The Effects of Different Storage Conditions and Repeated Freeze/Thaw Cycles on the Concentration, Purity and Integrity of Genomic DNA.

The crucial requirement of molecular genetic methods is high-quality input material. The key question is "how to preserve DNA during long-term storage." Biobanks are recommended to aliquot isolated DNA into provided volumes. The aim of this study was to analyse the effect of repeated freezing and thawing on the genomic DNA integrity, quality and concentration. The aliquoted DNA isolated from blood cells using the automatic MagNA system and manual salting out method underwent freeze/thaw cycles at different storage conditions (-20 °C, -80 °C and liquid nitrogen). The average initial concentrations were 270.6 ng/µl (salting out method) and 125.0 ng/µl (MagNA). All concentration deviations relative to the concentration after the first freeze/ thaw cycle were less than 5 % for -20 °C and -80 °C cycling with both isolation methods. The average percentage differences of liquid nitrogen samples were higher, and the MagNA isolation method showed significant differences. There were no significant changes in the DNA purity or quality. The repeating freeze/ thaw up to 100 cycles (through -20 °C and -80 °C, respectively) did not significantly influence the integrity, concentration, or purity of genomic DNA, suggesting that storage of samples in high-volume pools without multiple aliquoting is possible. Storage in a freezer seems to be the most suitable way of long-term DNA preservation, because liquid nitrogen storage leads to formation of DNA clumps.

The effect of surgery on the levels of matrix metalloproteinases in patients with inguinal hernia.

Matrix metalloproteinases (MMPs) are associated with the alteration of extracellular matrix. The purpose of this study was to investigate how the levels of matrix metalloproteinases and their inhibitors - TIMPs are influenced by the presence of inguinal hernia as well as by its surgical treatment. The studied group consisted of 25 patients with inguinal hernia and 21 healthy controls for comparison. Two blood samples - before and after the treatment were collected from patients. Serum concentrations of MMPs and TIMPs were analysed by multiplex immunoassays. There was a difference in circulating levels of MMPs in patients before the surgery compared to healthy controls - the concentrations of MMP-2 and MMP-9 were significantly lower (p=0.026, p=0.018, respectively). After the surgery, the levels of MMPs, especially MMP-2 (p<0.0001), were significantly decreased in patients compared to the preoperative values, apart from MMP-9. On the contrary, MMP-9 showed significant increase after the surgery (p<0.0001). Circulation levels of TIMP-2 in patients were significantly decreased in comparison with controls (p=0.004), whereas levels of TIMP-1 were similar to controls. Both tested metalloproteinase inhibitors showed a significant decrease in detected levels (TIMP-1 p=0.0004; TIMP-2 p<0.0001) after the procedure compared to the preoperative values. The levels of MMPs, especially MMP-2 and MMP-9, and their inhibitors TIMP-1 and TIMP-2 are involved by the presence of inguinal hernia as well as are influenced by the surgery.

Representative sample survey on factors determining the Czech physicians' awareness of generic drugs and substitution.

BACKGROUND: Generic drugs and generic substitution belong to the tools by which healthcare costs may be reduced. However, low awareness and reluctance among healthcare professionals towards generic drugs may negatively affect the rational use of generic substitution. METHODS: The study aimed to analyze opinions and attitudes towards generic drugs and generic substitution among Czech physicians including their understanding of generic substitution legislative rules and the physicians´ previous experience in this field. Using random allocation, 1551 physicians practicing in the Czech Republic were asked to participate in the sociological representative survey conducted from November to December 2016, through face-to-face structured interviews comprising 19 items. Factor analysis as well as reliability analysis of items focused on legal rules in the context of physicians' awareness were applied with p-value of < 0.05 as statistically significant. RESULTS: Of a total of 1237 (79.8%) physicians (43.7% males; mean age 47.5 ± 11.6 years, 46.3% general practitioners) 24.8% considered generic drugs to be less safe, especially those with specialized qualification (p < 0.01). However, only 4.4% of the physicians noticed any drug-related problems, including adverse drug reactions associated with generic substitution. The majority of physicians felt neutrally about performing generic substitution in pharmacies, nor they expressed any opinion on characteristics of generics, even though a better understanding of the legislation and higher need of accordance of substituted drugs were associated with more positive attitudes towards generic substitution (p < 0.05). Physicians showed low knowledge score of legislative rules (mean 3.9 ± 1.6 from maximum 9), nevertheless they overestimated the law, as they considered some rules valid, even if the law does not require them. Cronbach alpha of all legislative rules that regulate generic substitution increased from 0.318 to 0.553 if two optional rules (physician consent and strength equivalence) would be taken into account. CONCLUSIONS: There is no sufficient awareness of generic drugs and generic substitution related issues among Czech physicians, although a deeper knowledge of legislation improves their perception about providing generic substitution.

