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1.
Metabolic syndrome (MetS) is a cluster of interrelated common clinical disorders. The role of resistin in insulin sensitivity and MetS is controversial till date. So, the aim of the present study was to investigate the relationship of plasma resistin levels with markers of the MetS in Indian subjects. In a case control study, total 528 subjects were selected for the study. 265 (194 male and 71 female) were cases (with MetS) and 263 (164 male and 99 female) were controls (without MetS). Required anthropometric measurements and calculations were carried out accordingly. All the Biochemical estimations were carried out according to standard protocol. Resistin level was measured by the standard protocol (By ELISA i.e. enzyme linked immunosorbent assay) as illustrated in the kit. Insulin level was also measured by the standard protocol as illustrated in the kit and insulin resistance was calculated by the standard procedures. Plasma resistin levels were significantly higher in cases compared with controls (male = 13.05 ± 4.31 vs. 7.04 ± 2.09 ng/ml; p ≤ 0.001 and female = 13.53 ± 4.14 vs. 7.42 ± 2.30 ng/ml; p ≤ 0.001). Plasma resistin levels were well correlated with waist circumference, glucose, triglycerides, waist/hip ratio, systolic and diastolic blood pressure, high density lipoprotein, total cholesterol, serum low density lipoprotein, serum very low density lipoprotein, insulin and insulin resistance. Plasma resistin levels were elevated in presence of the MetS and were associated with increased metabolic risk factors.  相似文献   

2.

Introduction

Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing.

Materials and methods

A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination.

Results

The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample.

Conclusions

The characteristics established in our study are in concordance with the manufacturer’s specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization.Key words: lab-on-a-chip devices, capillary electrophoresis, multiplex PCR, circulating tumour cells, Agilent DNA 1000 kit  相似文献   

3.
BackgroundRhizoctonia solani (teleomorph: Thanatephorus cucumeris) is one of the most important pathogens of rice (Oryza sativa L.) that causes severe yield losses in all rice-growing regions. Sclerotia, formed from the aggregation of hyphae, are important structures in the life cycles of R. solani and contain a large quantity of polysaccharides, lipids, proteins and pigments. In order to extract high-quality total RNA from the sclerotia of R. solani, five methods, including E.Z.N.A.™ Fungal RNA Kit, sodium dodecyl sulfate (SDS)–sodium borate, SDS–polyvinylpyrrolidone (PVP), guanidinium thiocyanate (GTC) and modified Trizol, were compared in this study.ResultsThe electrophoresis results showed that it failed to extract total RNA from the sclerotia using modified Trizol method, whereas it could extract total RNA from the sclerotia using other four methods. Further experiments confirmed that the total RNA extracted using SDS–sodium borate, SDS–PVP and E.Z.N.A.™ Fungal RNA Kit methods could be used for RT-PCR of the specific amplification of GAPDH gene fragments, and that extracted using GTC method did not fulfill the requirement for above-mentioned RT-PCR experiment.ConclusionIt is concluded that SDS–sodium borate and SDS–PVP methods were the better ones for the extraction of high-quality total RNA that could be used for future gene cloning and expression studies, whereas E.Z.N.A.™ Fungal RNA Kit was not taken into consideration when deal with a large quantity of samples because it is expensive and relatively low yield.  相似文献   

4.

Introduction:

Zonulin is a eukaryotic protein structurally similar to Vibrio cholerae’s zonula occludens toxin. It plays an important role in the opening of small intestine tight junctions. The loss of gut wall integrity during sepsis might be pivotal and has been described in various experimental as well as human studies. Increased levels of zonulin could be demonstrated in diseases associated with increased intestinal inflammation, such as celiac disease and type 1 diabetes. We therefore investigated the role of plasma levels of zonulin in patients with sepsis as a non-invasive marker of gut wall integrity.

Materials and methods:

Plasma level of zonulin was measured in 25 patients with sepsis, severe sepsis or septic shock according to ACCP/SCCM criteria at the first day of diagnosed sepsis. 18 non-septic post-surgical ICU-patients and 20 healthy volunteers served as control. Plasma levels were determined by using commercially available ELISA kit. Data are given as median and interquartile range (IQR).

Results:

Significantly higher plasma concentration of zonulin were found in the sepsis group: 6.61 ng/mL (IQR 3.51–9.46), as compared to the to the post-surgical control group: 3.40 ng/mL (IQR 2.14–5.70) (P = 0.025), as well as to the healthy group: 3.55 ng/mL (IQR 3.14–4.14) (P = 0.008).

