The dynamics of telomere length in primary and metastatic colorectal cancer lesions.

Telomeric sequences, the structures comprised of hexanucleotide repeats and associated proteins, play a pivotal role in chromosome end protection and preservation of genomic stability. Herein we address telomere length (TL) dynamics in primary colorectal cancer (CRC) tumour tissues and corresponding liver metastases. TL was measured by multiplex monochrome real-time qPCR in paired samples of primary tumours and liver metastases along with non-cancerous reference tissues obtained from 51 patients diagnosed with metastatic CRC. Telomere shortening was observed in the majority of primary tumour tissues compared to non-cancerous mucosa (84.1%, p < 0.0001). Tumours located within the proximal colon had shorter TL than those in the rectum (p < 0.05). TL in liver metastases was not significantly different from that in primary tumours (p = 0.41). TL in metastatic tissue was shorter in the patients diagnosed with metachronous liver metastases than in those diagnosed with synchronous liver metastases (p = 0.03). The metastatic liver lesions size correlated with the TL in metastases (p < 0.05). Following the neoadjuvant treatment, the patients with rectal cancer had shortened telomeres in tumour tissue than prior to the therapy (p = 0.01). Patients with a TL ratio between tumour tissue and the adjacent non-cancerous mucosa of ≥ 0.387 were associated with increased overall survival (p = 0.01). This study provides insights into TL dynamics during progression of the disease. The results show TL differences in metastatic lesions and may help in clinical practice to predict the patient's prognosis.

Impact of Newborn Screening and Early Dietary Management on Clinical Outcome of Patients with Long Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency and Medium Chain Acyl-CoA Dehydrogenase Deficiency-A Retrospective Nationwide Study.

Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD/MTPD) and medium chain acyl-CoA dehydrogenase deficiency (MCADD) were included in the expanded neonatal screening program (ENBS) in Czechia in 2009, allowing for the presymptomatic diagnosis and nutritional management of these patients. The aim of our study was to assess the nationwide impact of ENBS on clinical outcome. This retrospective study analysed acute events and chronic complications and their severity in pre-ENBS and post-ENBS cohorts. In total, 28 children (12 before, 16 after ENBS) were diagnosed with LCHADD/MTPD (incidence 0.8/100,000 before and 1.2/100,000 after ENBS). In the subgroup detected by ENBS, a significantly longer interval from birth to first acute encephalopathy was observed. In addition, improvement in neuropathy and cardiomyopathy (although statistically non-significant) was demonstrated in the post-ENBS subgroup. In the MCADD cohort, we included 69 patients (15 before, 54 after ENBS). The estimated incidence rose from 0.7/100,000 before to 4.3/100,000 after ENBS. We confirmed a significant decrease in the number of episodes of acute encephalopathy and lower proportion of intellectual disability after ENBS (p < 0.0001). The genotype-phenotype correlations suggest a new association between homozygosity for the c.1528C > G variant and more severe heart involvement in LCHADD patients.

ELISA kit for mustard protein determination: interlaboratory study.

An interlaboratory study in 12 laboratories was performed to prove the validation of the ELISA method developed for the quantitative determination of mustard protein in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. This kit did not produce any false-positive results or cross-reactivity with in-house validation for a broad range of food matrixes with no detectable mustard protein. All participants obtained the Mustard ELISA kit with standard operational procedures, a list of samples, samples, and a protocol for recording test results. The study included 15 food samples and two spiked samples. Seven food matrix samples of zero mustard content and four samples with mustard declared as an ingredient showed mustard protein content lower than that of the first standard (0.42 mg/kg). Four samples with mustard declared as an ingredient revealed mustard protein content above 12.5 mg/kg (the highest standard). The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as an LOQ (1.8 mg mustard proteins/kg) and LOD (0.5 mg mustard proteins/kg), for the kit were calculated.

ELISA kit for casein determination: interlaboratory study.

An interlaboratory study was performed in eight laboratories to validate an ELISA method developed for quantitative determination of casein in foods. The ELISA kit used is based on rabbit polyclonal antibodies. The kit is quite specific; no false-positive results or cross-reactivities were obtained for a broad range of food matrixes with zero content of milk proteins. All participants in the study received the casein kit, which included a standard operating procedure, a list of the samples, the samples, and a protocol for recording test results. The study included nine food samples: wheat flour, buckwheat flour, instant potato purée with milk, instant coffee with sugar and cream, a mixture for fancy bread, salami, liver paté, chocolate muesli with nuts, and a mixture for gluten-free bread. Three food samples with zero content of milk proteins showed a casein content lower than the lowest casein standard (1.0 mg CAS/kg) in most laboratories and measurements (64%). In 98% of the cases, the casein content was lower than the estimated LOQ. Two food samples with no dairy ingredient declared on the ingredient list contained casein levels higher than the second casein standard (3.0 mg CAS/kg) and the third standard (10.0 mg CAS/kg), respectively. Four food samples containing milk as an ingredient tested positive, and three showed casein contents higher than the highest standard (30.0 mg CAS/kg). The statistical tests (Cochran, Dixon) and analysis of variance were used for evaluation of the interlaboratory study results. Repeatability and reproducibility limits as well as LOQ (1.8 mg CAS/kg) and LOD (0.5 mg CAS/kg) for the kit were calculated from the results of the interlaboratory study.

