Engineering Saccharomyces cerevisiae-based biosensors for copper detection

Heavy metals, that is Cu(II), are harmful to the environment. There is an increasing demand to develop inexpensive detection methods for heavy metals. Here, we developed a yeast biosensor with reduced-noise and improved signal output for potential on-site copper ion detection. The copper-sensing circuit was achieved by employing a secondary genetic layer to control the galactose-inducible (GAL) system in Saccharomyces cerevisiae. The reciprocal control of the Gal4 activator and Gal80 repressor under copper-responsive promoters resulted in a low-noise and sensitive yeast biosensor for copper ion detection. Furthermore, we developed a betaxanthin-based colorimetric assay, as well as 2-phenylethanol and styrene-based olfactory outputs for the copper ion detection. Notably, our engineered yeast sensor confers a narrow range switch-like behaviour, which can give a ‘yes/no’ response when coupled with a betaxanthin-based visual phenotype. Taken together, we envision that the design principle established here might be applicable to develop other sensing systems for various chemical detections.     

Genistein protects intervertebral discs from degeneration via Nrf2-mediated antioxidant defense system: An in vitro and in vivo study

Oxidative stress has been reported to be closely associated with the development of intervertebral disc degeneration (IDD). IDD is one of the major causes of low back pain. Genistein (GES), one of the main isoflavones of soybean, has been shown to exert multiple biological functions on different diseases. Here, we tested the therapeutic potential of GES for IDD. In vitro experiments, we confirmed GES was nontoxic to rat nucleus pulposus cells (NPCs) within the concentration of 100 μM. Furthermore, GES was able to suppress apoptosis in tert-butyl hydroperoxide (TBHP)-treated NPCs. In the aspect of extracellular matrix (ECM), GES not only reduced metalloproteinase-13 (MMP-13) and a disintegrin-like and MMP thrombospondin type 1 motif 5 expression, but also increased aggrecan and type II collagen levels. Also, we found GES might rescue TBHP-induced NPCs degeneration by enhancing Nrf2-mediated antioxidant defense system. Silencing Nrf2 partly abolished the protective effects of GES on apoptosis and ECM disruption in TBHP-treated NPCs. Correspondingly, GES ameliorated IDD in a rat model by preserving morphology of degenerative intervertebral discs and promoting Nrf2 expression. To sum up, our study suggests that GES exerts protective effects in NPCs against degeneration and reveals the underlying mechanism of GES on Nrf2 activation in NPCs.     

The group size effect and synchronization of vigilance in the Tibetan wild ass

Vigilance behavior is considered as an effective strategy for prey species to detect predators.An individual benefits from living in a group by reducing the time spent being vigilant without affecting the probability of detecting a predator.However,the mechanism producing a decrease in vigilance with increasing group size is unclear.Many models of vigilance assume that group members scan independently of one another.Yet in recent studies,the other 2 patterns of vigilance,coordination and synchronization,were reported in some species.In 2 summers(2018 and 2019),we studied the group-size effect on vigilance and foraging of Tibetan wild ass in Chang Tang Nature Reserve of Tibet.We also tested whether individuals scan the environment independently,tend to coordinate their scans,or tend to synchronize their vigilance.The results showed that individuals decreased the time spent on vigilance with increasing group size,while increased the time spent foraging.Group members scanned the environment at the same time more frequently and there was a positive correlation between group members'behaviors,indicating that Tibetan wild asses tend to synchronize their vigilance.     

氯氟氰菊酯与阿维菌素增效混配制剂对美洲斑潜蝇的防治效果

低毒化学杀虫剂氯氟氰菊酯与生物源农药阿维菌素混配,通过其对美洲斑潜蝇室内毒力实验,测定共毒系数CTC为161～232,处于明显增效范围内。据此确定最佳配比,配制此增效混剂2%渗透型可湿性粉剂。在北京、山东两地防治美洲斑潜蝇幼虫的田间试验表明药效优良,制剂用量50 g/667m²药后3、7、11天两地区校正防效分别为85.26%～90.76%和86.74%～94.02%,制剂用量25g/667m2两地区相应防效分别为75.28%～85.17%、79.96%～88.68%。该增效混剂防治斑潜蝇速效性和持效性皆佳,成本有所下降,使用可湿粉与乳油相比较,可减少投放入环境的化学品数量,以减少环境污染。     

