholesterolemia genetics   MeSH
• Introns genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Chromosomes, Human, Pair 2 MeSH
• Molecular Sequence Data MeSH
• Polymerase Chain Reaction MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Deoxyribonucleases, Type II Site-Specific MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Aged MeSH
• Enhancer Elements, Genetic genetics   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Geographicals
• Czechoslovakia MeSH
• Norway MeSH

Východiská: Odpoveďou onkoproteínu p53 na poškodenie DNA je indukcia zastavenia bunkového delenia (apoptózy). Mutácia génu TP53 (gén, ktorý kóduje proteín p53) je veľmi častá v humánnych malignitách a mení pôsobenie proteínu p53. Výsledkom je stabilná forma p53, ktorá sa hromadí v jadrách nádorových buniek súčasne so stratou jeho funkcie. Mutovaná p53 je stabilizovaná a ľahko imunohistochemicky (IHC) detegovateľná. Hladina p53 je v zdravých bunkách výrazne determinovaná proteínom MDM2 (murine double minute‑2). Tento spolupôsobí pri degradácii proteínu p53, a zároveň ho považujeme za potenciálny marker funkčnosti p53. Nadexpresia proteínu MDM2 predstavuje mechanizmus, ktorý môže počas tumorigenézy rušiť funkciu p53. Súbor a metódy: Stotridsaťšesť vzoriek karcinómu pľúc sme získali od pacientov, ktorí podstúpili radikálnu resekciu (lobektómiu alebo pulmonektómiu a lymfadektómiu). Patologická dia­gnóza bola stanovená na základe WHO kritérií. V našej štúdii sme sledovali expresiu proteínu p53 a proteínu MDM2, ktorý môže slúžiť ako IHC marker pre p53 status. Oba proteíny sme imunohistochemicky detegovali. Stanovili sme ich expresiu, ďalej sme porovnali ich výskyt s klinicko‑patologickými parametrami. Výsledky: Vo vzorkách p53 a MDM2 pozitívnych sme pozorovali silne tmavohnedo sfarbené jadrá nádorových buniek. Väčšina p53 pozitívnych vzoriek predstavovala skvamocelulárny typ (55 %), nasledovali vzorky veľkobunkového typu (53 %) a 26 % p53 pozitívnych vzoriek predstavoval adenokarcinóm. Ani jedna vzorka z iných typov neexprimovala p53. Ďalej sme porovnali výskyt p53 a stupeň malignity nádoru (grade). Zistili sme, že expresia p53 stúpa so stupňom malignity nádoru. Na štatistické vyhodnotenie sme použili chi‑kvadrátový test, ktorým sme dokázali štatistickú významnosť medzi expresiou p53 na jednej strane, a typom nádoru a stupňom malignity na strane druhej (p = 0,000425; p = 0,00157). S ohľadom na expresiu p53 a MDM2 sme zistili nasledovné: len deväť vzoriek bolo simultánne p53 a MDM2 pozitívnych. V 46 prípadoch (34 %) zvýšená hladina p53 bola kombinovaná s MDM2 negativitou. Ostatné vzorky boli buď negatívne na oba proteíny (71/52 %), alebo p53 negatívne a MDM2 pozitívne v počte 10/7 %. Záver: Vo väčšine štúdií je absencia p53 imunoreaktivity dávaná do súvisu s absenciou p53 mutácií. Platí to aj naopak, a síce, pozitívna expresia p53 je považovaná za znak mutácie p53, ktorý súvisí so stratou funkcie. V našej štúdii 34 % prípadov vykazovalo vysokú hladinu p53 bez expresie MDM2. Tieto nádory považujeme za tumory s inaktivujúcimi mutáciami, ktoré stabilizujú proteín p53. Na druhej strane nádory, ktoré exprimujú vysoké hladiny stabilnej p53 a sú súčasne pozitívne na MDM2 (v našom prípade 9 vzoriek/7 %) predstavujú skupinu s funkčnou p53. U tejto skupiny pacientov by sme mohli očakávať lepšiu prognózu. Kľúčové slová: onkoproteín p53 – MDM2 proteín – karcinóm pľúc – imunohistochémia