Sclerostin levels predict cardiovascular mortality in long-term hemodialysis patients: A prospective observational cohort study.

Sclerostin is a protein which is involved in bone metabolism and probably also in vessel wall function. This prospective observational cohort study evaluated the prognostic significance of sclerostin in hemodialysis (HD) patients. In total, 106 HD patients and 25 healthy controls participated in the study. HD patients were prospectively followed up for five years. Sclerostin was measured in serum using standard ELISA kits by Biomedica. Sclerostin concentrations in serum were higher in HD patients compared to the controls (89.2±40.3 pmol/l vs. 32.8±13.0 pmol/l, p<0.001). Sclerostin levels were significant for cardiovascular mortality but not for overall mortality and mortality due to infection. A higher cardiovascular risk was connected to sclerostin concentrations above the median (>84 pmol/l), HR (95 % CI): 2.577 (1.0002-10.207), p=0.04. When sclerostin was evaluated together with residual diuresis in Kaplan-Meier analysis the worst prognosis due to cardiovascular events was observed in the group with high sclerostin and zero residual diuresis compared to all other patients (p=0.007). In summary, serum sclerostin levels in HD patients were increased when compared to healthy subjects. High sclerostin levels were demonstrated as a risk factor for cardiovascular mortality. Further studies are required to clarify the pathophysiological mechanisms of sclerostin action in patients with renal failure before therapeutic measures can be established.

Patterns in anticoagulant utilization in the Czech Republic during 2007-2017.

Direct oral anticoagulants (DOACs) have gradually entered the Czech market as alternatives to vitamin K antagonists and parenteral anticoagulants since 2008. Considering the eventual changes, the aim was to evaluate drug use and expenditure patterns on anticoagulants in the Czech Republic. A retrospective utilization study was conducted retrieving data from the State Institute for Drug Control database, including reports on drug supplies from distributors with anatomical therapeutic chemical classification (ATC) codes B01AA, B01AB, B01AE, B01AF, and B01AX. The utilization on national level was expressed as the ratio of the number of defined daily doses per 1000 inhabitants per day (DDD/TID). Expenditures on all anticoagulants were also assessed. Data was analyzed using PASW (version 18.0). Between January 2007 and December 2017, the national anticoagulant utilization rate increased continuously from 14.15 to 27.67 DDD/TID. The use of DOACs was 0.002 DDD/TID in 2008, increased to 6.04 DDD/TID in 2017. Warfarin utilization, after a small decrease in 2008, has shown nearly stable levels in the recent years (70.9% of all anticoagulants; mean 11.55 DDD/TID over the last 5 years), while its increase was halted by the spread of DOACs utilization (p < 0.05). In 2017, over half of the expenditures (51.1%) were due to oral anticoagulants, whereof 47.6% was related to DOACs. The results reflected a growing utilization and increasing costs of all anticoagulants, especially in DOACs at the expense of warfarin. Still, additional information regarding patient persistence and prescribing patterns is needed for a better understanding of oral anticoagulant utilization.

[Prenatal growth curves of the Czech population]. / Prenatální rustové krivky ceské populace.

