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1.
Front Cell Infect Microbiol ; 11: 634215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34381737

RESUMO

Bloodstream infections (BSIs), the presence of microorganisms in blood, are potentially serious conditions that can quickly develop into sepsis and life-threatening situations. When assessing proper treatment, rapid diagnosis is the key; besides clinical judgement performed by attending physicians, supporting microbiological tests typically are performed, often requiring microbial isolation and culturing steps, which increases the time required for confirming positive cases of BSI. The additional waiting time forces physicians to prescribe broad-spectrum antibiotics and empirically based treatments, before determining the precise cause of the disease. Thus, alternative and more rapid cultivation-independent methods are needed to improve clinical diagnostics, supporting prompt and accurate treatment and reducing the development of antibiotic resistance. In this study, a culture-independent workflow for pathogen detection and identification in blood samples was developed, using peptide biomarkers and applying bottom-up proteomics analyses, i.e., so-called "proteotyping". To demonstrate the feasibility of detection of blood infectious pathogens, using proteotyping, Escherichia coli and Staphylococcus aureus were included in the study, as the most prominent bacterial causes of bacteremia and sepsis, as well as Candida albicans, one of the most prominent causes of fungemia. Model systems including spiked negative blood samples, as well as positive blood cultures, without further culturing steps, were investigated. Furthermore, an experiment designed to determine the incubation time needed for correct identification of the infectious pathogens in blood cultures was performed. The results for the spiked negative blood samples showed that proteotyping was 100- to 1,000-fold more sensitive, in comparison with the MALDI-TOF MS-based approach. Furthermore, in the analyses of ten positive blood cultures each of E. coli and S. aureus, both the MALDI-TOF MS-based and proteotyping approaches were successful in the identification of E. coli, although only proteotyping could identify S. aureus correctly in all samples. Compared with the MALDI-TOF MS-based approaches, shotgun proteotyping demonstrated higher sensitivity and accuracy, and required significantly shorter incubation time before detection and identification of the correct pathogen could be accomplished.


Assuntos
Bacteriemia , Infecções Estafilocócicas , Bacteriemia/diagnóstico , Candida albicans , Escherichia coli , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus
2.
Proc Natl Acad Sci U S A ; 118(28)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34244434

RESUMO

This article presents the beginning of a metric functional analysis. A major notion is metric functionals which extends that of horofunctions in metric geometry. Applications of the main tools are found in a wide variety of subjects such as random walks on groups, complex dynamics, surface topology, deep learning, evolution equations, and game theory, thus branching well outside of pure mathematics. In several cases, linear notions fail to describe linear phenomena that are naturally captured by metric concepts. An extension of the mean ergodic theorem testifies to this. A general metric fixed-point theorem is also proved.

3.
Sci Adv ; 7(16)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33863724

RESUMO

Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.

4.
PLoS One ; 15(5): e0232397, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365096

RESUMO

Biometric ratios of the relative length of the rays in the hand have been analyzed between primate species in the light of their hand function or phylogeny. However, how relative lengths among phalanges are mechanically linked to the grasping function of primates with different locomotor behaviors remains unclear. To clarify this, we calculated cross and triple-ratios, which are related to the torque distribution, and the torque generation mode at different joint angles using the lengths of the phalanges and metacarpal bones in 52 primates belonging to 25 species. The torque exerted on the finger joint and traction force of the flexor tendons necessary for a cylindrical grip and a suspensory hand posture were calculated using the moment arm of flexor tendons measured on magnetic resonance images, and were compared among Hylobates spp., Ateles sp., and Papio hamadryas. Finally, the torques calculated from the model were validated by a mechanical study detecting the force exerted on the phalanx by pulling the digital flexor muscles during suspension in these three species. Canonical discriminant analysis of cross and triple-ratios classified primates almost in accordance with their current classification based on locomotor behavior. The traction force was markedly reduced with flexion of the MCP joint parallel to the torque in brachiating primates; this was notably lower in the terrestrial quadrupedal primates than in the arboreal primates at mild flexion. Our mechanical study supported these features in the torque and traction force generation efficiencies. Our results suggest that suspensory or terrestrial quadrupedal primates have hand structures that can exert more torque at a suspensory posture, or palmigrade and digitigrade locomotion, respectively. Furthermore, our study suggests availability of the cross and triple-ratios as one of the indicators to estimate the hand function from the skeletal structure.


