Sound transmission across locally resonant honeycomb sandwich meta-structures with large spatial periodicity.

Honeycomb sandwich structures have been widely used in the field of engineering owing to their outstanding mechanical properties. However, for a honeycomb sandwich structure with large spatial periodicity, there is a low-frequency sound insulation valley. Here, the sound transmission across locally resonant honeycomb sandwich meta-structures was investigated to overcome this sound-insulation valley. An analytical model was developed based on the space-harmonic approach and the low-frequency sound insulation valley was determined analytically and numerically. The results indicate that the resonator distributed at the center of the face panel has a significant impact on the sound transmission performance of the honeycomb sandwich structure, whereas the resonator distributed on the wall of the honeycomb core does not contribute to overcoming this sound-insulation valley. Based on the research results, a design strategy for overcoming this sound-insulation valley was determined by tuning the damping parameter and constructing graded resonators. Moreover, sound transmission under the excitation of oblique incidence sound waves was also investigated. Compared with the method of filling porous materials, the proposed design method is more effective, and more importantly, the mass of the resonator is only 1.23% of that of the porous materials.

Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity.

Escherichia coli, one of the most efficient expression hosts for recombinant proteins (RPs), is widely used in chemical, medical, food and other industries. However, conventional expression strains are unable to effectively express proteins with complex structures or toxicity. The key to solving this problem is to alleviate the host burden associated with protein overproduction and to enhance the ability to accurately fold and modify RPs at high expression levels. Here, we summarize the recently developed optimization strategies for the high-level production of RPs from the two aspects of host burden and protein activity. The aim is to maximize the ability of researchers to quickly select an appropriate optimization strategy for improving the production of RPs.

Developing a dynamic equilibrium system in Escherichia coli to improve the production of recombinant proteins.

The combination of Escherichia coli BL21 (DE3) and the pET expression system is used extensively for the expression of various recombinant proteins (RPs). However, RP overexpression often introduces a growth burden for the host, especially in the case of toxic proteins. The key to solving this problem is to reduce the host burden associated with protein overproduction, which is often achieved by regulating the expression or activity of T7 RNAP or growth-decoupled systems. However, these strategies mainly relieve or interrupt the robbing of host resources, and do not eliminate other types of host burdens in the production process. In this study, we constructed a production system based on a dynamic equilibrium to precisely relieve the host burden and increase the RP production. The system is composed of three modules, including the overexpression of basic growth-related genes (rRNA, RNAP core enzyme, sigma factors), prediction and overexpression of key proteins using the enzyme-constrained model ec_iECBD_1354, and dynamic regulation of growth-related and key protein expression intensity based on a burden-driven promoter. Using this system, the production of many high-burden proteins, including autolysis protein and E. coli membrane proteins, was increased to varying degrees. Among them, the cytosine transporter protein (CodB) was most significantly improved, with a 4.02-fold higher production compared to the wild strain. This system can effectively reduce the optimizing costs, and is suitable for developing various types of RP expression hosts rapidly. KEY POINTS: • The basic growth-related resources can relieve the host burden from recombinant protein. • The enzyme-constrained model can accurately predict key genes to improve yield. • The expression intensity can be dynamically adjusted with changes in burden.

Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production.

Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.

Direct Liquid Phase Exfoliation of Graphite to Produce Few-Layer Graphene by Microfluidization.

Graphene has attracted a great number of attentions due to the excellent physical and chemical properties. For the convenience of investigations and applications, it is crucial to produce the grapheme with high-quality and high-yield by an easy-obtained method. In this research, a promising method is demonstrated to produce a high-concentration few-layer graphene (FLG) dispersion by direct microfluidization in water/surfactant systems. The effects of surfactant selection, chamber pressure and microfluidization cycles on the graphitic material exfoliation efficiency are systematically studied. The FLG concentration and the quality of the as-prepared FLG were determined by a series of characterizations. The graphene dispersions, with an average lateral size of 0.6 µm and a few-layer structure, were stabilized by surfactants at a high concentration of up to 1.7 mg/mL and exhibited a relatively high quality (ID/IG = 0.07-0.56, C/O ~ 19.36) within a processing time of a few hours. This method should facilitate the mass production of high-quality graphene by liquid-phase exfoliation and promote the industrial application of graphene.

MMP-14 overexpression correlates with poor prognosis in non-small cell lung cancer.

