Rederivation of transgenic mice from iPS cells derived from frozen tissue

In mice, induced pluripotent stem (iPS) cells with embryonic stem (ES)-like characteristics have been derived by ectopic expression of four transcription factors in somatic cells: Sox2, Oct3/4, Klf4 and/or c-Myc. To date, iPS cells have only be made from freshly harvested tissues and cells. However, if iPS cells could be derived from frozen tissues and cells, then cryopreservation of tissues such as mouse tails could conceivably become a reliable alternative to the more traditional formats, like germplasm and ES cells, for the archiving of genetically altered mouse lines. To test this hypothesis, we sought to demonstrate that a live transgenic mouse line could be recovered from transgenic iPS cells derived from cryopreserved mouse tissues. Tails and tail-derived fibroblasts from a DsRED transgenic mouse were cryopreserved in the presence of 5% dimethylsulfoxide (DMSO) in liquid nitrogen for 1 week and 1 month, respectively. Afterward, tissues and cells were thawed and underwent nuclear reprogramming by molecular transfection to derive iPS cells which generated germline confirmed transgenic mice. Our results demonstrate for the first time that iPS cells can be efficiently derived from frozen-stored-thawed tail tissue and fibroblasts and used to re-establish a transgenic mouse line. Therefore, this study provides conclusive evidence that, as a practical matter, frozen tails and fibroblasts can be used as an effective and reliable alternative to frozen germplasm and ES cells for the storage, maintenance, and distribution of genetically-altered mutant mice

Circulating CD133+VEGFR2+ and CD34+VEGFR2+ cells and arterial function in patients with beta-thalassaemia major

Arterial dysfunction has been documented in patients with beta-thalassaemia major. This study aimed to determine the quantity and proliferative capacity of circulating CD133+VEGFR2+ and CD34+VEGFR2+ cells in patients with beta-thalassaemia major and those after haematopoietic stem cell transplantation (HSCT), and their relationships with arterial function. Brachial arterial flow-mediated dilation (FMD), carotid arterial stiffness, the quantity of these circulating cells and their number of colony-forming units (CFUs) were determined in 17 transfusion-dependent thalassaemia patients, 14 patients after HSCT and 11 controls. Compared with controls, both patient groups had significantly lower FMD and greater arterial stiffness. Despite having increased CD133+VEGFR2+ and CD34+VEGFR2+ cells, transfusion-dependent patients had significantly reduced CFUs compared with controls (p = 0.002). There was a trend of increasing CFUs across the three groups with decreasing iron load (p = 0.011). The CFUs correlated with brachial FMD (p = 0.029) and arterial stiffness (p = 0.02), but not with serum ferritin level. Multiple linear regression showed that CFU was a significant determinant of FMD (p = 0.043) and arterial stiffness (p = 0.02) after adjustment of age, sex, body mass index, blood pressure and serum ferritin level. In conclusion, arterial dysfunction found in patients with beta-thalassaemia major before and after HSCT may be related to impaired proliferation of CD133+VEGFR2+ and CD34+VEGFR2+ cells

Uncoupling Protein-4 (UCP4) Increases ATP Supply by Interacting with Mitochondrial Complex II in Neuroblastoma Cells

Mitochondrial uncoupling protein-4 (UCP4) protects against Complex I deficiency as induced by 1-methyl-4-phenylpyridinium (MPP+), but how UCP4 affects mitochondrial function is unclear. Here we investigated how UCP4 affects mitochondrial bioenergetics in SH-SY5Y cells. Cells stably overexpressing UCP4 exhibited higher oxygen consumption (10.1%, p<0.01), with 20% greater proton leak than vector controls (p<0.01). Increased ATP supply was observed in UCP4-overexpressing cells compared to controls (p<0.05). Although state 4 and state 3 respiration rates of UCP4-overexpressing and control cells were similar, Complex II activity in UCP4-overexpressing cells was 30% higher (p<0.05), associated with protein binding between UCP4 and Complex II, but not that of either Complex I or IV. Mitochondrial ADP consumption by succinate-induced respiration was 26% higher in UCP4-overexpressing cells, with 20% higher ADP:O ratio (p<0.05). ADP/ATP exchange rate was not altered by UCP4 overexpression, as shown by unchanged mitochondrial ADP uptake activity. UCP4 overexpression retained normal mitochondrial morphology in situ, with similar mitochondrial membrane potential compared to controls. Our findings elucidate how UCP4 overexpression increases ATP synthesis by specifically interacting with Complex II. This highlights a unique role of UCP4 as a potential regulatory target to modulate mitochondrial Complex II and ATP output in preserving existing neurons against energy crisis