Assuntos
Mãos/anatomia & histologia , Mãos/fisiologia , Locomoção/fisiologia , Primatas/anatomia & histologia , Primatas/fisiologia , Animais , Atelinae/anatomia & histologia , Atelinae/fisiologia , Fenômenos Biomecânicos , Falanges dos Dedos da Mão/anatomia & histologia , Falanges dos Dedos da Mão/diagnóstico por imagem , Falanges dos Dedos da Mão/fisiologia , Análise de Elementos Finitos , Mãos/diagnóstico por imagem , Força da Mão/fisiologia , Humanos , Hylobates/anatomia & histologia , Hylobates/fisiologia , Imageamento por Ressonância Magnética , Ossos Metacarpais/anatomia & histologia , Ossos Metacarpais/diagnóstico por imagem , Ossos Metacarpais/fisiologia , Fenômenos Fisiológicos Musculoesqueléticos , Sistema Musculoesquelético/anatomia & histologia , Papio hamadryas/anatomia & histologia , Papio hamadryas/fisiologia , Especificidade da Espécie , Tomografia Computadorizada por Raios X , Torque
5.
Prev Vet Med ; 177: 104959, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32240886

RESUMO

By avoiding animal transportation, mobile slaughter may have the potential to reduce animal stress. In a cross-sectional study with elements of cohort design, we investigated relationships between animal handling and stress-related animal behaviours in connection with slaughter at two Swedish slaughter plants: a newly started small-scale on-farm mobile abattoir and a relatively large-scale stationary slaughterhouse. To the stationary plant, the animals were transported on average 99 km from farms, and one third of these animals spent one night in lairage before slaughter. Data were collected during processing of 298 animals at both plants during one year. Stockperson actions and animal behaviours were observed in the driveways (2.4-7.3 m long) to the stun box. Data on season, hour of day, air temperature, animal breed, animal category, animal age, carcass weight and stockperson category (plant or farm) were also collected. We used Spearman rank correlation, principal-component analysis and confirmatory factor analysis to develop a final generalised structural equation model, which contained six variables that represented observed animal backing; turning; slipping; eliminating; vocalising; and violent behaviour, regressed on a latent variable representing animal stress level. Stress level and animal behaviours were also regressed on five variables representing observed stockperson actions (touching, patting or hitting with hand; touching, patting or hitting with a tool; pushing; tail-twisting; and prodding with an electric goad) and on background variables including plant identity. The animal behaviours were modelled as counts per driveway length, clustered on farm identity. Marginal effects of stockperson actions and predictive margins of plants were calculated. The animals displayed backing and violent behaviour (kicking, goring or violent fighting) at significantly lower frequencies at the mobile plant than at the stationary one. In general, stockperson actions were significantly positively associated with animal behaviours, i.e. increased actions were associated with more frequent behaviours. Stockperson moving with tool, pushing and using electric goad were indirectly significantly associated with all animal behaviours via animal stress level. This study shows the importance of adequate cattle handling to limit pre-slaughter stress.


Assuntos
Matadouros/estatística & dados numéricos , Criação de Animais Domésticos/métodos , Bem-Estar do Animal , Bovinos/fisiologia , Estresse Fisiológico , Animais , Bovinos/psicologia , Estudos Transversais , Feminino , Masculino , Suécia , Transportes
6.
BMC Microbiol ; 20(1): 80, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32264835