Conclusion:

We were able demonstrate elevated levels of plasma zonulin, a potential marker of intestinal permeability in septic patients. Increased zonulin may serve as an additional mechanism for the observed increased intestinal permeability during sepsis and SIRS.  相似文献   

5.
IntroductionThe aim of this study was to perform a comprehensive verification of a 6-part differential haematology analyser Siemens Advia 2120i (Erlangen, Germany), prior to its routine implementation.Materials and methodsOur verification protocol included: precision (within- and between-run), estimated bias (%) as measure of trueness, which was calculated from observed and manufacturers’ declared value, analytical measuring interval (AMI), carryover, confirmation of a limit of blank (LoB), determination of a limit of detection (LoD) and limit of quantitation (LoQ). The K2 ethylenediaminetetraacetic acid (EDTA) patients’ leftover samples were used for verification of analyser Advia 2021i. Acceptance criteria were based on manufacturer technical specifications (Siemens), 2016 state-of-the-art criteria (Vis and Huisman), and EFLM Biological Variation Database.ResultsThe within- and between-run precision were acceptable for all parameters and the lowest coefficients of variation were observed for mean corpuscular volume (MCV) (0.3% and 0.6%, respectively). Estimated bias was within the acceptance criteria for all parameters except for MCV (the estimated bias was 2.2% (acceptance criteria 2.0%). AMI was confirmed for all tested parameters (r > 0.99). The carryover estimates ranged from 0.1% for platelet count (Plt) to 0.6% for red blood cell count and were within the manufacturers’ specifications (≤ 1%). Manufacturers’ claims for LoB were confirmed for leukocytes, erythrocytes, haemoglobin, and platelets. The estimated LoD and LoQ were 0.05 x109/L and 0.1 x109/L for white blood cell count, while for Plt values were 2 x109/L and 3 x109/L, respectively.ConclusionsAnalytical performance of the Siemens Advia 2120i meets predefined quality goals and is suitable for routine use in a clinical laboratory.Key words: verification, haematology analyser, haematology, blood cell count  相似文献   

6.
BackgroundSmall ribonucleic acids represent an important repertoire of mobile molecules that exert key roles in several cell processes including antiviral defense. Small RNA based repertoire includes both small interfering RNA (siRNA) and microRNA (miRNA) molecules. In the Prunus genus, sharka disease, caused by the Plum pox virus (PPV), first occurred on European plum (Prunus domestica) and then spread over among all species in this genus and thus classified as quarantine pathogen. Next-generation sequencing (NGS) was used for the study of siRNA/miRNA molecules; however, NGS relies on adequate extraction protocols. Currently, knowledge of PPV-Prunus interactions in terms of siRNA populations and miRNA species is still scarce, and siRNA/miRNA extraction protocols are limited to species such as peach, almond, and sweet cherry.ResultsWe describe a reliable procedure for siRNA/miRNA purification from Prunus salicina trees, in which previously used protocols did not allow adequate purification. The procedure was based on a combination of commercially available RNA purification kits and specific steps that yielded high quality purifications. The resulting molecules were adequate for library construction and NGS, leading to the development of a pipeline for analysis of both siRNAs and miRNAs in the PPV–P. salicina interactions. Results showed that PPV infection led to altered siRNA profiles in Japanese plum as characterized by decreased 24-nt and increased 21- and 22-nt siRNAs. Infections showed miR164 and miR160 generation and increased miR166, miR171, miR168, miR319, miR157, and miR159.ConclusionWe propose this protocol as a reliable and reproducible small RNA isolation procedure for P. salicina and other Prunus species.  相似文献   

7.

Introduction

Centrifugation is an essential step for plasma preparation to remove residual elements in plasma, especially platelets and platelet-derived microparticles (PMPs). Our working hypothesis was that centrifugation as a preanalytical step may influence some coagulation parameters.

Materials and methods

Healthy young men were recruited (N = 17). For centrifugation, two protocols were applied: (A) the first centrifugation at 2500 x g for 15 min and (B) at 2500 x g for 20 min at room temperature with a light brake. In protocol (A), the second centrifugation was carried out at 2500 x g for 15 min, whereas in protocol (B), the second centrifugation involved a 10 min spin at 13,000 x g. Thrombin-antithrombin (TAT) and plasmin-antiplasmin (PAP) complexes concentrations were determined by enzyme-linked immunosorbent assays. PMPs were stained with CD41 antibody and annexin V, and analyzed by flow cytometry method. Procoagulant activity was assayed by the Calibrated Automated Thrombogram method as a slope of thrombin formation (CAT velocity).