Calpain-1 Cleaves and Activates Caspase-7

Caspase-7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase-7 activation by granzyme B and caspase-3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase-7 is not known. Here, we present that recombinant caspase-7 is directly cleaved by calpain-1 within the large subunit of caspase-7 to produce two novel products, large subunit p18 and p17. This new form of caspase-7 has a 6-fold increase in V_max when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase-7 product (p17) is 18-fold more active than either the caspase-3-cleaved product (p20) or the larger calpain-1 product of caspase-7 (p18). Mass spectrometry and site-directed mutagenesis identified the calpain cleavage sites within the caspase-7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase-7 p18 and p17 subunits in cortical neurons undergoing Ca²⁺ dysregulation. Further, cleavage at amino acid 45/47 of caspase-7 by calpain results in a reduction in nuclear localization when compared with the caspase-3 cleavage product of caspase-7 (p20). Our studies suggest the calpain-activated form of caspase-7 has unique enzymatic activity, localization, and binding affinity when compared with the caspase-activated form.Apoptosis is a well-defined cellular destruction pathway that primarily utilizes a family of cysteine proteases, the caspases (1, 2). This cell death program can be initiated by cell death receptor activation (extrinsic pathway) or a variety of drugs or cellular stresses (intrinsic pathway) leading to activation of apical caspase-8, -9, and/or -10 (1, 3, 4). These initiator caspases in turn directly activate the executioner caspases, caspase-3 and -7, which through proteolysis of defined substrates are responsible for the dismantling of the cell and subsequent death (3, 4). Granzyme B, released by cytotoxic T lymphocytes to protect the host from pathogens and tumor cells, can also initiate this apoptotic cascade and therefore is considered an apical caspase mimic (5–7). All caspases, as well as granzyme B, preferentially cleave after aspartic acid residues, with many having well-defined consensus sequences, making substrate cleavage sites easy to predict and establish (3, 4, 7, 8).Caspases exist in a latent form prior to activation. Both the initiator and executioner caspases are synthesized as a single chain protein, which require proteolytic cleavage to become active. Procaspase-7 is expressed as a 303-amino acid residue polypeptide chain. The activation and regulation of executioner caspase-7 by caspases and granzyme B has been extensively studied. Caspase-7 requires cleavage by caspase-3 and caspase-8/-10 or granzyme B, for activation (6, 9). Current evidence suggests that caspase-3 initially cleaves off the first 23 amino acids (propeptide, 2 kDa), followed by caspase-8/-10 or granzyme B cleaving between the large (20 kDa) and small (12 kDa) subunit after amino acid 198 to activate the enzyme. The large subunit containing the catalytic His-237 and Cys-285 (caspase-1 numbering convention), and the small subunit are involved in the formation of the substrate-binding region. In vitro, granzyme B can also activate caspase-7 independently of caspase-3, but this does not appear to occur in vivo (5, 6). Currently, there is no evidence that other classes of proteases play a role in activating or modulating caspase-7 activity.Changes in intracellular Ca²⁺ levels influence apoptosis in a number of cell types (10–13). Because in many of these apoptotic cell models the Ca²⁺-dependent cysteine proteases, calpains, are activated upstream of caspases (14–16), it is possible that calpains may activate and/or modulate caspase activity via direct cleavage. Studies directed at understanding calpains with respect to caspase activation are limited. Calpain-2 was shown to cleave procaspase-9, decreasing its activity (17). In the same study, calpain-2 treatment cleaved procaspase-7 to produce a single, novel fragment, but in this case the effect on enzymatic activity was not investigated (17). To improve our understanding of calpains and the role of calcium in cell death, we carried out studies directed at understanding how calpains activate or modulate caspase activity. We found that calpain treatment produced a large increase in caspase-7 activity. Calpain cleaves procaspase-7 to produce two large subunits of 18.5 and 17.2 kDa, the smaller of which has a robust increase in activity relative to the 20-kDa large subunit produced by caspase-3 cleavage of caspase-7. Both calpain cleavage sites in caspase-7 are identified using mass spectrometry. N-methyl-d-aspartate-induced Ca²⁺-dependent cell death in primary cortical neurons produced calpain-derived caspase-7 cleavage products in vivo. Lastly, the strictly cytosolic localization of the smaller calpain fragment confirms that a previously identified nuclear localization signal (18) is involved in caspase-7 cytosolic/nuclear distribution. Our data suggest that increases in Ca²⁺ leading to activation of calpains may significantly modulate caspase-7 activity and thus, apoptosis.     

林跳鼠亚科的系统学研究述评

林跳鼠亚科分布在亚洲和北美洲,虽然种类及数量都不多,但对该亚科的分类和系统发育关系一直存在争议,近几年对林跳鼠哑科分子系统学的研究,产生了一些与形态学、生物地理学等研究不同的结果.本文对林跳鼠亚科系统学研究的一些工作加以述评,归纳出林跳鼠亚科在系统学方面还存在的问题,并对林跳鼠亚科系统学的进一步研究提出了一些建议.     

Expression of the calcium-activated potassium channel in upper and lower segment human myometrium during pregnancy and parturition

Background  

Large conductance calcium-activated potassium channel (BKCa) plays an important role in the control of uterine contractility during pregnancy. The change from uterine quiescence to enhanced contractile activity may be associated with the spatial and temporal expression of BKCa within myometrium. The objectives of this study were to examine the expression of BKCa alpha- and beta-subunit in upper segment (US) and lower segment (LS) regions of uterus, and to investigate for the possibly differential expression of these proteins in US and LS myometrium obtained from three functional states: (1) non-pregnant (NP); (2) term pregnant not in labour (TNL) and (3) term pregnant in labour (TL).