The infectious disease tuberculosis is one of the fifteen most common causes of death worldwide (according to the WHO). About every fourth person is infected with the main causative agent ...Mycobacterium tuberculosis (Mb). A characteristic of the pathogen is its entrance into a dormant state in which a phenotypic antibiotic resistance is achieved. To target resistant strains, novel dormancy-specific targets are very promising. Such a possible target is the Mb "fatty acid-CoA ligase 6" (MbFACL6), which activates fatty acids and thereby modulates the accumulation of triacylglycerol-containing lipid droplets that are used by Mb as an energy source during dormancy. We investigated the membrane association of MbFACL6 in E. coli and its specific activity towards different substrates after establishing a novel MbFACL6 activity assay. Despite a high homology to the mammalian family of fatty acid transport proteins, which are typically transmembrane proteins, our results indicate that MbFACL6 is a peripheral membrane-attached protein. Furthermore, MbFACL6 tolerates a broad spectrum of substrates including saturated and unsaturated fatty acids (C12-C20), some cholic acid derivatives, and even synthetic fatty acids, such as 9(E)-nitrooleicacid. Therefore, the substrate selectivity of MbFACL6 appears to be much broader than previously assumed.
Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic
maturation of mouse oocytes in vitro. We therefore compared ...FF-MAS-induced maturation of naked mouse oocytes arrested in prophase
I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame
the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively.
We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes
by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a
barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small
cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that
in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations
suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a
delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly
and by delaying CG release, which is known to contribute to reduced fertilization.
The intercellular binding of desmosomal junctions is mediated by cadherins of the desmoglein (Dsg) and desmocollin (Dsc) type. Dsg2 mutant mice with deletion of a substantial segment of the ...extracellular EC1-EC2 domain, which is believed to participate in homo- and heterophilic desmosomal cadherin interactions, develop cardiac fibrosis and ventricular dilation. Widening of the intercellular cleft and complete intercalated disc ruptures can be observed in the hearts of these mice. Since a reduced litter size of homozygous Dsg2 mutant mice was noted and a functional correlation between desmosomes and embryo implantation has been deduced from animal studies, we looked for an alteration of desmosomes in uterine endometrial epithelium. Shape and number of desmosomes as well as the expression of Dsg2 and the desmosomal plaque protein desmoplakin (Dsp) were investigated by electron microscopy and immunohistochemistry in 12 oestrous-dated mice (7 wild type and 5 homozygous Dsg2 mutant mice) at the age of 9–17 weeks. The immunohistochemical detection of Dsg2 was diminished in the mutants and the number of desmosomes was significantly reduced as revealed by electron microscopy. In addition, the intercellular desmosomal space measured in electron micrographs was considerably widened in the Dsg2 mutants. The increased intercellular spacing can be explained by the partial deletion of the extracellular EC1–EC2 domain of Dsg2. Whether these changes explain the reduced number of offspring of homozygous Dsg2 mutant mice remains to be further investigated.
Mice carrying a deletion of the adhesive extracellular domain of the desmosomal cadherin desmoglein 2 develop an arrhythmogenic right ventricular cardiomyopathy-like phenotype with ventricular ...dilation, fibrosis and arrhythmia. To unravel the sequence of myocardial alterations and to identify potential pathomechanisms, histological analyses were performed on mutant hearts from the juvenile to the adult state, i.e., between 2 and 13 weeks. At an age of 2 weeks 30% of mutants presented lesions, which were visible as white plaques on the heart surface or in the septum. From 4 weeks onwards, all mutants displayed a cardiac phenotype. Dying cardiomyocytes with calcification were found in lesions of all ages. But lesions of young mutant animals contained high amounts of CD45+ immune cells and little collagen fibers, whereas lesions of the older animals were collagen-rich and harbored only a small but still significantly increased number of CD45+ cells. Electron microscopy further showed that distinct desmosomes cannot be distinguished in intercalated discs of mutant hearts. Widening of the intercellular cleft and even complete dissociation of intercalated discs were often observed close to lesions. Disturbed sarcomer structure, altered Z-discs, multiple autophagic vacuoles and swollen mitochondria were other prominent pathological features. Taken together, the following scenario is suggested: mutant desmoglein 2 cannot fully support the increased mechanical requirements placed on intercalated disc adhesion during postnatal heart development, resulting in compromised adhesion and cell stress. This induces cardiomyocyte death, aseptic inflammation and fibrotic replacement. The acute stage of scar formation is followed by permanent impairment of the cardiac function.
To develop a method of freezing small amounts of spermatozoa in polymerized alginic acid drops, which can be liquified after thawing for recovery of the spermatozoa.
Prospective clinical study.
...Medical School, RWTH Aachen, Aachen Germany.
None.
Validation of the encapsulation method with bovine sperm; cryopreservation of human spermatozoa in alginic capsules.
We optimized the cryopreservation method by testing different parameters influencing the freezing procedure, such as concentration of alginic acid, size of drops, time of polymerization, and culture media.
The final protocol was as follows: encapsulation by 7.3 mg/mL alginic acid forming 10-muL drops polymerized for 30 seconds and liquefied for 2.5 minutes in sodium citrate. Cryopreservation of human spermatozoa by this protocol resulted in a decreased motility of 18.3% compared with standard protocols but a 19.9% higher vitality of the immotile spermatozoa.
No difference in viability of spermatozoa after both sperm-freezing procedures could be observed. Further investigation will be undertaken to reduce the amount of immotile but viable sperm after microencapsulation in alginic acid.
Circulating tumor cells (CTC) released into blood from primary cancers and metastases reflect the current status of tumor genotypes, which are prone to changes. Here, we conducted the first ...comprehensive genomic profiling of CTCs using array-comparative genomic hybridization (CGH) and next-generation sequencing. We used the U.S. Food and Drug Administration-cleared CellSearch system, which detected CTCs in 21 of 37 patients (range, 1-202/7.5 mL sample) with stage IV colorectal carcinoma. In total, we were able to isolate 37 intact CTCs from six patients and identified in those multiple colorectal cancer-associated copy number changes, many of which were also present in the respective primary tumor. We then used massive parallel sequencing of a panel of 68 colorectal cancer-associated genes to compare the mutation spectrum in the primary tumors, metastases, and the corresponding CTCs from two of these patients. Mutations in known driver genes e.g., adenomatous polyposis coli (APC), KRAS, or PIK3CA found in the primary tumor and metastasis were also detected in corresponding CTCs. However, we also observed mutations exclusively in CTCs. To address whether these mutations were derived from a small subclone in the primary tumor or represented new variants of metastatic cells, we conducted additional deep sequencing of the primary tumor and metastasis and applied a customized statistical algorithm for analysis. We found that most mutations initially found only in CTCs were also present at subclonal level in the primary tumors and metastases from the same patient. This study paves the way to use CTCs as a liquid biopsy in patients with cancer, providing more effective options to monitor tumor genomes that are prone to change during progression, treatment, and relapse.
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