Results

Median TAT and PAP concentrations did not differ between the centrifugation protocols. The high speed centrifugation reduced the median (IQR) PMP count in plasma from 1291 (841-1975) to 573 (391-1010) PMP/µL (P = 0.001), and CAT velocity from 2.01 (1.31-2.88) to 0.97 (0.82-1.73) nM/min (P = 0.049). Spearman’s rank correlation analysis showed correlation between TAT and PMPs in the protocol A plasma which was (rho = 0.52, P < 0.050) and between PMPs and CAT for protocol A (rho = 0.74, P < 0.050) and protocol B (rho = 0.78, P < 0.050).

Conclusion

Centrifugation protocols do not influence the markers of plasminogen (PAP) and thrombin (TAT) generation but they do affect the PMP count and procoagulant activity.Key words: cell-derived microparticles, blood coagulation tests, centrifugation, preanalytical phase  相似文献   

8.
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9.
Spatially varied surface treatment of a fluorescently labeled Bovine Serum Albumin (BSA) protein, on the walls of a closed (sealed) microchannel is achieved via a well-defined gradient in plasma intensity. The microchips comprised a microchannel positioned in-between two microelectrodes (embedded in the chip) with a variable electrode separation along the length of the channel. The channel and electrodes were 50 μm and 100 μm wide, respectively, 50 μm deep, and adjacent to the channel for a length of 18 mm. The electrode separation distance was varied linearly from 50 μm at one end of the channel to a maximum distance of 150, 300, 500, or 1000 μm to generate a gradient in helium plasma intensity. Plasma ignition was achieved at a helium flow rate of 2.5 ml/min, 8.5 kVpk-pk, and 10 kHz. It is shown that the plasma intensity decreases with increasing electrode separation and is directly related to the residual amount of BSA left after the treatment. The plasma intensity and surface protein gradient, for the different electrode gradients studied, collapse onto master curves when plotted against electrode separation. This precise spatial control is expected to enable the surface protein gradient to be tuned for a range of applications, including high-throughput screening and cell-biomolecule-biomaterial interactions.  相似文献   

10.
IntroductionThe measurement of serum free light chain (FLC) represents a fundamental aspect on the assessment of patients with monoclonal gammopathies (MG). Different analytical methods for FLC have become available with the possibility to obtain different value with a substantial impact on the assessment of patients with MG. This study aimed to evaluate FLC results obtained with two different assays and how the difference value obtained can impact in the patient’s assessment.Materials and methodsNinety-three patient serum samples that underwent analysis for FLC with two different methods, Serum Freelite (The Binding Site, Birmingham, UK) and N-Latex FLC (Siemens, Marburg, Germany), were included in this retrospective study. Statistical analysis was performed to evaluate correlation, difference, and the grade of concordance between the results obtained with the two methods.ResultsSignificant statistical differences between the results obtained from the two methods were found (P < 0.05). A good correlation was found (0.99 for κ FLC, 0.95 for λ FLC, and 0.94 for the κ/λ ratio, respectively). We found a weighted kappa value of 0.65 for κ/λ ratio, 0.65 for λ FLC and 0.90 for κ FLC. A positive bias found with the Bland-Altman plot mirrors overestimation of κ FLC and κ/λ ratio with Freelite compared to N-Latex, whilst a negative bias underscores underestimation of λ FLC by Freelite compared to N-Latex.ConclusionAlthough in general the concordance between Freelite and N-Latex appears satisfactory, several discrepancies could be evidenced and consequently the two assays are not interchangeable.  相似文献   