Background: The oncoprotein p53 protein induces cell growth arrest (apoptosis) in response to endo‑ or exogenous stimuli. Mutation of TP53 (gene encoding the p53 protein) is common in human malignancies and alters the conformation of p53. The result is a more stable protein which accumulates in nuclei of tumor cells with loss of function. Mutant p53 is stabilized, and it is possible to detect this form very clearly by immunohistochemistry (IHC). Expression of the MDM2 protein is used as a potential marker of p53 function. P53 levels in normal cells are highly determined by the MDM2 protein (murine double minute‑2) – mediated degradation of p53. MDM2 overexpression represents at least one mechanism by which p53 function can be abrogated during tumorigenesis. Material and Methods: Lung carcinoma samples were obtained from patients, who underwent radical resection (lobectomy or pulmonectomy and lymphadectomy). Pathological dia­gnosis was based on the WHO criteria. In our study, we investigated the expression of p53 and MDM2 protein that might improve IHC as a marker for p53 status. Proteins were IHC detected in 136 samples of primary lung carcinoma. Immunostaining results of p53 positive samples were compared to IHC expression of MDM2 positive and MDM2 negative samples. Results: Strong brown nuclear staining was visible in p53 and MDM2 positive cells. The most p53 positive cases were samples of squamocellular carcinoma (55%), then samples of large cell carcinoma (53%) and 26% adenocarcinoma samples showed the p53 immunoreactivity. No one sample of different types was p53 positive. When we compared the p53 expression and grade of tumor, we found that the p53 expression increased with the grade of tumor. For statistical evaluation, the chi‑square test was used. The relationship between p53 expression and type of tumor, also the p53 expression and grade of tumor was statistically significant (p = 0.000425; p = 0.00157). Regarding p53 and MDM2 expression, only nine samples (7%) were simultaneously p53 and MDM2 positive. In 46 (34%) cases, elevation of p53 was combined with MDM2 negative expression. Other tumor samples were either negative for both proteins (71/52%), or p53 negative and MDM2 positive in 10 (7%) tumor samples. Conclusion: Absence of p53 staining in most studies indicates absence of p53 mutation, and on the contrary, positive expression of p53 is a sign of p53 mutations with loss of function. In our study, 34% of cases with extensively high level of p53 without increased level of MDM2 were identified. We suppose that these are tumors with inactivating mutations that stabilize p53. On the other hand, tumors with high level of stabilized wild‑type p53 protein and simultaneously with increased MDM2 staining (9 samples/7%) represent group with functional p53. In this group of patients, we could expect better prognosis with regard to function of p53 protein. Key words: oncoprotein p53 – MDM2 protein – lung cancer – immunohistochemistry This work was supported by the Grant Agency of the Ministry of Education of the Slovak Republic VEGA: 1/0224/12; 1/0925/11; 1/0928/11. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 13. 6. 2013 Accepted: 23. 7. 2013

• MeSH
• Point Mutation MeSH
• Gene Expression * MeSH
• Genes, p53 * physiology   genetics   MeSH
• Histology MeSH
• Immunohistochemistry MeSH
• Laboratories MeSH
• Humans MeSH
• Microscopy MeSH
• Antibodies, Monoclonal MeSH
• Biomarkers, Tumor MeSH
• Lung Neoplasms MeSH
• Proto-Oncogene Proteins c-mdm2 * physiology   genetics   MeSH
• Neoplasm Staging MeSH
• Statistics as Topic MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Cell Biology MeSH
• Genetic Predisposition to Disease MeSH
• Molecular Biology MeSH
• Neoplastic Processes MeSH
• Neoplasms MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Publication type
• monografie

Wound healing is a process regulated by a complex interaction of multiple growth factors including fibroblast growth factor 2 (FGF2). Although FGF2 appears in several tissue engineered studies, its applications are limited due to its low stability both in vitro and in vivo. Here, this shortcoming is overcome by a unique nine-point mutant of the low molecular weight isoform FGF2 retaining full biological activity even after twenty days at 37 °C. Crosslinked freeze-dried 3D porous collagen/chitosan scaffolds enriched with this hyper stable recombinant human protein named FGF2-STAB® were tested for in vitro biocompatibility and cytotoxicity using murine 3T3-A31 fibroblasts, for angiogenic potential using an ex ovo chick chorioallantoic membrane assay and for wound healing in vivo with 3-month old white New Zealand rabbits. Metabolic activity assays indicated the positive effect of FGF2-STAB® already at very low concentrations (0.01 µg/mL). The angiogenic properties examined ex ovo showed enhanced vascularization of the tested scaffolds. Histological evaluation and gene expression analysis by RT-qPCR proved newly formed granulation tissue at the place of a previous skin defect without significant inflammation infiltration in vivo. This work highlights the safety and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® protein. Moreover, these sponges could be used as scaffolds for growing cells for dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration.

• Publication type
• Journal Article MeSH