Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.     

The magnetosome membrane protein, MmsF, is a major regulator of magnetite biomineralization in Magnetospirillum magneticum AMB-1

Magnetotactic bacteria (MTB) use magnetosomes, membrane-bound crystals of magnetite or greigite, for navigation along geomagnetic fields. In Magnetospirillum magneticum sp. AMB-1, and other MTB, a magnetosome gene island (MAI) is essential for every step of magnetosome formation. An 8-gene region of the MAI encodes several factors implicated in control of crystal size and morphology in previous genetic and proteomic studies. We show that these factors play a minor role in magnetite biomineralization in vivo. In contrast, MmsF, a previously uncharacterized magnetosome membrane protein encoded within the same region plays a dominant role in defining crystal size and morphology and is sufficient for restoring magnetite synthesis in the absence of the other major biomineralization candidates. In addition, we show that the 18 genes of the mamAB gene cluster of the MAI are sufficient for the formation of an immature magnetosome organelle. Addition of MmsF to these 18 genes leads to a significant enhancement of magnetite biomineralization and an increase in the cellular magnetic response. These results define a new biomineralization protein and lay down the foundation for the design of autonomous gene cassettes for the transfer of the magnetic phenotype in other bacteria.     

Biogenesis and subcellular organization of the magnetosome organelles of magnetotactic bacteria

Bacterial cells, like their eukaryotic counterparts, are capable of constructing lipid-based organelles that carry out essential biochemical functions. The magnetosomes of magnetotactic bacteria are one such compartment that is quickly becoming a model for exploring the process of organelle biogenesis in bacteria. Magnetosomes consist of a lipid-bilayer compartment that houses a magnetic crystal. By arranging magnetosomes into chains within the cell, magnetotactic bacteria create an internal compass that is used for navigation along magnetic fields. Over the past decade, a number of studies have elucidated the possible factors involved in the formation of the magnetosome membrane and biomineralization of magnetic minerals. Here, we highlight some of these recent advances with a particular focus on the cell biology of magnetosome formation.     

Ghrelin regulates Bax and PCNA but not Bcl-2 expressions following scrotal hyperthermia in the rat

More recently, we have reported the beneficial effects of ghrelin in improvement of histopathological features of the rat testis following local heat exposure. However, the exact mechanism and the precise role of apoptosis- and proliferation-specific proteins in this regeneration process remained to be explored. Thus, thirty adult male Wistar rats were allotted for the experiment and subdivided equally into three groups: control-saline (CS), heat-saline (HS) and heat-ghrelin (HG). The scrota of HS and HG groups were immersed once in water bath at 43 °C for 15 min. HG animals received 2 nmol of ghrelin subcutaneously immediately after heating every other day until day 60 and the other groups were given physiological saline using the same method. The testes of all groups were taken after rat killing on days 30 and 60 after heat treatment for immunocytochemical detection of pro-apoptotic factor Bax, anti-apoptotic protein Bcl-2 and proliferation-associated peptide PCNA in the germ cells. Ghrelin could significantly suppress the Bax expression in spermatocytes compared to the HS group at day 30 (P < 0.05). Likewise, the mean percentages of spermatogonia containing Bax substance were lower in ghrelin-exposed animals, however the differences were not statistically significant. There were immunoreactive cells against Bcl-2 in each germ cell neither in the control nor in the heated animals of experimental groups. In contrast, the number of PCNA immunolabeling cells were higher in HG group in compared to HS or CS animals on both experimental days (P < 0.001). Down-regulation of Bax expression concurrent with overexpression of PCNA in HG group indicates the ability of ghrelin in acceleration of testicular germ cells regeneration following heat stress. These findings indicate that ghrelin may be used as a novel and efficient antioxidant agent to induce resumption of spermatogenesis upon environmental heat exposure.     

The effect of ghrelin pretreatment on epididymal sperm quality and tissue antioxidant enzyme activities after testicular ischemia/reperfusion in rats