11.
IntroductionThe Fourth Universal Definition of Myocardial Infarction Global Taskforce recommends the use of high sensitive troponin (hs-Tn) assays in the diagnosis of acute myocardial infarction. We evaluated the analytical performance of the Atellica IM High-sensitivity Troponin I Assay (hs-TnI) (Siemens Healthcare Diagnostics Inc., Tarrytown, USA) and compared its performance to other hs-TnI assays (Siemens Advia Centaur, Dimension Vista, Dimension EXL, and Abbott Architect (Wiesbaden, Germany)) at one or more sites across Europe.Materials and methodsPrecision, detection limit, linearity, method comparison, and interference studies were performed according to Clinical and Laboratory Standards Institute protocols. Values in 40 healthy individuals were compared to the manufacturer’s cut-offs. Sample turnaround time (TAT) was examined.ResultsImprecision repeatability CVs were 1.1–4.7% and within-lab imprecision were 1.8–7.6% (10.0–25,000 ng/L). The limit of blank (LoB), detection (LoD), and quantitation (LoQ) aligned with the manufacturer’s values of 0.5 ng/L, 1.6 ng/L, and 2.5 ng/L, respectively. Passing-Bablok regression demonstrated good correlations between Atellica IM analyser with other systems; some minor deviations were observed. All results in healthy volunteers fell below the 99th percentile URL, and greater than 50% of each sex demonstrated values above the LoD. No interference was observed for biotin (≤ 1500 µg/L), but a slight bias at 5.0 g/L haemoglobin and 50 ng/L Tn was observed. TAT from was fast (mean time = 10.9 minutes) and reproducible (6%CV).ConclusionsReal-world analytical and TAT performance of the hs-TnI assay on the Atellica IM analyser make this assay fit for routine use in clinical laboratories.  相似文献   

12.

Introduction

Measurements of blood ethanol concentrations must be accurate and reliable. The most important factors affecting blood ethanol stability are temperature and storage time. In this study, we aimed to compare ethanol stability in plasma samples at -20 °C for the different storage periods.

Materials and methods

Blood samples were collected from intoxicated drivers (N = 80) and initial plasma ethanol concentrations were measured immediately. Plasma samples were then stored at -20 °C and re-assessed after 2, 3, 4, or 5 months of storage. Differences between the initial and stored ethanol concentrations in each group (N = 20) were analyzed using Wilcoxon matched-pairs test. The deviation from the initial concentration was calculated and compared with Clinical Laboratory Improvement Amendments (CLIA’88) Proficiency Testing Limits. Relationships between the initial concentrations and deviations from initial concentrations were analyzed by Spearman’s correlation analysis. For all statistical tests, differences with P values of less than 0.05 were considered statistically significant.

Results

Statistically significant differences were observed between the initial and poststorage ethanol concentrations in the overall sample group (P < 0.001). However, for the individual storage duration groups, analytically significant decreases were observed only for samples stored for 5 months, deviations from the initial concentrations exceeded the allowable total error (TEa). Ethanol decreases in the other groups did not exceed the TEa.

Conclusion

According to our results, plasma ethanol samples can be kept at -20 °C for up to 3-4 months until re-analysis. However, each laboratory should also establish its own work-flow rules and criterion for reliable ethanol measurement in forensic cases.Key words: preanalytical phase, ethanol, stability, storage temperature  相似文献   

13.
IntroductionThe aim of this study was to investigate lipoprotein particle distributions and the likelihood of achieving cholesterol homeostasis in the remission phase of nephrotic syndrome (NS) in paediatric patients. We hypothesized that lipoprotein particle distributions moved toward less atherogenic profile and that cholesterol homeostasis was achieved.Materials and methodsThirty-three children, 2 to 9 years old with NS were recruited. Blood sampling took place both in the acute phase and during remission. Serum low-density lipoprotein particles (LDL) and high-density lipoprotein particles (HDL) were separated using non-denaturing polyacrylamide gradient gel (3-31%) electrophoresis. Serum non-cholesterols sterols (NCSs), desmosterol, lathosterol, 7-dehydrocholesterol (7-DHC), campesterol and β-sitosterol were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).ResultsAll patients had desirable serum HDL cholesterol concentrations during remission. The dominant lipoprotein diameters and LDL subclass distribution did not change significantly during follow-up. In contrast, HDL lipoprotein particle distribution shifted towards larger particles. The absolute concentration of desmosterol was significantly lower during remission (P = 0.023). β-sitosterol concentration markedly increased during remission (P = 0.005). Desmosterol/β-sitosterol (P < 0.001) and 7-DHC/β-sitosterol (P = 0.005) ratios significantly declined during disease remission.ConclusionsFavourable changes in the serum lipid profiles, HDL particle subclass distribution and cholesterol metabolism in paediatric patients with NS during remission took place. For the first time, we found that cholesterol homeostasis changed in favour of increased cholesterol absorption during disease remission. Nevertheless, complete cholesterol homeostasis was not achieved during disease remission.  相似文献   