Matrix metalloproteinase-14 (MMP-14) has been demonstrated to play an important role in tumor progression. The aim of this study was to analyze the correlation between MMP-14 expression and clinicopathologic features and its prognostic significance in non-small cell lung cancer (NSCLC). Immunohistochemical staining for MMP-14 protein was performed in 104 patients with NSCLC. High levels of MMP-14 protein were positively correlated with the status of clinical stage (I-II vs. III-IV; P < 0.001), N classification (N0-N1 vs. N2-N3; P < 0.001), distant metastasis (no vs. yes; P = 0.014), and differentiated degree (high vs. low or undifferentiated; P = 0.001). The patients with higher MMP-14 expression of protein had shorter survival time than patients with low MMP-14 expression. Multivariate analysis indicated that the level of MMP-14 expression was an independent prognostic indicator (P < 0.001) for the survival of patients with NSCLC. In conclusion, MMP-14 is a potential unfavorable prognostic factor for patients with NSCLC.

Rapid construction of Escherichia coli chassis with genome multi-position integration of isopentenol utilization pathway for efficient and stable terpenoid accumulation.

The isopentenol utilization pathway (IUP) is potential in terpenoids synthesis. This study aimed to construct IUP-employed Escherichia coli chassis for stably synthesizing terpenoids. As to effectiveness, promotor engineering strategy was employed to regulate IUP expression level, while ribosome-binding site (RBS) library of the key enzyme was constructed for screening the optimal RBS, followed by optimization of concentration of inducer and substrates, the titer of reporting production, lycopene, from 0.087 to 8.67 mg OD600 -1 . As about stability, the IUP expression cassette was integrated into the genome through transposition tool based on CRISPR-associated transposases. Results showed that the strain with 13 copies produced 1.78-fold lycopene titer that of the controlled strain with IUP-harbored plasmid, and it exhibited stable expression after ten successions while the plasmid loss was observed in the controlled strain in the 3rd succession. This strategy provides valuable information for rapid construction of highly effective and stable chassis employing IUP for terpenoids production.

Differential gene expression of Wnt signaling pathway in benign, premalignant, and malignant human breast epithelial cells.

To gain insight into the role of gene expression alterations in breast cancer progression, we conducted a comprehensive gene expression analysis of a series of cell lines derived from MCF10A, which include benign MCF10A cells, premalignant AT, and malignant CA1a tumor cells. We analyzed gene expression variation using the Agilent Human Genome Oligo Microarray with the goal of identifying gene-specific expression change events. In addition to a previously noted overexpression in oncogene MDM2, HRAS, and PCNA, our studies identified overexpression of Wnt signaling pathway in malignant breast cell lines. The Kaplan-Meier plot showed that high c-Myc expression in breast cancer was associated with tumor progression and the patient's poor survival. This study showed that the Wnt pathway has further provided a basis for the development of potential biomarker for breast cancer prognosis.

Assessing the potential of Schizochytrium sp. HX-308 for microbial lipids production from corn stover hydrolysate.

Schizochytrium sp. has received increasing attention as promising commercial resource for the sustainable production of lipids, due to their fast growth rate and high lipid content. However, the price of glucose represents a significant proportion of the total substrate cost. Therefore, in this study, the lignocellulosic hydrolysate of corn stover hydrolysate (CSH) was used as low-cost culture medium to replace glucose in Schizochytrium sp. fermentation. When Schizochytrium sp. HX-308 was fermented with 20% glucose from CSH and 80% of glucose from pure glucose, the lipid production reached 21.2 g L-1 , which is lower than that of using 100% of pure glucose. However, the shifts of fatty acid composition indicated that CSH has great potential to enhance the percentage of polyunsaturated fatty acids (PUFAs) in total lipids. However, as the second largest carbon source in CSH, xylose was not utilized by the Schizochytrium sp. HX-308, and further analysis showed that probably because it does not possess a functional xylulose kinase. In addition, the degradation products in lignocellulosic hydrolysate have a strong inhibitory effect on cell growth, so it is necessary to investigate the tolerance of Schizochytrium sp. HX-308 to degradation products. Here, the effects of five typical degradation products on the growth and lipid synthesis were further investigated. Schizochytrium sp. HX-308 showed good tolerance to furan derivatives and organic acids, but low tolerance to phenolic compounds. Furthermore, in order to improve the lipid accumulation using CSH, the two-stage fermentation strategy was developed, resulting in a 54.8% increase compared to that of the one-stage strategy. In summary, this study provides a reference for further fermentation engineering with cheap lignocellulosic biomass as substrate.

