Nonviral vector-based gene transfection of primary human skeletal myoblasts

Low-level transgene efficiency is one of the main obstacles in ex vivo nonviral vector-mediated gene transfer into primary human skeletal myoblasts (hSkMs). We optimized the cholesterol:N-[1-(2, 3-dioleoyloxy)propyl]-N, N, N-trimethylammonium methylsulfate liposome (CD liposome) and 22-kDa polyethylenimine (PEI22)- and 25-kDa polyethylenimine (PEI25)-mediated transfection of primary hSkMs for angiogenic gene delivery. We found that transfection efficiency and cell viability of three nonviral vectors were cell passage dependent: early cell passages of hSkMs had higher transfection efficiencies with poor cell viabilities, whereas later cell passages of hSkMs had lower transfection efficiencies with better cell viabilities. Trypsinization improved the transfection efficiency by 20% to 60% compared with adherent hSkMs. Optimum gene transfection efficiency was found with passage 6 trypsinized hSkMs: transfection efficiency with CD lipoplexes was 6.99 +/- 0.13%, PEI22 polyplexes was 18.58 +/- 1.57%, and PEI25 polyplexes was 13.32 +/- 0.88%. When pEGFP (a plasmid encoding the enhanced green fluorescent protein) was replaced with a vector containing human vascular endothelial growth factor 165 (phVEGF(165)), the optimized gene transfection conditions resulted in hVEGF(165) expression up to Day 18 with a peak level at Day 2 after transfection. This study demonstrated that therapeutic angiogenic gene transfer through CD or PEI is feasible and safe after optimization. It could be a potential strategy for treatment of ischemic disease for angiomyogenesis.     

Ultraviolet screening by slug tissue and tight packing of plastids protect photosynthetic sea slugs from photoinhibition

Photosynthesis Research - One of the main mysteries regarding photosynthetic sea slugs is how the slug plastids handle photoinhibition, the constant light-induced damage to Photosystem II of...     

Quantitative determination of dehydroepiandrosterone fatty acyl esters in human female adipose tissue and serum using mass spectrometric methods

Dehydroepiandrosterone-fatty acyl esters (DHEA-FAE) are naturally occurring water-insoluble metabolites of DHEA, which are transported in plasma exclusively by lipoproteins. To find out whether DHEA, like estradiol, might be stored in adipose tissue in FAE form, we set up a mass spectrometric method to quantify DHEA-FAE and free DHEA in human adipose tissue and serum. The method consists of chromatographic purification steps and final determination of hydrolyzed DHEA-FAE and free DHEA, which was carried out by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our results showed that no detectable amounts of DHEA-FAE could be found in adipose tissue although 32-178 pmol/g of free DHEA were determined by GC-MS and LC-MS/MS. The DHEA-FAE concentrations in serum quantified by GC-MS were 1.4±0.7 pmol/ml in premenopausal women (n=7), and 0.9±0.4 pmol/ml in postmenopausal women (n=5). Correspondingly, the free DHEA concentrations were 15.2±6.3 pmol/ml and 6.8±3.0 pmol/ml. In addition, the mean proportions of DHEA-FAE of total DHEA (DHEA-FAE+free DHEA) in serum were 8.6% and 11.2% in pre- and postmenopausal women, respectively. Serum DHEA-FAE concentration was below quantification limit for LC-MS/MS (signal-to-noise ratio, S/N=10), while free DHEA concentrations varied between 5.8 and 23.2 pmol/ml. In conclusion, the proportion of DHEA-FAE of total DHEA in serum was approximately 9%. However, in contrast to our previous findings for estradiol fatty acid esters in adipose tissue which constituted about 80% of total estradiol (esterified+free), the proportion of DHEA-FAE of total DHEA was below 5%. Four to ten times higher concentrations of free DHEA were quantified in adipose tissue compared to those in serum.     

A trade-off between current and future sex allocation revealed by maternal energy budget in a small mammal

Sex-allocation theories generally assume differential fitness costs of raising sons and daughters. Yet, experimental confirmation of such costs is scarce and potential mechanisms are rarely addressed. While the most universal measure of physiological costs is energy expenditure, only one study has related the maternal energy budget to experimentally controlled offspring sex. Here, we experimentally test this in the bank vole (Myodes glareolus) by simultaneously manipulating the litter's size and sex ratio immediately after birth. Two weeks after manipulation, when mothers were at the peak of lactation and were pregnant with concurrent litters, we assessed their energy budget. We found that maternal food consumption and daily energy expenditure increased with the size of the litters being lactated. Importantly, the effects of offspring sex on energy budget depended on the characteristics of the simultaneously gestating litters. Specifically, the mothers nursing all-male litters and concurrently pregnant with male-biased litters had the highest energy expenditure. These had consequences for the next generation, as size of female offspring from the concurrent pregnancy of these mothers was compromised. Our study attests a higher cost of sons, consequently leading to a lower investment in them, and reveals the significance of offspring sex in moulding the trade-off between current and future maternal investment.     