14.
IntroductionEpithelial cells (ECs) are structures regularly observed during urine microscopy analysis. The correct identification of EC subtypes can be useful since renal tubular epithelial cells (RTECs) are clinically relevant. We investigate the urinary ECs report and the judgement of its clinical importance by Brazilian laboratories.Materials and methodsA survey with four questions was made available to participants of the Urinalysis External Quality Assessment Program (EQAP) from Controllab. Laboratories composed 3 groups: (1) differentiating ECs subtypes: “squamous”, “transitional” and “RTECs”; (2) differentiating ECs subtypes: “squamous” or “non-squamous” cells; (3) without ECs subtype identification. Participants did not necessarily answer to all questions and the answers were evaluated both within the same laboratory’s category and within different categories of laboratories.ResultsA total of 1336 (94%) laboratories answered the survey; Group 1, 119/140 (85%) reported that ECs differentiation is important to the physician and 62% want to be evaluated by EQAP, while in Group 3, 455/1110 (41%) reported it is useful to them, however only 25% want be evaluated by EQAP. Group 2 laboratories 37/51 (73%) reported that the information is important, but only 13/52 (25%) are interested in an EQAP with differentiation of the 3 ECs subtypes.ConclusionMost of the laboratories do not differentiate ECs in the three subtypes, despite the clinical importance of RTECs. Education of laboratory staff about the clinical significance of urinary particles should be considered a key priority.  相似文献   

15.
The negative interference by bilirubin in serum creatinine estimation by Jaffe’s kinetic method is well known. Several approaches have been suggested to overcome this interference. In this article three different creatinine kits (Jaffe’s kinetic method) have been tested for bilirubin interference and its rectification using two simple approaches. The performance of three kits (A, B and C) supplied by three different manufacturers was tested using IQC and EQAS sera and pooled serum with added bilirubin. To overcome the bilirubin interference two approaches viz. NaOH preincubation and TCA precipitation were used. Bilirubin did interfere in creatinine estimation after a certain level (2.3 mg/dl). However, both NaOH preincubation and TCA precipitation approach rectified this interference. The performance of kit A was better than kit B and C. All the three kits showed bilirubin interference upon increasing the bilirubin concentration but kit A performed better than kit B and C. However, NaOH incubation and TCA precipitation methods overcame this interference to a great extent.  相似文献   

16.
Nicotine, responsible for the addictive properties of tobacco, is widely used in nicotine replacement therapy for tobacco use cessation. We investigated the time-dependent effect of treatment with nicotine on the tumor suppressor, DNA repair and immune responses. Swiss Albino mice (laca strain) of both sexes received nicotine dissolved at a dose of 100 µg/ml in 2% sucrose for 24 weeks, by oral gavage, while age- and gender-matched controls received only 2% sucrose for the same period. Nicotine-treated and control mice were sacrificed 6, 16 and 24 weeks post-treatment, and their tissues evaluated for alterations in histology, oxidative stress, TNF-α levels, nitric oxide (NO) and myeloperoxidase (MPO) release, tumor suppressor response and DNA repair response. Statistical significance of results was determined using Students’ t test. The tissues of nicotine treated mice exhibited a large number of multinucleated and binucleated cells, enlarged nuclei and non-uniform distribution of cells, significant increase in expression of TNF-α gene and serum TNF-α, and time-dependent significant increase in lipid peroxidation, protein carbonylation, NO and MPO release when compared to age-and gender-matched controls. The mRNA expression of the tumor suppressor gene p53, its primary regulator Mdm2, and the DNA repair genes Brca2 and Ape1 were significantly elevated, but the corresponding protein levels remained largely unaltered. In conclusion, treatment with nicotine caused oxidative stress and inflammation which can cause widespread cellular damage from the very onset of treatment, without subverting the tumor suppressor and DNA repair responses.Electronic supplementary materialThe online version of this article (10.1007/s12291-020-00903-8) contains supplementary material, which is available to authorized users.  相似文献   

17.
IntroductionWhile early commenting on studies is seen as one of the advantages of preprints, the type of such comments, and the people who post them, have not been systematically explored.Materials and methodsWe analysed comments posted between 21 May 2015 and 9 September 2019 for 1983 bioRxiv preprints that received only one comment on the bioRxiv website. The comment types were classified by three coders independently, with all differences resolved by consensus.ResultsOur analysis showed that 69% of comments were posted by non-authors (N = 1366), and 31% by the preprints’ authors themselves (N = 617). Twelve percent of non-author comments (N = 168) were full review reports traditionally found during journal review, while the rest most commonly contained praises (N = 577, 42%), suggestions (N = 399, 29%), or criticisms (N = 226, 17%). Authors’ comments most commonly contained publication status updates (N = 354, 57%), additional study information (N = 158, 26%), or solicited feedback for the preprints (N = 65, 11%).ConclusionsOur results indicate that comments posted for bioRxiv preprints may have potential benefits for both the public and the scholarly community. Further research is needed to measure the direct impact of these comments on comments made by journal peer reviewers, subsequent preprint versions or journal publications.  相似文献   