RESUMO

BACKGROUND: The skin commensal Staphylococcus haemolyticus is an emerging nosocomial pathogen. Despite its clinical relevance, published information about S. haemolyticus virulence factors is scarce. In this study, the adhesive and biofilm forming properties of ten clinical and ten commensal S. haemolyticus strains were examined using standard adhesion and biofilm assays. One of the clinical strains was used to identify expressed surface proteins using bacterial surface shaving. Protein abundance was examined by a comparative analysis between bacterial protein expression after human keratinocyte (HaCaT) colonization and growth in cell culture media supplemented with serum. Relative protein quantification was performed by labeling peptides with tandem mass tags (TMT) prior to Mass Spectrometry analysis. Surface proteins can be used as novel targets for antimicrobial treatment and in diagnostics. RESULTS: Adherence to fibronectin, collagen and plastic was low in all tested strains, but with significantly higher adhesion to fibronectin (p = 0.041) and collagen (p = 0.001) in the commensal strains. There was a trend towards higher degree of biofilm formation in the clinical strains (p = 0.059). By using surface shaving, 325 proteins were detected, of which 65 were classified as surface proteins. Analyses showed that the abundance of nineteen (5.8%) proteins were significantly changed following HaCaT colonization. The bacterial Toll/interleukin-1 like (TIRs) domain containing protein (p = 0.04), the transglycosylase SceD (p = 0.01), and the bifunctional autolysin Atl (p = 0.04) showed a 1.4, 1.6- and 1.5-fold increased abundance. The staphylococcal secretory antigen (SsaA) (p = 0.04) was significantly downregulated (- 1.5 fold change) following HaCaT colonization. Among the 65 surface proteins the elastin binding protein (Ebps), LPXAG and LPXSG domain containing proteins and five LPXTG domain containing proteins were identified; three Sdr-like proteins, the extracellular matrix binding protein Embp and a SasH-like protein. CONCLUSIONS: This study has provided novel knowledge about expression of S. haemolyticus surface proteins after direct contact with eukaryotic cells and in media supplemented with serum. We have identified surface proteins and immune evasive proteins previously only functionally described in other staphylococcal species. The identification of expressed proteins after host-microbe interaction offers a tool for the discovery and design of novel targets for antimicrobial treatment.


Assuntos
Técnicas Bacteriológicas/métodos , Proteínas de Membrana/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus haemolyticus/classificação , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Colágeno/metabolismo , Fibronectinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Espectrometria de Massas , Plásticos/química , Staphylococcus haemolyticus/crescimento & desenvolvimento , Staphylococcus haemolyticus/isolamento & purificação , Staphylococcus haemolyticus/patogenicidade , Simbiose
7.
Mol Cell Proteomics ; 19(3): 518-528, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31941798

RESUMO

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.


Assuntos
Proteínas de Bactérias/metabolismo , Haemophilus influenzae/metabolismo , Moraxella catarrhalis/metabolismo , Peptídeos/metabolismo , Staphylococcus aureus/metabolismo , Streptococcus pneumoniae/metabolismo , Biomarcadores/metabolismo , Haemophilus influenzae/isolamento & purificação , Humanos , Moraxella catarrhalis/isolamento & purificação , Sistema Respiratório/microbiologia , Infecções Respiratórias/microbiologia , Especificidade da Espécie , Staphylococcus aureus/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Espectrometria de Massas em Tandem
8.
J Sep Sci ; 43(4): 696-707, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31724311

RESUMO

An updated and improved method for analysis of omeprazole/esomeprazole and related substances on core-shell columns was developed using Fusion LC Method Development™. The method was optimized with respect to column type, column temperature, mobile phase pH level, and gradient time. Four different core-shell columns were examined to develop a method suitable for both high performance- and ultra-high performance liquid chromatography using a Quality by Design approach. The final method offers two alternative columns: Poroshell EC C18 (3.0 × 100 mm, 2.7 µm) or Poroshell HPH (3.0 × 100 mm, 2.7 µm) with the same gradient elution condition and mobile phase composition. Total run time is 18 min with 12 min of gradient elution. Phosphate buffer (15 mM, pH 7.8) is selected as the aqueous mobile phase and acetonitrile as the organic mobile phase. Column temperature is set at 40°C and ultraviolet detection at 302 nm. Furthermore, by studying parameters in a systematic way, an understanding of the effect of the input parameters enhances the method robustness and should allow for regulatory flexibility in terms of post-approval changes. Compared to the current United States Pharmacopeia method, the updated method is faster, more efficient and performs well above acceptance criteria.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Esomeprazol/isolamento & purificação , Omeprazol/isolamento & purificação , Cromatografia Líquida de Alta Pressão/instrumentação , Esomeprazol/análise , Omeprazol/análise , Temperatura
9.
Eur J Radiol ; 114: 185-191, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31005171