Autologous stem cells for the treatment of post-mastectomy lymphedema: a pilot study

BACKGROUND AIMS. Lymphedema is a common complication with breast cancer treatment that does not have a definite cure. Our objective was to determine the efficacy of autologous stem cells (ASC) in the treatment of lymphedema secondary to mastectomy and axillary lymphadenectomy in comparison with traditional decongestive treatment with compression sleeves. METHODS. A prospective study including 20 women with lymphedema secondary to breast cancer surgery with axillary lymphadenectomy was conducted. Women were assigned at random to one of two groups. One group of 10 women was injected with ASC in the affected arm, whereas the other 10 women comprised the control group and received traditional compression sleeve therapy (CST). The follow-up for both groups was 12 weeks. Pain, sensitivity and mobility were assessed before and after therapy. RESULTS. There was improvement in the volume of lymphedema in both groups, with no significant difference. In the ASC group there was an overall volume reduction during the follow-up, whereas in the CST group lymphedema recurred after the compression sleeve was removed. CONCLUSIONS. Our findings suggest that ASC injection for patients with lymphedema can be an effective treatment. It reduces arm volume and associated co-morbidities of pain and decreased sensitivity. Traditional CST was also effective for lymphedema reduction, but it was dependent on continuous use of the treatment.     

New insights into Alzheimer's disease progression: a combined TMS and structural MRI study

Background:

Combination of structural and functional data of the human brain can provide detailed information of neurodegenerative diseases and the influence of the disease on various local cortical areas.

Methodology and Principal Findings:

To examine the relationship between structure and function of the brain the cortical thickness based on structural magnetic resonance images and motor cortex excitability assessed with transcranial magnetic stimulation were correlated in Alzheimer''s disease (AD) and mild cognitive impairment (MCI) patients as well as in age-matched healthy controls. Motor cortex excitability correlated negatively with cortical thickness on the sensorimotor cortex, the precuneus and the cuneus but the strength of the correlation varied between the study groups. On the sensorimotor cortex the correlation was significant only in MCI subjects. On the precuneus and cuneus the correlation was significant both in AD and MCI subjects. In healthy controls the motor cortex excitability did not correlate with the cortical thickness.

Conclusions:

In healthy subjects the motor cortex excitability is not dependent on the cortical thickness, whereas in neurodegenerative diseases the cortical thinning is related to weaker cortical excitability, especially on the precuneus and cuneus. However, in AD subjects there seems to be a protective mechanism of hyperexcitability on the sensorimotor cortex counteracting the prominent loss of cortical volume since the motor cortex excitability did not correlate with the cortical thickness. Such protective mechanism was not found on the precuneus or cuneus nor in the MCI subjects. Therefore, our results indicate that the progression of the disease proceeds with different dynamics in the structure and function of neuronal circuits from normal conditions via MCI to AD.     

Severe acute respiratory syndrome coronavirus fails to activate cytokine-mediated innate immune responses in cultured human monocyte-derived dendritic cells

Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-alpha], IFN-beta, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-alpha/beta-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.     

Localization of plasma membrane t-SNAREs syntaxin 2 and 3 in intracellular compartments

Background  

Membrane fusion requires the formation of a complex between a vesicle protein (v-SNARE) and the target membrane proteins (t-SNAREs). Syntaxin 2 and 3 are t-SNAREs that, according to previous over-expression studies, are predominantly localized at the plasma membrane. In the present study we investigated localization of the endogenous syntaxin 2 and 3.     

Evidence for the role of the oxygen-evolving manganese complex in photoinhibition of Photosystem II

Photoinhibition of PSII occurs at the same quantum efficiency from very low to very high light, which raises a question about how important is the rate of photosynthetic electron transfer in photoinhibition. We modulated electron transfer rate and light intensity independently of each other in lincomycin-treated pea leaves and in isolated thylakoids, in order to elucidate the specific effects of light and PSII electron transport on photoinhibition. Major changes in the rate of electron transport caused only small changes in the rate of photoinhibition, suggesting the existence of a significant photoinhibitory pathway that contains an electron-transfer-independent phase. We compared the action spectrum of photoinhibition with absorption spectra of PSII components that could function as photoreceptors of the electron-transfer-independent phase of photoinhibition and found that the absorption spectra of Mn(III) and Mn(IV) compounds resemble the action spectrum of photoinhibition, showing a steep decrease from UV-C to blue light and a low visible-light tail. Our results show that the release of a Mn ion to the thylakoid lumen is the earliest detectable step of both UV- and visible-light-induced photoinhibition. After Mn release from the oxygen-evolving complex, oxidative damage to the PSII reaction center occurs because the Mn-depleted oxygen-evolving complex cannot reduce P680+ normally.