18.
Errors in laboratory medicine occur in the preanalytical, analytical, and postanalytical phases. The errors are mostly detected in the preanalytical period. However, analytical errors are still an important source of error, despite their frequency is reduced significantly in years thanks to developments in laboratories. In this case, an analytical error was noticed during the verification of a patient’s results. The direct bilirubin of a 66-year-old male patient admitted to the emergency department was higher than the total bilirubin. The patient’s symptoms were fatigue and dyspnoea. Albumin and haemoglobin (Hb) concentrations of the patient were significantly low. After considering the patient’s demographics and laboratory results, the laboratory specialist suspected a paraproteinemia interference. Total protein was performed as a reflective test. The albumin/globulin ratio was reversed. Thereafter, serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE) were performed as another reflective tests, respectively. SPEP and IFE results were in favour of monoclonal gammopathy. The patient was directed to a haematologist, underwent a bone marrow biopsy, and the result was reported as Waldenstrom’s macroglobulinemia with plasma cell differentiation expressing IgM-Kappa. The patient went on a chemotherapy protocol, and his condition has been improved in subsequent months. Detection of analytical errors is of great importance, like in our case, and may be used as a tool to identify patients who have not yet been diagnosed. The laboratory specialist must dominate the entire process of each test in the laboratory, be aware of the limitations of tests, and turn these disadvantages into advantages when necessary.  相似文献   

19.
BackgroundThe acidic subunit of amarantin (AAC)—the predominant amaranth seed storage protein—has functional potential and its third variable region (VR) has been modified with antihypertensive peptides to improve this potential. Here, we modified the C-terminal in the fourth VR of AAC by inserting four VY antihypertensive peptides. This modified protein (AACM.4) was expressed in Escherichia coli. In addition, we also recombinantly expressed other derivatives of the amarantin protein. These include: unmodified amarantin acidic subunit (AAC); amarantin acidic subunit modified at the third VR with four VY peptides (AACM.3); and amarantin acidic subunit doubly modified, in the third VR with four VY peptides and in the fourth VR with the RIPP peptide (AACM.3.4).ResultsE. coli BL21-CodonPlus (DE3)-RIL was the most favorable strain for the expression of proteins. After 6 h of induction, it showed the best recombinant protein titer. The AAC and AACM.4 were obtained at higher titers (0.56 g/L) while proteins modified in the third VR showed lower titers: 0.44 g/L and 0.33 g/L for AACM.3 and AACM.3.4, respectively. As these AAC variants were mostly expressed in an insoluble form, we applied a refolding protocol. This made it possible to obtain all proteins in soluble form. Modification of the VR 4 improves the thermal stability of amarantin acidic subunit; AAC manifested melting temperature (Tm) at 34°C and AACM.4 at 37.2°C. The AACM.3 and AACM.3.4 did not show transition curves.ConclusionsModifications to the third VR affect the thermal stability of amarantin acidic subunit.  相似文献   

20.
IntroductionThe interest in quality management tools/methodologies is gradually increasing to ensure quality and accurate results in line with international standards in clinical laboratories. Six Sigma stands apart from other methodologies with its total quality management system approach. However, the lack of standardization in tolerance limits restricts the advantages for the process. Our study aimed both to evaluate the applicability of analytical quality goals with Roche Cobas c 702 analyser and to determine achievable goals specific to the analyser used.Materials and methodsThe study examined under two main headings as Sigmalaboratory and Sigmaanalyser. Sigmalaboratory was calculated using internal and external quality control data by using Roche Cobas c 702 analyser for 21 routine biochemistry parameters and, Sigmaanalyser calculation was based on the manufacturer data presented in the package inserts of the reagents used in our laboratory during the study. Sigma values were calculated with the six sigma formula.ResultsConsidering the total number of targets achieved, Sigmaanalyser performed best by meeting all CLIA goals, while Sigmalaboratory showed the lowest performance relative to biological variation (BV) desirable goals.ConclusionsThe balance between the applicability and analytical assurance of “goal-setting models” should be well established. Even if the package insert data provided by the manufacturer were used in our study, it was observed that almost a quarter of the evaluated analytes failed to achieve even “acceptable” level performance according to BV-based goals. Therefore, “state-of-the-art” goals for the Six Sigma methodology are considered to be more reasonable, achievable, and compatible with today’s technologies.  相似文献   

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