RESUMO

PURPOSE: The Pre-Operative MRI of the Breast (POMB) trial was a randomized, prospective, multicenter trial evaluating the impact of pre-operative breast MRI on treatment regimens and short-term surgical outcomes in women up to 56 years of age with breast cancer. The purpose of this study was to evaluate the performance of pre-operative breast MRI in the POMB trial with respect to incremental MRI findings - over conventional breast imaging methods - and their concordance with histopathology. PATIENTS AND METHODS: Two-hundred and ten patients (n = 210) participating in the POMB trial underwent pre-operative breast MRI at two Swedish breast units. Positive predictive values (PPV) for the incremental MRI findings were calculated for three subgroups of patients with: 1. alteration/alterations of treatment plan; 2. no alteration of treatment plan; and, 3. MRI-related conversion from BCS to mastectomy. Area under the receiver operating characteristic curve (AUC) was calculated using in-breast BI-RADS based ratings for the whole MRI group. RESULTS: After exclusions a total number of 99 incremental findings in 78 patients were eligible for statistical analysis resulting in a PPV = 74%: (95% CI 60-84%) in 39 patients with MRI related alterations of initial treatment plans and 27%: (95% CI 14-44%) in 39 patients without. Positive predictive values of incremental findings decisive for specific treatment alteration/s were 83% (95% CI 68-92%) in patients with any alteration of initial treatment plans and 91% (95% CI 70-98%) for patients (n = 20/22) with conversion from breast conserving surgery to mastectomy. The empirical AUC for the incremental findings in the whole MRI group was 85% (95% CI 78-91%). CONCLUSION: Breast MRI, performed and evaluated together with conventional breast imaging methods can provide relevant information at a high degree of accuracy in the pre-operative setting.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Mama/diagnóstico por imagem , Mama/patologia , Imageamento por Ressonância Magnética , Cuidados Pré-Operatórios , Adulto , Idoso , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Mastectomia/métodos , Pessoa de Meia-Idade , Estudos Prospectivos , Reprodutibilidade dos Testes , Suécia
10.
Int J Biol Macromol ; 131: 812-820, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30880057

RESUMO

Hyaluronic acid (HA) cross-linked with 1,4-butanediol diglycidyl ether (BDDE) are hydrogels with many biomedical applications. Degree of substitution, cross-linking and substitution position of the cross-linker might influence the properties of the hydrogels. We showed earlier that the most common substitution position of the cross-linker on the hyaluronan chain was the 4-hydroxyl of N-acetylglucosamine. This result has led us to investigate unsulfated chondroitin (CN) which only differ from HA in the primary structure by the configuration at C4 of the aminoglycan. In the present study, we have investigated (i) the substitution positions of the cross-linker in CN using NMR and LC-MS and compared the results to the data obtained for HA (ii) the effect of alkali on the 13C and 1H chemical shifts in CN and HA (iii) the temperature coefficients and chemical shifts of hydroxyl protons in CN and HA. In CN, the 2-hydroxyl of glucuronic acid and 6-hydroxyl of N-acetylgalactosamine were found to be the major sites of substitution by BDDE. Moreover, while chondroitinase was not able to cleave HA tetrasaccharide substituted at the 4-hydroxyl GlcNAc reducing end by BDDE, it is able to degrade CN-BDDE down to disaccharide units.


Assuntos
Butileno Glicóis/química , Condroitina/química , Ácido Hialurônico/química , Cromatografia Líquida , Reagentes para Ligações Cruzadas/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
11.
J Poult Sci ; 56(4): 308-317, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-32055229

RESUMO

The aim of this study was to determine the changes in chicken breast meat quality (water-holding capacity, color, texture, myofibrillar fragmentation index (MFI), total protein solubility, thiobarbituric acid reactive substances (TBARS), total viable count (TVC), and lactic acid bacteria (LAB) count) due to storage under superchilling conditions (-1.3°C) and fluctuating temperatures (ranging from -20°C to -5°C) as compared to the quality of meat stored at chilled (2-4°C) and frozen (-20°C) temperatures, respectively. Results indicated that the TVC and LAB count of the chilled and superchilled breast meat increased with storage time. TVC of the chilled and superchilled breast meat reached the safety level of 7 log cfu/g at approximately day 8 and18, respectively. This suggested that the superchilling method extended the storage duration by 10 days. Weight loss and TBARS of the chilled and superchilled samples tended to increase with increasing storage time. The color, texture, protein solubility, and MFI were stable throughout the entire storage period of the chilled (9 days) and superchilled (28 days) samples. Results indicated that while three cycles of storage temperature fluctuation influenced the weight loss and dry matter of the meat, they did not affect the TVC, LAB count, texture, color, pH, MFI, and protein solubility. The superchilling technique (-1.3°C) could extend the shelf-life of meat and maintain the quality of chicken breast meat. Fluctuations in temperature during frozen storage decreased the water-holding capacity of chicken breast meat, indicating that temperature stability should be maintained during frozen storage.