Pharmaceuticals and personal care products' (PPCPs) impact on enriched nitrifying cultures

The impact of pharmaceutical and personal care products (PPCPs) on the performance of biological wastewater treatment plants (WWTPs) has been widely studied using whole-community approaches. These contaminants affect the capacity of microbial communities to transform nutrients; however, most have neither honed their examination on the nitrifying communities directly nor considered the impact on individual populations. In this study, six PPCPs commonly found in WWTPs, including a stimulant (caffeine), an antimicrobial agent (triclosan), an insect repellent ingredient (N,N-diethyl-m-toluamide (DEET)) and antibiotics (ampicillin, colistin and ofloxacin), were selected to assess their short-term toxic effect on enriched nitrifying cultures: Nitrosomonas sp. and Nitrobacter sp. The results showed that triclosan exhibited the greatest inhibition on nitrification with EC 50 of 89.1 μg L −1. From the selected antibiotics, colistin significantly affected the overall nitrification with the lowest EC 50 of 1 mg L −1, and a more pronounced inhibitory effect on ammonia-oxidizing bacteria (AOB) compared to nitrite-oxidizing bacteria (NOB). The EC 50 of ampicillin and ofloxacin was 23.7 and 12.7 mg L −1, respectively. Additionally, experimental data suggested that nitrifying bacteria were insensitive to the presence of caffeine. In the case of DEET, moderate inhibition of nitrification (<40%) was observed at 10 mg L −1. These findings contribute to the understanding of the response of nitrifying communities in presence of PPCPs, which play an essential role in biological nitrification in WWTPs. Knowing specific community responses helps develop mitigation measures to improve system resilience

Reconfigerable FPGA-based switching path frequency-domain echo canceller with applications to voice control device

Acoustic echo control is of vital interest for hands-free operation of telecommunications equipment. An important property of an acoustic echo canceller is its capability to handle double-talk and be able to operate in real time. When it is applied to intelligent voice control device, it is important to suppress the speech from the device and enhance the speech of the user for speech recognition, where double-talk situation is frequently occurred. In this paper, we propose a novel hardware architecture to support a robust adaptive algorithm in combination with a switching path model to tackle the double-talk situation. The proposed switching path model avoids adapting two filters at the same time during double-talk and prevents the disadvantage of the conventional two-path model. In order to achieve computational efficiency and to meet the rigorous timing requirements, the echo canceller is operated in the frequency domain and its computing power is raised by a hardware accelerator implemented in the FPGA fabric surrounding a PowerPC on a Xilinx XUP V2P platform. Results obtained show the echo canceller is successful in handling double-talk situation and the sub-band implementation has improved convergence significantly. An overall improvement by 82.5 times is achieved when a hardware accelerator is used to perform the critical part of the algorithm over a pure software implementation running on a 300 MHz embedded PowerPC processor

Investigation of Retinal Spatial Interaction Using mfERG Stimulation

Introduction: Adaptation is one of the key characteristic of our vision which can maximize the visual function. It applies to both spatial and temporal characteristics. The fast flickering stimulation characteristics of the multifocal electroretinogram (mfERG) can be applied to analyze retinal interactions between flashes and to investigate retinal temporal processing mechanism. Besides, its localized stimulus pattern can also be used as a tool for investigation of retinal spatial interaction. Methods: The mfERG recordings were obtained from 13 eyes of 9, normal, six-week-old Yorkshire pigs. The control mfERG was measured using the pattern consisting of 103 nonscaled hexagons, where each hexagon will follow a pre-set m-sequence. Nine isolated hexagons from the 103 nonscaled pattern were chosen in the masking mfERG stimulation, where the remaining hexagons were kept at constant luminance. First-order and the second-order kernel responses were analyzed, which represent the outer and inner retinal responses, respectively. Results: The second-order kernel response amplitude from the visual streak region showed a significant enhancement under the masking stimulation. Conclusions: The enhancement found under the masking condition indicates that the retinal signal will be suppressed under surrounding flicker stimulation, and this spatial inhibitory mechanism may originate from the inner retina