Multiplex Expression Cassette Assembly: A flexible and versatile method for building complex genetic circuits in conventional vectors.

The manipulation of multiple transcription units for simultaneous and coordinated expression is not only key to building complex genetic circuits to accomplish diverse functions in synthetic biology, but is also important in crop breeding for significantly improved productivity and overall performance. However, building constructs with multiple independent transcription units for fine-tuned and coordinated regulation is complicated and time-consuming. Here, we introduce the Multiplex Expression Cassette Assembly (MECA) method, which modifies canonical vectors compatible with Golden Gate Assembly, and then uses them to produce multi-cassette constructs. By embedding the junction syntax in primers that are used to amplify functional elements, MECA is able to make complex constructs using only one intermediate vector and one destination vector via two rounds of one-pot Golden Gate assembly reactions, without the need for dedicated vectors and a coherent library of standardized modules. As a proof-of-concept, we modified eukaryotic and prokaryotic expression vectors to generate constructs for transient expression of green fluorescent protein and ß-glucuronidase in Nicotiana benthamiana, genome editing to block monoterpene metabolism in tomato glandular trichomes, production of betanin in tobacco and synthesis of ß-carotene in Escherichia coli. Additionally, we engineered the stable production of thymol and carvacrol, bioactive compounds from Lamiaceae family plants, in glandular trichomes of tobacco. These results demonstrate that MECA is a flexible, efficient and versatile method for building complex genetic circuits, which will not only play a critical role in plant synthetic biology, but also facilitate improving agronomic traits and pyramiding traits for the development of next-generation elite crops.

Regulation of apoptosis by Pacific oyster Crassostrea gigas reveals acclimation strategy to CO2 driven acidification.

Ocean acidification (OA) has posed formidable threats to marine calcifiers. In response to elevated CO2 levels, marine calcifiers have developed multiple strategies to survive, such as taking advantage of apoptosis, but its regulation mechanism remains largely unknown. Here, we used the Pacific oyster Crassostrea gigas as model to understand the apoptotic responses and regulation mechanism at short- (7 d) to long-term (56 d) CO2 exposure (pH = 7.50). The apoptosis of hemocytes was significantly induced after short-term treatment (7-21 d) but was suppressed under long-term CO2 exposure (42-56 d). Similarly, caspase-3 and caspase-9 were also increased post short-term exposure and fell back to normal levels after long-term exposure. These data together indicated diverse regulation mechanisms of apoptosis through different exposure periods. Through analysis of the B-cell lymphoma 2 (Bcl-2) family mitochondrial apoptosis regulators, we showed that only CgBcl-XL's expression kept at high levels after 42- and 56-day CO2 exposure. CgBcl-XL shared sequence, and structural similarity with its mammalian counterpart, and knockdown of CgBcl-XL in hemocytes via RNA interference promoted apoptosis. The protein level of CgBcl-XL was significantly increased after long-term CO2 exposure (28-56 d), and its distribution in hemocytes became more concentrated and dense. Therefore, CgBcl-XL serves as an essential anti-apoptotic protein for tipping the balance of cell apoptosis, which may play a key role in survival under long-term CO2 exposure. These results reveal a potential adaptation strategy of oysters towards OA and the variable environment changes through the modulation of apoptosis.

The involvement of a novel calmodulin-like protein isoform from oyster Crassostrea gigas in transcription factor regulation provides new insight into acclimation to ocean acidification.

Marine organisms need to adapt to improve organismal fitness under ocean acidification (OA). Recent studies have shown that marine calcifiers can achieve acclimation by stimulating calcium binding/signaling pathways. Here, a CaM-like gene (CgCaLP-2) from oyster Crassostrea gigas which typically responded to long-term CO2 exposure (two months) rather than short-term exposure (one week) was characterized. The cloned cDNA was 678 bp and was shorter than the retrieved sequence from NCBI (1125 bp). The two sequences, designated as CgCaLP-2-v1 and CgCaLP-2-v2, were demonstrated to be different splice variants by the genome sequence analysis. Western blotting analysis revealed two bands of 23 kD and 43 kD in mantle and hemocytes, corresponding to predicted molecular weight of CgCaLP-2-v1 and CgCaLP-2-v2, respectively. The isoform CgCaLP-2-v1 (the 23 kD band) was highly stimulated in response to long-term CO2 exposure (42-day and 56-day treatment) in hemocytes and mantle tissue. The fluorescence signal of CgCaLP-2 in mantle and hemocytes became more intensive after long-term CO2 exposure. Besides, in hemocytes, CgCaLP-2 presented a higher localization on the nuclear membrane after long-term CO2 exposure (56 d). The target gene network of CgCaLP-2 was predicted, and a transcription factor (TF) gene annotated as Homeobox protein SIX4 (CgSIX4) showed a similar expressive trend to CgCaLP-2 during CO2 exposure. Suppression of CgCaLP-2 via RNA interference significantly reduced the mRNA expression of CgSIX4. The results suggested that CgCaLP-2 might mediate the Ca2+-CaLP-TF signal transduction pathway under long-term CO2 exposure. This study serves as an example to reveal that alternative splicing is an important mechanism for generation multiple protein isoforms and thus shape the plastic responses under CO2 exposure, providing new insight into the potential acclimation ability of marine calcifiers to future OA.