12.
PLoS One ; 13(12): e0208804, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30532202

RESUMO

A range of methodologies may be used for analyzing bacteria, depending on the purpose and the level of resolution needed. The capability for recognition of species distinctions within the complex spectrum of bacterial diversity is necessary for progress in microbiological research. In clinical settings, accurate, rapid and cost-effective methods are essential for early and efficient treatment of infections. Characterization and identification of microorganisms, using, bottom-up proteomics, or "proteotyping", relies on recognition of species-unique or associated peptides, by tandem mass spectrometry analyses, dependent upon an accurate and comprehensive foundation of genome sequence data, allowing for differentiation of species, at amino acid-level resolution. In this study, the high resolution and accuracy of MS/MS-based proteotyping was demonstrated, through analyses of the three phylogenetically and taxonomically most closely-related species of the Mitis Group of the genus Streptococcus: i.e., the pathogenic species, Streptococcus pneumoniae (pneumococcus), and the commensal species, Streptococcus pseudopneumoniae and Streptococcus mitis. To achieve high accuracy, a genome sequence database used for matching peptides was created and carefully curated. Here, MS-based, bottom-up proteotyping was observed and confirmed to attain the level of resolution necessary for differentiating and identifying the most-closely related bacterial species, as demonstrated by analyses of species of the Streptococcus Mitis Group, even when S. pneumoniae were mixed with S. pseudopneumoniae and S. mitis, by matching and identifying more than 200 unique peptides for each species.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas de Tipagem Bacteriana , Proteoma/metabolismo , Proteômica , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/metabolismo , Espectrometria de Massas em Tandem
13.
Chromatographia ; 81(6): 851-860, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29887619

RESUMO

The overreaching purpose of this study is to evaluate new approaches for determining the optimal operational and column conditions in chromatography laboratories, i.e., how best to select a packing material of proper particle size and how to determine the proper length of the column bed after selecting particle size. As model compounds, we chose two chiral drugs for preparative separation: omeprazole and etiracetam. In each case, two maximum allowed pressure drops were assumed: 80 and 200 bar. The processes were numerically optimized (mechanistic modeling) with a general rate model using a global optimization method. The numerical predictions were experimentally verified at both analytical and pilot scales. The lower allowed pressure drop represents the use of standard equipment, while the higher allowed drop represents more modern equipment. For both compounds, maximum productivity was achieved using short columns packed with small-particle size packing materials. Increasing the allowed backpressure in the separation leads to an increased productivity and reduced solvent consumption. As advanced numerical calculations might not be available in the laboratory, we also investigated a statistically based approach, i.e., the Taguchi method (empirical modeling), for finding the optimal decision variables and compared it with advanced mechanistic modeling. The Taguchi method predicted that shorter columns packed with smaller particles would be preferred over longer columns packed with larger particles. We conclude that the simpler optimization tool, i.e., the Taguchi method, can be used to obtain "good enough" preparative separations, though for accurate processes, optimization, and to determine optimal operational conditions, classical numerical optimization is still necessary.

14.
Meat Sci ; 136: 79-84, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29107866

RESUMO

The tenderness of pork loins from castrates, entire males and females was quantified with sensory analysis and measurement of instrumental texture during ageing. Furthermore, the effects of intramuscular fat (IMF), collagen content and solubility, hot carcass weights and meat percentages on tenderness were examined. Meat from castrates was significantly (p=0.043) more tender than meat from entire males and females as assessed by trained sensory panellists. Tenderness scores were positively affected by IMF content (p=0.008) and hot carcass weight (p<0.001), but no effect of collagen content and solubility was found. Meat from all three genders had the same tenderisation rate during ageing (two, five, seven and ten days). It is therefore suggested that meat from entire males and females should be aged for longer than meat from castrates to obtain the same level of tenderness.


