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.     

Limited DNA elimination from the irradiated potato parent in fusion products of albino Lycopersicon esculentum and Solanum tuberosum

Summary This paper describes the analysis of the elimination of potato DNA from potato-tomato somatic cell hybrids. The hybrids were obtained by fusion of protoplasts of a cytoplasmic albino tomato genotype with leaf mesophyll protoplasts of a potato genotype carrying the -glucuronidase (GUS) gene of Escherichia coli. The potato protoplasts were either isolated from unirradiated plants or from plants irradiated with 50 or 500 Gy of -rays. Green calli were selected as putative fusion products. The hybridity of these calli was confirmed by isoenzyme analysis. All of the green calli tested contained a potato-specific chloroplast DNA restriction fragment, and most of the calli analysed were positive for -glucuronidase activity. In 72 of the hybrid calli we determined the percentage of potato nuclear DNA using species-specific probes. All of the tested green calli contained a considerable amount of potato genomic DNA, irrespective of the dose of irradiation of the potato parent. The limited degree of potato DNA elimination and the absence of true cybrids are discussed.     

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.     

Living on the edge: substrate competition explains loss of robustness in mitochondrial fatty-acid oxidation disorders

Background

Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders.

Results

First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients’ metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach.

Conclusion

We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.

     

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.     

<Emphasis Type="Italic">TRAF1/C5</Emphasis>polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies

Introduction  

Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients.     

Mesenchymal stem cells in autoimmune diseases: hype or hope?

Intervention with mesenchymal stem cells (MSCs) represents a promising therapeutic tool in treatment-refractory autoimmune diseases. A new report by Schurgers and colleagues in a previous issue of Arthritis Research & Therapy sheds novel mechanistic insight into the pathways employed by MSCs to suppress T-cell proliferation in vitro, but, at the same time, indicates that MSCs do not influence T-cell reactivity and the disease course in an in vivo arthritis model. Such discrepancies between the in vitro and in vivo effects of potent cellular immune modulators should spark further research and should be interpreted as a sign of caution for the in vitro design of MSC-derived interventions in the setting of human autoimmune diseases.     

Pentacycloundecane lactam vs lactone norstatine type protease HIV inhibitors: binding energy calculations and DFT study

Background

Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC₅₀ values of less than 1.00 μM. A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC₅₀) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.

Results

The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide (IC₅₀ = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC₅₀ = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆G_solv) were also considered.

Conclusions

A general trend is observed that the lactam species appears to have a larger negative charge distribution around the heteroatoms, larger quadrupole constant, dipole moment and better solvation energy, in comparison to the PCU-lactone model. It can be argued that these characteristics will ensure better eletronic interaction between the lactam and the receptor, corresponding to the observed HIV protease activities in terms of experimental IC₅₀ data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0115-5) contains supplementary material, which is available to authorized users.     

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.