Protective reaction of the myocardium in poisoning

Intensive synthesis of collagen-like substance was revealed in the rabbit myocardium during experimental diphtheria intoxication. It was more marked in the right ventricle 24 hours after the injection of diphtheria toxin. Since similar changes (the substance was mainly formed around blood vessels) have been observed in other cases of toxic myocardial alterations (i.e. ethanol intoxication, injection of pharmacological agents, etc.), it can be assumed that it is a standard protective reaction of the altered heart to the penetration of toxic agents from the blood into the myocardial tissue.     

Long-term platinum retention after treatment with cisplatin and oxaliplatin

Background  

The aim of this study was to evaluate long-term platinum retention in patients treated with cisplatin and oxaliplatin.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.     

Fibrinogen alpha chain O-glycopeptides as possible markers of urinary tract infection

Urinary tract infection (UTI) is the most common bacterial infection leading to substantial morbidity and considerable health care expenditures across all ages. Here we present an exploratory UPLC-MS study of human urine in the context of febrile, complicated urinary tract infection aimed to reveal and identify possible markers of a host response on infection. A UPLC-MS based workflow, taking advantage of Ultra High Resolution (UHR) Qq-ToF-MS, and multivariate data handling were applied to a carefully selected group of 39 subjects with culture-confirmed febrile Escherichia coli UTI. Using a combination of unsupervised and supervised multivariate modeling we have pinpointed a number of peptides specific for UTI. An unequivocal structural identification of these peptides, as O-glycosylated fragments of the human fibrinogen alpha 1 chain, required MS2 and MS3 experiments on two different MS platforms: ESI-UHR-Qq-ToF and ESI-ion trap, a blast search and, finally, confirmation was achieved by matching experimental tandem mass spectra with those of custom synthesized candidate-peptides.In conclusion, exploiting non-targeted UPLC-MS based approach for the investigation of UTI related changes in urine, we have identified and structurally characterized unique O-glycopeptides, which are, to our knowledge, the first demonstration of O-glycosylation of human fibrinogen alpha 1-chain.     

Risk stratification by residual enzyme activity after newborn screening for medium-chain acyl-CoA dehyrogenase deficiency: Data from a cohort study

ABSTRACT: BACKGROUND: Since the introduction of medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency in population newborn bloodspot screening (NBS) programs, subjects have been identified with variant ACADM (gene encoding MCAD enzyme) genotypes that have never been identified in clinically ascertained patients. It could be hypothesised that residual MCAD enzyme activity can contribute in risk stratification of subjects with variant ACADM genotypes. METHODS: We performed a retrospective cohort study of all patients identified upon population NBS for MCAD deficiency in the Netherlands between 2007-2010. Clinical, molecular, and enzymatic data were integrated. RESULTS: Eighty-four patients from 76 families were identified. Twenty-two percent of the subjects had a variant ACADM genotype. In patients with classical ACADM genotypes, residual MCAD enzyme activity was significantly lower (median 0%, range 0-8%) when compared to subjects with variant ACADM genotypes (range 0-63%; 4 cases with 0%, remainder 20-63%). Patients with (fatal) neonatal presentations before diagnosis displayed residual MCAD enzyme activities <1%. After diagnosis and initiation of treatment, residual MCAD enzyme activities <10% were associated with an increased risk of hypoglycaemia and carnitine supplementation. The prevalence of MCAD deficiency upon screening was 1/8,750 (95% CI 1/7,210-1/11,130). CONCLUSIONS: Determination of residual MCAD enzyme activity improves our understanding of variant ACADM genotypes and may contribute to risk stratification. Subjects with variant ACADM genotypes and residual MCAD enzyme activities <10% should be considered to have the same risks as patients with classical ACADM genotypes. Parental instructions and an emergency regimen will remain principles of the treatment in any type of MCAD deficiency, as the effect of intercurrent illness on residual MCAD enzyme activity remains uncertain. There are, however, arguments in favour of abandoning the general advice to avoid prolonged fasting in subjects with variant ACADM genotypes and 10% residual MCAD enzyme activity.     

Hypertension Is a Conditional Factor for the Development of Cardiac Hypertrophy in Type 2 Diabetic Mice

Background

Type 2 diabetes is frequently associated with co-morbidities, including hypertension. Here we investigated if hypertension is a critical factor in myocardial remodeling and the development of cardiac dysfunction in type 2 diabetic db/db mice.

Methods

Thereto, 14-wks-old male db/db mice and non-diabetic db/+ mice received vehicle or angiotensin II (AngII) for 4 wks to induce mild hypertension (n = 9–10 per group). Left ventricular (LV) function was assessed by serial echocardiography and during a dobutamine stress test. LV tissue was subjected to molecular and (immuno)histochemical analysis to assess effects on hypertrophy, fibrosis and inflammation.

Results

Vehicle-treated diabetic mice neither displayed marked myocardial structural remodeling nor cardiac dysfunction. AngII-treatment did not affect body weight and fasting glucose levels, and induced a comparable increase in blood pressure in diabetic and control mice. Nonetheless, AngII-induced LV hypertrophy was significantly more pronounced in diabetic than in control mice as assessed by LV mass (increase +51% and +34%, respectively, p<0.01) and cardiomyocyte size (+53% and +31%, p<0.001). This was associated with enhanced LV mRNA expression of markers of hypertrophy and fibrosis and reduced activation of AMP-activated protein kinase (AMPK), while accumulation of Advanced Glycation End products (AGEs) and the expression levels of markers of inflammation were not altered. Moreover, AngII-treatment reduced LV fractional shortening and contractility in diabetic mice, but not in control mice.

Conclusions

Collectively, the present findings indicate that type 2 diabetes in its early stage is not yet associated with adverse cardiac structural changes, but already renders the heart more susceptible to hypertension-induced hypertrophic remodeling.     

Stimulation by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine of rat hepatic polyamine biosynthesis in vivo

Intraperitoneal injection of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), resulted in a rapid and transient induction of rat hepatic ornithine decarboxylase (ODC) activity. Maximal activity was found about 5 hr after application. The levels of putrescine and spermidine increased accordingly, reaching a maximum at 7 and 12 hr following injection, respectively, while the concentration of spermine remained almost constant. The implications of these findings are discussed in relation to the mechanism of induction of ornithine decarboxylase and concomitant polyamine biosynthesis.