Assuntos
Castração/veterinária , Carne Vermelha/análise , Fatores Sexuais , Suínos/fisiologia , Tecido Adiposo , Animais , Composição Corporal , Colágeno/análise , Feminino , Manipulação de Alimentos/métodos , Humanos , Masculino , Músculo Esquelético , Fatores de Tempo
15.
Meat Sci ; 130: 1-6, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28342329

RESUMO

Different types of cheese powder were added to meat emulsion sausages in order to address its influence on chemical composition, volatile compounds profile and sensory properties, and its potential to reduce salt content through boosting saltiness. Addition of cheese powder to emulsion sausages modified their profile of volatile compounds. Blue cheese increased some ketones, alcohols, and esters, while brown cheese brought typical Maillard reaction compounds. Overall, addition of cheese powders to sausages enhanced the intensity of flavour traits. A mixture of hard and blue cheese powder showed the highest effect on boosting saltiness, while brown cheese powder showed the strongest umami and meat flavour boosting effect, and sausages with added blue cheese powder showed a more intense aftertaste. Hardness significantly increased due to the addition of blue cheese powder. Addition of cheese powder to emulsion sausages might be an interesting tool to boost flavour and reduce salt content in cooked sausages with no negative effect on saltiness or overall flavour.


Assuntos
Queijo , Produtos da Carne , Paladar , Compostos Orgânicos Voláteis/análise , Animais , Emulsões , Tecnologia de Alimentos , Humanos , Cloreto de Sódio na Dieta , Suínos
16.
BMC Microbiol ; 17(1): 11, 2017 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-28061865

RESUMO

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. RESULTS: Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the ß-Barrel Assembly Machinery (BAM) complex were up-regulated. CONCLUSIONS: ETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.


Assuntos
Escherichia coli Enterotoxigênica/metabolismo , Proteínas de Escherichia coli/análise , Proteômica , Adenosina Trifosfatases , Aminoácidos/metabolismo , Proteínas da Membrana Bacteriana Externa/análise , Toxinas Bacterianas/análise , Toxinas Bacterianas/metabolismo , Regulação para Baixo , Transporte de Elétrons , Escherichia coli Enterotoxigênica/crescimento & desenvolvimento , Escherichia coli Enterotoxigênica/patogenicidade , Enterotoxinas/análise , Enterotoxinas/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Redes e Vias Metabólicas , Óperon , Biossíntese de Proteínas , Espectrometria de Massas em Tandem/métodos , Transcrição Genética , Tripsina/metabolismo , Regulação para Cima
17.
Front Microbiol ; 7: 1757, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27891114

RESUMO

Helicobacter pylori, a gastroenteric pathogen believed to have co-evolved with humans over 100,000 years, shows significant genetic variability. This motivates the study of different H. pylori strains and the diseases they cause in order to identify determinants for disease evolution. In this study, we used proteomics tools to compare two H. pylori strains. Nic25_A was isolated in Nicaragua from a patient with intestinal metaplasia, and P12 was isolated in Europe from a patient with duodenal ulcers. Differences in the abundance of surface proteins between the two strains were determined with two mass spectrometry-based methods, label-free quantification (MaxQuant) or the use of tandem mass tags (TMT). Each approach used a lipid-based protein immobilization (LPITM) technique to enrich peptides of surface proteins. Using the MaxQuant software, we found 52 proteins that differed significantly in abundance between the two strains (up- or downregulated by a factor of 1.5); with TMT, we found 18 proteins that differed in abundance between the strains. Strain P12 had a higher abundance of proteins encoded by the cag pathogenicity island, while levels of the acid response regulator ArsR and its regulatory targets (KatA, AmiE, and proteins involved in urease production) were higher in strain Nic25_A. Our results show that differences in protein abundance between H. pylori strains can be detected with proteomic approaches; this could have important implications for the study of disease progression.