[Effect of miR-22 Targeting FMNL2 on Cell Migration and Apoptosis in Childhood Acute Myeloid Leukemia].

OBJECTIVE: To investigate the effect of miR-22 targeting formin-like protein 2 (FMNL2) on the migration and apoptosis of childhood acute myeloid leukemia (AML) cells. METHOD: Peripheral blood samples from 11 children with AML, 10 children with immune thrombocytopenia, human AML cell lines TF-1a, HL-60, THP-1 and human bone marrow stromal cells HS-5 were used as the research objects. UniCel DxH 800 automatic hematology analyzer detected platelet count, hemoglobin, and white blood cell count in peripheral blood samples, and RT-qPCR detected miR-22 expression in peripheral blood samples and AML cells. HL-60 cells were transfected with LipofectamineTM 2000 kit, the experiments were divided into seven groups: blank (no cells transfected), miR-NC, miR-22 mimics, si-NC, si-FMNL2 , miR-22 mimics+OE-NC and miR-22 mimics+OE-FMNL2 . RT-qPCR was used to detect the expression of miR-22 in each group. Transwell was used to detect cell migration. Flow cytometry was used to detect cell apoptosis. Dual-luciferase reporter gene detection experiments verified the targeting relationship between miR-22 and FMNL2 . Western blot was used to detect the expression of FMNL2 protein. RESULTS: Compared with the control group, the number of leukocytes in the peripheral blood of children with AML was significantly increased (P <0.001), while the concentration of hemoglobin and the number of platelets were significantly decreased P <0.001). The expression level of miR-22 in peripheral blood of children with AML was significantly lower than that in control group (P <0.001). Compared with HS-5 cells, the expression levels of miR-22 in TF-1a, HL-60, and THP-1 cells were significantly decreased (P <0.05), and in HL-60 cells was the lowest. Therefore, HL-60 cells were selected for subsequent experiments. Up-regulation of miR-22 or silencing of FMNL2 could reduce the number of migrating cells and increase apoptosis rate (P <0.05). MiR-22 targeted and negatively regulated the expression of FMNL2 . FMNL2 overexpression reversed the effects of up-regulated miR-22 on migration and apoptosis of HL-60 cells. CONCLUSION: MiR-22 can inhibit the migration and promote apoptosis of HL-60 cells by down regulating the expression of FMNL2 .

A calcification-related calmodulin-like protein in the oyster Crassostrea gigas mediates the enhanced calcium deposition induced by CO2 exposure.

Calcium transportation and homeostasis are essential for marine bivalves to maintain basic metabolism and build their shells. Calmodulin-like proteins (CaLPs) are important calcium sensors and buffers and can respond to ocean acidification (OA) in marine calcifiers. However, no further study of their physiological function in calcium metabolism under elevated CO2 has been performed. Here, we identified a novel CaLP (designated CgCaLP) in the Pacific oyster Crassostrea gigas and demonstrated its participation in the calcification process: the mRNA expression level of CgCaLP peaked at the trochophore larval stage and remained high at stages when shells were shaped; the mRNA and protein of CgCaLP were more highly expressed in mantle tissue than in other tissues. Under elevated CO2 levels, the protein expression level of CgCaLP in hemocytes increased, while in contrast, significantly decreased protein levels were detected in gill and mantle tissues. Shell dissolution caused the imbalance of calcium in hemocytes and decreased calcium absorption and transportation demand in gill and mantle tissues, inducing the molecular function allocation of CgCaLP under CO2 exposure. Despite the decreased protein level in mantle tissue, CgCaLP was found to translocate to outer mantle epithelium (OME) cells where condensed calcium-rich deposits (CRDs) were detected. We further demonstrated that CgCaLP mRNA and protein expression levels could respond to seawater Ca2+ availability, suggesting that the calcium deposition capacity of oysters might be enhanced to fight against shell dissolution problems and that CgCaLP might serve as an essential participator of the process. In summary, CgCaLP might enhance calcium deposition under CO2 exposure and thus play a significant and flexible molecular function involved in a compensation strategy of oysters to fight against the acidified ocean.

