Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

Summary A major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen–thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (≈98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 °C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4 °C, with >90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen–thawed hES cells after incubation at 37 °C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze–thawing with conventional slow-cooling protocols.     

Bioactivity assay of extracts from Calocedrus macrolepis var. formosana bark

Alcoholic extracts from bark of Calocedrus macrolepis var. formosana Florin (Cupressaceae) were extracted successively using n-hexane, dichloromethane, ethyl acetate, 1-butanol and water, which gave 34.8%, 34.1%, 24.1%, 3.3% and 3.7% soluble fractions, respectively. Antioxidation activity of these fractions by DPPH assay and dissimilar IC50 values of the DPPH showed that ethyl acetate fraction had the best antioxidant activity; its IC50 was 2.6 microg/ml. Analyses of the composition and anti-inflammatory activity of the subfractions from n-C6H14 fraction showed that the T3 and H5ppt had the best anti-inflammatory activity in LPS-stimulated murine macrophage J774A. 1 cells, respectively; moreover, their major constituent was sugiol (T3 37.1%, H5ppt 81.1%), which at dosages of 10 microg/ml inhibited proIL-1beta protein production completely. Furthermore, the T1 also exhibited anti-inflammatory activity, and its major constituent was ferruginol (above 85.6%).     

Improved efficiency of separate hexose and pentose fermentation from steam-exploded corn stalk for butanol production using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

Water extract of steam-exploded corn stalk (SECS) was detoxified and used as feed for acetone–butanol–ethanol (ABE) fermentation using Clostridium beijerinckii. Utilization of water extract improved the total ABE yield (g ABE/g dry SECS). Separated fermentation showed higher fermentability (0.078 g ABE/g dry SECS) over typical fermentation (0.058 g ABE/g dry SECS). Furthermore, the final ABE yields (g ABE/g utilized sugar) from water extract neutralized by Ca(OH)₂, NaOH, and Na₂SO₃ were 0.16, 0.1 and 0.07, respectively, suggesting that Ca(OH)₂ had the best detoxification effect.     

TNF-α和IL-13对人肺成纤维细胞胶原蛋白生成的影响

以胶原蛋白过量沉积为主要特征的纤维化是临床肺部疾患常见的病理现象。该研究利用RT-PCR技术检测不同剂量TNF-α和IL-13对人肺成纤维细胞IL-13Rα1、IL-13Rα2和Ⅰ型胶原蛋白转录水平的影响;ELISA检测细胞培养上清sIL-13Rα2分泌量;羟脯氨酸法定量分析各组肺成纤维细胞胶原蛋白生成情况。结果发现:在实验剂量条件下,TNF-α和IL-13对人肺成纤维细胞IL-13Rα1的表达无显著影响;两者均能不同程度地上调IL-13Rα2的表达;与对照组相比,TNF-α对胶原蛋白的表达有下调作用,IL-13则无显著影响。     

Neural responses in the primary auditory cortex of freely behaving cats while discriminating fast and slow click-trains

Repeated acoustic events are ubiquitous temporal features of natural sounds. To reveal the neural representation of the sound repetition rate, a number of electrophysiological studies have been conducted on various mammals and it has been proposed that both the spike-time and firing rate of primary auditory cortex (A1) neurons encode the repetition rate. However, previous studies rarely examined how the experimental animals perceive the difference in the sound repetition rate, and a caveat to these experiments is that they compared physiological data obtained from animals with psychophysical data obtained from humans. In this study, for the first time, we directly investigated acoustic perception and the underlying neural mechanisms in the same experimental animal by examining spike activities in the A1 of free-moving cats while performing a Go/No-go task to discriminate the click-trains at different repetition rates (12.5-200 Hz). As reported by previous studies on passively listening animals, A1 neurons showed both synchronized and non-synchronized responses to the click-trains. We further found that the neural performance estimated from the precise temporal information of synchronized units was good enough to distinguish all 16.7-200 Hz from the 12.5 Hz repetition rate; however, the cats showed declining behavioral performance with the decrease of the target repetition rate, indicating an increase of difficulty in discriminating two slower click-trains. Such behavioral performance was well explained by the firing rate of some synchronized and non-synchronized units. Trial-by-trial analysis indicated that A1 activity was not affected by the cat's judgment of behavioral response. Our results suggest that the main function of A1 is to effectively represent temporal signals using both spike timing and firing rate, while the cats may read out the rate-coding information to perform the task in this experiment.     

Profiling of Substrate Specificity of SARS-CoV 3CLpro

Background

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection.

Methodology/Principal Findings

To obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3'' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1'' position prefers small residues, while P2'' and P3'' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3'' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence.

Conclusions/Significance

Our results demonstrated a strong structure-activity relationship between the 3CL^pro and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors.     

The tricks plants use to reach appropriate light

The perception of ambient light signals that produce a relevant response to ensure exposure to appropriate levels of light energy is vital for plants. In response to this, intricate molecular mechanisms to mediate light signaling have evolved in plants. Among the responses induced by light, seedling extension is a determining event for plant survival in darkness, especially in the initial stage of plant growth. Here we review previous studies and recent progress towards an understanding of light signaling that regulates seedling elongation. We focus on the three regions of the sunlight spectrum that primarily control seedling elongation, namely red/far-red light, blue/UV-A light and UV-B light, and summarize the four signaling pathways that correspond to the three effective spectra.     

A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products

Novel directional cloning and expression vectors were developed for blunt-end ligation of PCR products that are suitable for high-throughput cloning and simplifying the screening procedure. The PCR products, without further processing, are cloned into vectors digested with SchI and, following transformation, the desired recombinants give typical blue colonies on selectable plates. The principle of this selection strategy is that the construction also generates a full-length ideal lacO gene. To the best of our knowledge, this is the first time that this lacO reconstruction strategy has been applied in the selection of recombinants.