Ghrelin, the endogenous ligand for growth hormone secretagogue receptor, has been reported to prevent ischemia/reperfusion (I/R) injury in various tissues by its antioxidant activity. Therefore, this study was aimed to investigate the effect of ghrelin on sperm quality and antioxidant enzyme activity in a rat testicular ischemia/reperfusion injury model. Forty-two male Wistar rats were divided into groups control, I/R, and I/R plus ghrelin. The right testes were rotated 720° for 1 h and were allowed to reperfuse for 4 h and 30 days thereafter. Ghrelin (40 nmol/kg IP) or vehicle (physiological saline) was administrated 15 min before reperfusion. After 4 h of reperfusion, a right orchiectomy was performed to measure the biochemical parameters. In addition, the sperm was collected from the epididymis after 30 days of reperfusion, and sperm characteristics were examined. The malondialdehyde levels of the testis tissues were significantly increased, but a statistically significant decrease was found in the superoxide dismutase, glutathione peroxidase, and catalase activities in the I/R group as compared with the control, indicating I/R injury. The sperm evaluation showed a significant reduction in all characteristics resulted from I/R compared with the control. In the ghrelin-treated group, the malondialdehyde values were significantly lowered, and only enzyme activity of glutathione peroxidase showed significant increases compared with the I/R group. Ghrelin significantly enhanced sperm motility, movement, and concentration but did not prevent I/R-induced reduction in membrane integrity in the testes of rats compared to the I/R group. Our results suggest that ghrelin treatment has a protective role on IR-induced testicular injury, and this effect may be due to its antioxidant properties.     

Single tube real time PCR for detection of Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila from clinical samples of CAP

We designed a multiplex real time PCR for rapid, sensitive and specific detection of Streptococcus pneumoniae, Legionella pneumophila, Chlamydophila pneumoniae and Mycoplasma pneumoniae. The study cases consisted of 129 patients with community acquired pneumonia (CAP). Bacteriological techniques were implemented for detection of the cultivable organisms. DNA were extracted from sputa, throat swabs, bronchoalveolar lavages and tracheal aspirates and used as templates in real time PCR. The primers and probes were designed for cbpA (S. pneumoniae), p1adhesin (M. pneumoniae), mip (L. pneumophila) and ompA (C. pneumoniae). After optimization of real time PCR for every organism, the experiments were continued in multiplex in a single tube. Of 129 CAP specimens, the positive cultures included 14 (10.85%) for S. pneumoniae, 9 (6.98%) for L. pneumophila and 3 (2.33%) for M. pneumoniae. Four specimens (3.10%) were positive for C. pneumoniae by real time PCR. The sensitivity of our real time PCR was 100% for all selected bacteria. The specificity of the test was 98.26%, 98.34%, 100% and 100% for S. pneumoniae, L. pneumophila, M. pneumoniae and C. pneumoniae, respectively. This is the first report on the use of multiplex real time PCR for detection of CAP patients in the Middle East. The method covers more than 90% of the bacterial pathogens causing CAP.     

Response of high-sensitive C-reactive protein to catheter ablation of atrial fibrillation and its relation with rhythm outcome

Aims

This study investigated the possible association between hs-CRP as well as hs-CRP changes and rhythm outcome after AF catheter ablation.

Methods

We studied 68 consecutive patients with AF undergoing catheter ablation. hs-CRP levels were measured using commercially available assays before and 6 months after catheter ablation. Serial 7-day Holter ECGs were used to detect AF recurrences.

Results

Early AF recurrence (ERAF, within one week) was observed in 38%, while late AF recurrence (LRAF, between 3 and 6 months) occurred in 18% of the patients. None of the baseline clinical or echocardiographic variables was predictive of ERAF or LRAF. Baseline hs-CRP measured 2.07±1.1 µg/ml and was not associated with ERAF and LRAF. At 6 months, hs-CRP levels were comparable with baseline values (2.14±1.19 µg/ml, p = 0.409) and were also not related with LRAF. However, patients with LRAF showed an hs-CRP increase from 2.03±0.61 to 2.62±1.52 µg/ml (p = 0.028). Patients with an hs-CRP change in the upper tertile (>0.2 µg/ml) had LRAF in 32% as opposed to 11% (p = 0.042) in patients in the lower (<−0.3 µg/ml) or intermediate (−0.3–0.2 µg/ml) tertile.

Conclusions

Changes in hs-CRP but not baseline hs-CRP are associated with rhythm outcome after AF catheter ablation. This finding points to a link between an inflammatory response and AF recurrence in this setting.     

Variable patterns of programmed death-1 expression on fully functional memory T cells after spontaneous resolution of hepatitis C virus infection

The inhibitory receptor programmed death-1 (PD-1) is present on CD8(+) T cells in chronic hepatitis C virus (HCV), but expression patterns in spontaneously resolving infections are incompletely characterized. Here we report that PD-1 was usually absent on memory CD8(+) T cells from chimpanzees with resolved infections, but sustained low-level expression was sometimes observed in the absence of apparent virus replication. PD-1-positive memory T cells expanded and displayed antiviral activity upon reinfection with HCV, indicating conserved function. This animal model should facilitate studies of whether PD-1 differentially influences effector and memory T-cell function in resolved versus persistent human infections.