One-Step and Colorimetric Detection of Fish Freshness Indicator Hypoxanthine Based on the Peroxidase Activity of Xanthine Oxidase Grade I Ammonium Sulfate Suspension.

The global food waste problem, especially aquatic product spoilage, stimulates the accurate freshness analysis of food products. However, it still remains a great challenge to realize in-field determination of fish freshness at the time of use. In the present study, a colorimetric enzyme biosensor was developed for one-step detection of hypoxanthine (Hx), which is an important intermediate of adenosine triphosphate decomposition during fish storage. We demonstrate that xanthine oxidase grade I ammonium sulfate suspension (XOD-ASS) possesses peroxidase activity. It can oxidize different peroxidase substrates, including 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and o-phenylenediamine in the presence of H2O2, producing visible color reactions. Further experiments indicate that XOD-ASS displayed effective peroxidase activity and could be used for H2O2 detection. Based on this, a one-step Hx detection method was established using only XOD-ASS as the catalyst. The method displays a good linear relationship in the range from 20 to 100 µM with a detection limit of 6.93 µM. Additionally, we successfully applied this method in testing Hx accumulation in sea bass fish samples of different storage times. The recovery values range from 97.44 to 102.56%. It is exciting to note that, compared with other methods, our proposed method provides a robust advantage on the economic reaction system, ease of preparation, short time consumption, and moderate reaction temperature. We believe that this method shows good application prospects for on-site fish freshness determination.

Enhancement of phenol biodegradation: Metabolic division of labor in co-culture of Stenotrophomonas sp. N5 and Advenella sp. B9.

The aim of this work is to study the synergistic effect of Stenotrophomonas sp. N5 and Advenella sp. B9 co-culture (COC) on enhancement of phenol biodegradation. These two strains utilizing phenol as sole carbon and energy source were isolated from phenol-containing coking wastewater. The results of biodegradation experiment showed the COC of N5 and B9 has stronger capability to degrade phenol than either of mono-culture (MOC). Growth kinetics studies indicated inhibitory effect of phenol on COC was reduced by the interaction of N5 and B9 in COC. The RNA-Seq results demonstrated that phenol biodegradation was enhanced by metabolic division of labor (DOL) in COC based on the expression of key genes for phenol degradation. GO enrichment analysis of differentially expressed genes (DEGs) indicated DEGs between COC and MOC degradation systems are mainly concentrated in the synthesis of cell components, microbial growth and metabolism, and catalytic activity. The expression of 3 transcriptional factors (LysR, Two-component system response regulator, and TetR families) which can regulate degradation of aromatic compounds, was identified beneficial to phenol degradation.

[Ozone and carbon monoxide concentration distribution on Taishan Mountain].

The concentrations of ozone (O3) and carbon monoxide (CO) on the top of Taishan Mountain were monitored, and the variations and correlation were studied. The results show that the frequency of O3 hourly concentrations more than the first-degree of National Ambient Air Quality Standard(GB-3095-1996, NAAQS) was 15.81%, and the frequency of CO concentrations more than the first-degree of NAAQS was zero. The variation of O3 concentrations appears in a narrow scope, which indicates that there is scarcely influenced by the pollution of industry around. The diurnal variation of the concentrations of O3 and that of CO both present two peaks with the peaks of O3 in the behind of CO, which indicates that the concentration variations of O3 and its precursor CO are primarily controlled by local photochemical reaction process. The daily concentrations of O3 correlated well with CO.

[Ozone concentration distribution of urban].

The increase of ozone concentration in urban is one of the most important research topics on environmental science. With the increase of nitrogen oxides and hydrogen-carbon compounds which are exhausted from cars, the ozone concentration in urban is obviously increased on sunlight, and threat of photochemistry smog will be possible. Therefore, it is very important to monitor and study the ozone concentration distribution in urban. The frequency-distribution, diurnal variation and monthly variation of ozone concentration were studied on the campus of Shandong University during six months monitoring. The influence of solar radiation and weather conditions on ozone concentration were discussed. The frequency of ozone concentration less than 200 microg/m3 is 96.88%. The ozone concentration has an obvious diurnal variation. The ozone concentration in the afternoon is higher than in the morning and in the evening. The maximum appears in June, when it is the strong solar radiation and high air-temperature. The weather conditions also influence the ozone concentration. The ozone concentration in clear day is higher than in rainy and cloudy day.