OBJECTIVE: Determination of ultrasound biometric parameters of fetuses in the Czech population during pregnancy. METHODS: We retrospectively analyzed a data set of 20,566 pregnant women from the years 2008 to 2012 who met the following inclusion criteria: Caucasian ethnicity; measured biometric parameters biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), femur length (FL); body mass index (BMI) of the pregnant women < 35 kg/m2.We excluded pregnant women with multiple fibroids distorting the uterine cavity; pathologically developing fetuses; and pregnancies where we were not able to verify the correct dating. We did not carry out selection as to the method of getting pregnant. The pregnancy age was from 19+0 to 42+6 gestation weeks. As in all studies, the age of pregnancy refers to the completed gestation weeks (for instance 23+0 to 23+6 = 23rd gestation week). We then compared our newly derived growth curves with the curves currently used, for BPD, HC, AC, FL and EFW, in the 3rd or 5th percentile, 50th percentile and 95th or 97th percentile (according to data obtained from reference studies). RESULTS: The study included 40,806 observations. The number of observations exceeded 500 in all weeks except in the 42nd week. The median age of the pregnant patients was 31.9 years (min 14.9, max 52.54). In the studied group we found a lower degree of variability in all parameters. The biometric parameters had higher values for the gestation week than the reference parameters. The estimated weight of the fetuses in our group on the level of the 3rd percentile was significantly higher than the reference value. DISCUSSION: The study was based exclusively on sonographic measurements. Although we excluded from our group intrauterine pathologies that could affect the physiological growth of the fetus, we did not carry out an additional selection using information on the pathologies of fetuses obtained postnatally. However, as the study contains more than 40,000 measurements, we can assume that such additional selection would have had only a minimum effect on the resulting curve. A section of the fetuses that would have been considered eutrophic according to present reference studies could be considered hypotrophic or small for gestational age (SGA) according to our observations. CONCLUSION: We have obtained new prenatal ultrasound growth curves for the Czech population of healthy developing fetuses. In early gestation weeks, we have found statistically significant deviations from the reference studies. Fetuses in our study have significantly higher weight estimate and show a lower degree of variability compared to the reference studies. KEYWORDS: prenatal, ultrasound diagnosis, fetal biometry, percentile curves, biparietal diameter, head circumference, abdominal circumference, femur length, estimated fetal weight, Czech population.

Bone mineral density and body composition in men with multiple sclerosis chronically treated with low-dose glucocorticoids.

The aim of the study was to compare the bone mineral density (BMD) and body composition between ambulatory male MS patients and control subjects and to evaluate the relationships among body composition, motor disability, glucocorticoids (GC) use, and bone health. Body composition and BMD were measured by dual-energy X-ray absorptiometry in 104 ambulatory men with MS (mean age: 45.2 years) chronically treated with low-dose GC and in 54 healthy age-matched men. Compared to age-matched controls, MS patients had a significantly lower total body bone mineral content (TBBMC) and BMD at all measured sites except for the radius. Sixty five male MS patients (62.5 %) met the criteria for osteopenia and twenty six of them (25 %) for osteoporosis. The multivariate analysis showed a consistent dependence of bone measures (except whole body BMD) on BMI. The total leg lean mass % was as an independent predictor of TBBMC. The Expanded Disability Status Scale (EDSS), cumulative GC dose and age were independent determinants for BMD of the proximal femur. We conclude that decreasing mobility in male MS patients is associated with an increasing degree of osteoporosis and muscle wasting in the lower extremities. The chronic low-dose GC treatment further contributes to bone loss.

Effects of morning vs. evening teriparatide injection on bone mineral density and bone turnover markers in postmenopausal osteoporosis.