ELISA kit for determination of egg white proteins: interlaboratory study.

An interlaboratory study was conducted in 11 laboratories to validate an ELISA method developed for the quantitative determination of egg white proteins (EWPs) in foods. The ELISA kit used for this study is based on sheep polyclonal antibody. It does not produce any false-positive results or cross-reactivity in a broad food matrix range with zero EWP content. All participants obtained the Egg ELISA Kit-native with standard operational procedure and the list of samples, as well as the samples and a protocol for recording test results. The study included 10 food samples. Four samples of food matrix with zero EWP content showed EWP content lower than the first standard (EWP content 0.5 mg/kg). One sample of food matrix with zero EWP content revealed EWP content higher than standard 3 (1.5 mg EWP/kg). Five food samples containing EWP as an ingredient tested positive and one negative. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ (1.4 mg EWP/kg) and LOD (0.43 mg EWP/kg), were calculated for the kit.

Enzyme-linked immunosorbent assay kit for beta-lactoglobulin determination: interlaboratory study.

An interlaboratory study was performed in six laboratories to prove the validation of the ELISA method developed for quantitative determination of beta-lactoglobulin (BLG) in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. In-house validation of the kit did not produce false-positive results or cross-reactivity in a broad range of food matrixes containing no milk proteins. All participants obtained the BLG kit with a standard operational procedure, the list of the samples, samples, and a protocol for recording test results. The study included 14 food samples (extruded breakfast cereals, bread, two soy desserts, butter, chicken ham, chicken meat, wheat flour, long grain rice, jelly, two whey drinks, crackers, and bitter chocolate) and six spiked samples (two rice, two wheat flour, and two chicken meat). Nine samples of food matrixes containing no milk proteins showed BLG content lower than the first standard (0.15 mg/kg). Two samples of food matrixes with no milk proteins revealed BLG content higher than standard 3 (1.5 mg/100 g) and standard 4 (5.0 mg/100 g). Three food samples containing milk were tested as positive, and all spiked samples were evaluated as positive. The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as LOQ (0.22 mg BLG/kg) and LOD (0.07 mg BLG/kg), for the kit were calculated.

Substitutional mutations in the uncoupling protein-specific sequences of mitochondrial uncoupling protein UCP1 lead to the reduction of fatty acid-induced H+ uniport.

Mutants were constructed for mitochondrial uncoupling protein UCP1, with single or multiple substitutions within or nearby the UCP-signatures located in the first alpha-helix and second matrix-segment, using the QuickChange site directed mutagenesis protocol (Stratagene), and were assayed fluorometrically for kinetics of fatty acid (FA)-induced H+ uniport and for Cl- uniport. Their ability to bind 3H-GTP was also evaluated. The wild type UCP1 was associated with the FA-induced H+ uniport proportional to the added protein with a Km for lauric acid of 43 micro M and Vmax of 18 micro molmin(-1)(mg protein)(-1). Neutralization of Arg152 (in the second matrix-segment UCP-signature) led to approximately 50% reduction of FA affinity (reciprocal Km) and of Vmax for FA-induced H+ uniport. Halved FA affinity and 70% reduction of Vmax was found for the double His substitution outside the signature (H145L and H147L mutant). Neutralization of Asp27 in the first alpha-helix UCP-signature (D27V mutant) resulted in 75% reduction of FA affinity and approximately 50% reduction of Vmax, whereas the triple C24A and D27V and T30A mutant was fully non-functional (Vmax reduced by 90%). Interestingly, the T30A mutant exhibited only the approximately 50% reduced FA affinity but not Vmax. Cl- uniport and 3H-GTP binding were preserved in all studied mutants. We conclude that amino acid residues of the first alpha-helix UCP signature may be required to hold the intact UCP1 transport conformation. This could be valid also for the positive charge of Arg152 (second matrix-segment UCP signature), which may alternatively mediate FA interaction with the native protein.