18.
Chromatographia ; 79(19): 1283-1291, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27738352

RESUMO

The adsorption of the proton-pump inhibitor omeprazole was investigated using RP-LC with chemometric models combined with adsorption isotherm modelling to study the effect of pH and type of organic modifier (i.e., acetonitrile or methanol). The chemometric approach revealed that omeprazole was tailing with methanol and fronting with acetonitrile along with increased fronting at higher pH. The increased fronting with higher pH for acetonitrile was explored using a pH-dependent adsorption isotherm model that was determined using the inverse method and it agreed well with the experimental data. The model indicated that the peaks exhibit more fronting at high pH due to a larger fraction of charged omeprazole molecules. This model could accurately predict the shape of elution profiles at arbitrary pH levels in the studied interval. Using a two-layer adsorption isotherm model, the difference between acetonitrile and methanol was studied at the lowest pH at which almost all omeprazole molecules are neutral. Omeprazole had adsorbate-adsorbate interactions that were similar in strength for the acetonitrile and methanol mobile phases, while the solute-adsorbent interactions were almost twice as strong with methanol. The difference in the relative strengths of these two interactions likely explains the different peak asymmetries (i.e., tailing/fronting) in methanol and acetonitrile. In conclusion, thermodynamic modelling can complement chemometric modeling in HPLC method development and increase the understanding of the separation.

19.
J Sep Sci ; 39(23): 4520-4532, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27739648

RESUMO

In the last decade, core-shell particles have gained more and more attention in fast liquid chromatography separations due to their comparable performance with fully porous sub-2 µm particles and their significantly lower back pressure. Core-shell particles are made of a solid core surrounded by a shell of classic fully porous material. To embrace the developed core-shell column market and use these columns in pharmaceutical analytical applications, 17 core-shell C18 columns purchased from various vendors with various dimensions (50 mm × 2.1 mm to 100 mm × 3 mm) and particle sizes (1.6-2.7 µm) were characterized using Tanaka test protocols. Furthermore, four selected active pharmaceutical ingredients were chosen as test probes to investigate the batch to batch reproducibility for core-shell columns of particle size 2.6-2.7 µm, with dimension of 100 × 3 mm and columns of particle size 1.6 µm, with dimension 100 × 2.1 mm under isocratic elution. Columns of particle size 2.6-2.7 µm were also tested under gradient elution conditions. To confirm the claimed comparable efficiency of 2.6 µm core-shell particles as sub-2 µm fully porous particles, column performances of the selected core-shell columns were compared with BEH C18 , 1.7 µm, a fully porous column material as well.


Assuntos
Cromatografia Líquida , Preparações Farmacêuticas/análise , Tamanho da Partícula , Porosidade , Reprodutibilidade dos Testes
20.
J Pharm Biomed Anal ; 129: 273-281, 2016 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-27442889

RESUMO

Quality Control methods (QC-methods) play an important role in the overall control strategy for drug manufacturing. However, efficient life-cycle management and continual improvement are hindered due to a variety of post-approval variation legislations across territories and a lack of harmonization of the requirements. As a result, many QC-methods fall behind the technical development. Developing the QC-method in accordance with the Quality by Design guidelines gives the possibility to do continual improvements inside the original Method Operable Design Region (MODR). However, often it is necessary to do changes outside the MODR, e.g. to incorporate new technology that was not available at the time the original method was development. Here, we present a method enhancement concept which allows minor adjustments, within the same measuring principle, outside the original MODR without interaction with regulatory agencies. The feasibility of the concept is illustrated by a case study of a QC-method based on HPLC, assumed to be developed before the introduction of UHPLC, where the switch from HPLC to UHPLC is necessary as a continual improvement strategy. The concept relies on the assumption that the System Suitability Test (SST) and failure modes are relevant for other conditions outside the MODR as well when the same measuring principle is used. It follows that it should be possible to move outside the MODR as long as the SST has passed. All minor modifications of the original, approved QC-method must be re-validated according to a template given in the original submission and a statistical equivalence should be shown between the original and modified QC-methods. To summarize, revalidation is handled within the pharmaceutical quality control system according to internal change control procedures, but without interaction with regulating agencies.


Assuntos
Indústria Farmacêutica/normas , Preparações Farmacêuticas/normas , Cromatografia Líquida de Alta Pressão/métodos , Controle de Qualidade , Melhoria de Qualidade
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