UNLABELLED: A 12-month morning teriparatide (TPTD) administration resulted in a larger increase in the lumbar spine bone mineral density (BMD) than the evening application. The results indicate that the response of bone cells to teriparatide treatment depends on dosing time. INTRODUCTION: The aim of this study was to assess the long-term effects of the morning vs. the evening teriparatide administration on BMD and bone turnover markers (BTMs) in postmenopausal osteoporosis. METHODS: Fifty women with established postmenopausal osteoporosis were randomized to 12-month treatment with 20 µg of TPTD, administered daily in the morning or in the evening. The BMD and serum concentrations of C-terminal telopeptide of type I collagen, N-terminal propeptide of type I procollagen (PINP), and tartrate-resistant acid phosphatase isoform 5b (TRAP 5b) were measured at baseline, after 6 and 12 months. General linear model-repeated measurements were used to analyze the data. RESULTS: After 12 months, the lumbar spine BMD grew markedly (p < 0.001) with a significantly greater increase in the morning arm compared to the evening arm (9.1% vs. 4.8%, respectively, p < 0.05). The BMD at the distal radius significantly decreased (p < 0.001), with no differences between the arms. The BMD at proximal femur did not change significantly. After 6 months, the BTMs were significantly increased compared with baseline (p < 0.001). The increases in the evening arm vs. the morning arm, however, were more pronounced in PINP (+358% vs. +215%, respectively) and in TRAP 5b (+70% vs. +37%, respectively) (both p < 0.05). CONCLUSION: 12-month morning administration of TPTD resulted in a larger increase in the lumbar spine BMD than the evening application. The timing of TPTD administration may be important for its efficacy.

[Efficiency of vitrectomy in diabetic macular edema and morphometry of surgically removed of the internal limiting membrane]. / Význam vitrektomie u diabetického makulárního edému a morfometrie chirurgicky odstranené vnitrní limitující membrány.

AIM: To evaluate the efficiency of vitrectomy with peeling of the internal limiting membrane (ILM) in eyes with diffuse and/or cystoid nontractional diabetic macular edema (ME) refractory to macular laser photocoagulation. Histopathologic and morphometric analysis of the ILM in diabetic eyes was performed. Thickness of the ILM was correlated with the level of glykosylated hemoglobin (HbA1c) and other clinical factors. MATERIAL AND METHODS: The prospective study involved 56 eyes of 52 diabetic patients with a mean age of 63 +/- 7.6 years. Vitrectomy with trypan blue-assisted ILM peeling was performed in standard way. Mean follow-up period was 8.7 months (range 3 - 19 months). The ILM was fixed immediately after peeling in 2.5% glutaraldehyde and submitted for electron microscopic evaluation. The ILM was photographed in standard magnification (x 5000) with the scale of 1 microm in the shot. RESULTS: Statistical analysis of the postoperative visual acuity (VA) in the study group of 56 eyes proved a general improvement with prevalence of the resulted VA in intervals 0.1-0.2 and 0.5-1.0 related to ETDRS chart. The VA was improved by at least 2 lines in 29 eyes of 56 eyes (51.8%), one line in 6 eyes (10.7%) and remained unchanged in 11 eyes (19.6%). The postoperative VA deteriorated by one line in 2 eyes (3.6%) and at least 2 lines in 8 eyes (14.3%). Morphometric analysis demonstrated a significant thickening of the ILM in all eyes with a mean thickness of the ILM 3.61 +/- 1.22 micro m. It was found that a higher thickness of the ILM is related to elevated HbA1c by both types of diabetes mellitus (DM) (p = 0.040). We also found significant dependence of ILM thickness in relation to duration of DM by comparison of men and women (p = 0.026) and significant correlation between ILM thickness and the age of diabetic patients related to their gender (p = 0.029). CONCLUSIONS: Vitrectomy with peeling of the ILM in eyes with chronic diffuse and/or cystoid nontractional diabetic ME mildly improves the VA and extends a hope for its stabilization. We confirmed increased thickness of the surgically peeled ILM and statistically significant correlations to elevated HbA1c by both types of DM and to further clinical characteristics of case series. Morphometric and histopathologic analyses of the ILM contribute to more objective evaluation of ultrastructure of the vitreomacular interface.

Dissociation in non-clinical and clinical sample of Czech adolescents. Reliability and validity of the Czech version of the Adolescent Dissociative Experiences Scale.

OBJECTIVE: The purpose of this study was to examine psychometric properties of the Czech language version of the Adolescent Dissociative Experiences Scale (A-DES) [2]. METHOD: 653 non-clinical participants and 162 adolescent psychiatric inpatients completed Czech versions of the A-DES and the Somatoform Dissociation Questionnaire (SDQ-20), and provided further information (data regarding demographic variables, diagnoses, further psychopathology). RESULTS: The Czech A-DES has very good internal consistency, test-retest reliability and a good validity, though its predictive power is limited. The ADES scores significantly correlate with the measure of somatoform dissociation, a presence of clinician-observed dissociative symptoms, reported traumatic experiences, self injurious behavior, and polysymptomatic diagnostic picture. A-DES scores were significantly higher in ADHD group, but not in a group with a diagnosis of a dissociative disorder. CONCLUSION: The authors stress that all adolescent psychiatric patients who show more complex behavioral disturbances, have histories of trauma, show self-injurious behaviors or have ADHD diagnosis should be screened for dissociation.

Analysis of vacancy and interstitial nucleation kinetics in Si wafers during rapid thermal annealing.

The successful application of the rapid thermal annealing (RTA) process for creation of a magic denude zone in individual Czochralski silicon wafers is based on vacancy controlled oxygen precipitation. The kinetics of the vacancy and self-interstitial processes in Si wafers are studied in this paper. Detailed insight into nucleation processes, out-diffusion and vacancy-interstitial recombination during the RTA leads to a new model of interaction between vacancies and oxygen. The calculation of the distribution function of these defects follows from modified Becker-Döring equations transformed for vacancies and interstitials and extended by diffusion and recombination terms. The new model, which includes the vacancy influence on oxygen nucleation and which follows from this theoretical analysis, corresponds very well to the experimental properties of formation of bulk micro-defects during RTA processes.

Influence of latent toxoplasmosis on the secondary sex ratio in mice.

The sex ratio may be influenced by many factors, such as stress and immunosuppression, age of parents, parity and sex of preceding siblings. In animal systems, parasitism often changes the sex ratio of infected hosts, which can increase the probability of their transmission. The most common human protozoan parasite in developed countries, Toxoplasma gondii (prevalence 20%-80%), is known to change the behaviour of its intermediate hosts, thereby increasing the probability of transmission to its definitive host (the cat) by predation. The intermediate hosts, which under natural conditions are rodents, serve as the vector for Toxoplasma. Therefore, we speculate that Toxoplasma can alter the secondary sex ratio (i.e. male to female ratio in the offspring) of infected females to increase the proportion of (congenitally infected) male offspring, which are the more migratory sex in most rodent species. Here we studied the sex ratio of experimentally infected laboratory mice, expressed here as the proportion of males in the litter. In accordance with our hypothesis and results of previous retrospective cohort studies on human subjects, mice with toxoplasmosis produced a higher sex ratio than controls, in the early phase of latent infection. In the later phase of infection, mice with congenital toxoplasmosis had a lower sex ratio than controls, which is in accord with the Trivers-Willard hypothesis of sex ratio manipulation, suggesting that females in poor physical condition give birth to more female offspring.

The frequency of alleles of the Pro12Ala polymorphism in PPARgamma2 is different between healthy controls and patients with type 2 diabetes.

The aim of this initial case-control study was to determine the association between common Pro12Ala polymorphism in the PPARgamma2 gene and type 2 diabetes in the Czech Republic. Furthermore, the effect of this polymorphism on phenotypic characteristics and on levels of lipids (total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides) was studied. One hundred thirty-three patients with type 2 diabetes and 97 control subjects were investigated. PCR and RFLP analysis were used for identification of individual genotypes. In the group of patients, three samples (2.26%) were identified as homozygous for the Ala/Ala genotype and 99 samples (74.44%) were homozygotes for the Pro/Pro genotype. Thirty-one samples (23.31%) were identified as Pro12Ala heterozygous. In the control group, six samples (6.19%) were homozygous for the Ala/Ala genotype and 61 samples (62.89%) were homozygotes for the Pro/Pro genotype. Thirty samples (30.93%) were identified as Pro12Ala heterozygous. The allele frequency for the Ala allele was lower in the type 2 diabetic group than in the control group (13.91% vs. 21.43%, P = 0.022). There was no difference (at P < 0.05) between the phenotypic characteristics (BMI, sex) studied in the group of patients according to the Pro12Ala genotype. There was no significant effect of the Pro12Ala polymorphism on lipid levels.