Novel fluorescent molecular clips: selective recognition towards Fe3+ in aqueous solution.

In this paper, we report the synthesis, characterization and Fe(3+)-sensing properties of a series of new artificial fluorescent molecular clips, and structure of clip 1 is confirmed by X-ray crystallography. All these molecular clips had the ability to bind and sense Fe(3+) selectively through decrease fluorescence responses in THF-MeOH-Water. Fluorimetric titration experiment indicated that the quenching of these compounds' fluorescence upon Fe(3+) probably arises from the electron/energy transfer between Fe(3+) and the excited chemosensors. The limit of detection, linear concentration range and selectivity of the fluorescent molecular clips were evaluated in this study as well.

Developing GDi-CRISPR System for Multi-copy Integration in Saccharomyces cerevisiae.

In recent years, Saccharomyces cerevisiae has been widely used in the production of biofuels and value-added chemicals. To stably express the target products, it is necessary to integrate multiple target genes into the chromosome of S. cerevisiae. CRISPR multi-copy integration technology relying on delta sites has been developed, but it often requires the help of high-throughput screening or resistance markers, resulting in non-replicability and high cost. This study aims to develop a low-cost and easy-to-use multi-copy integration tool in S. cerevisiae. Firstly, twenty-one Cas proteins from different microorganisms were tested in S. cerevisiae to find the functional Cas proteins with optimal cleavage ability. Results showed that eight Cas proteins can complete gene editing. However, most of the transformants have low copy numbers, which may be caused by high cutting efficiency exceeding the repair rate. Therefore, the effect of donor translocation order was further investigated. Results showed that 4 copies were obtained by donor first translocation. Then, the gene drive delta site integration system by the CRISPR system (GDi-CRISPR) was developed by combining gene drive principle and CRISPR system. To be clear, the gRNA was put into donor fragments. Then, both of them were integrated into the genome, which can drive further cutting and repair due to increasing number of gRNA. Instead of high-throughput screening or resistance pressure, 6 copies were obtained in only 5-6 days using the GDi-CRISPR system. It is expected to further advance the development of S. cerevisiae multi-copy integration tools.

The complete chloroplast genome sequence of Lepidium perfoliatum L.

This study was based on the collection of the complete genome of Lepidium perfoliatum chloroplast (cp). The full cp genome is 154,264 bp long, containing 130 genes, in which 8 genes are specified for ribosomal RNA (rRNA), while 85 and 37 genes for protein-coding and transfer RNA (tRNA) respectively. Phylogenetic analyss revealed the closed cluster of Lepidium perfoliatum with other Lepidium species such as Lepidium apetalum, Lepidium sativum, Lepidium meyenii and Lepidium virginicum, which helps for the evaluation of how Lepidium perfoliatum is phylogenetically related to other species.

An emerging simple and effective approach to increase the productivity of thraustochytrids microbial lipids by regulating glycolysis process and triacylglycerols' decomposition.

BACKGROUND: The oleaginous microorganism Schizochytrium sp. is widely used in scientific research and commercial lipid production processes. However, low glucose-to-lipid conversion rate (GLCR) and low lipid productivity of Schizochytrium sp. restrict the feasibility of its use. RESULTS: Orlistat is a lipase inhibitor, which avoids triacylglycerols (TAGs) from hydrolysis by lipase. TAGs are the main storage forms of fatty acids in Schizochytrium sp. In this study, the usage of orlistat increased the GLCR by 21.88% in the middle stage of fermentation. Whereas the productivity of lipid increased 1.34 times reaching 0.73 g/L/h, the saturated fatty acid and polyunsaturated fatty acid yield increased from 21.2 and 39.1 to 34.9 and 48.5 g/L, respectively, indicating the advantages of using a lipase inhibitor in microbial lipids fermentation. Similarly, the system was also successful in Thraustochytrid Aurantiochytrium. The metabolic regulatory mechanisms stimulated by orlistat in Schizochytrium sp. were further investigated using transcriptomics and metabolomics. The results showed that orlistat redistributed carbon allocation and enhanced the energy supply when inhibiting the TAGs' degradation pathway. Therefore, lipase in Schizochytrium sp. prefers to hydrolyze saturated fatty acid TAGs into the ß-oxidation pathway. CONCLUSIONS: This study provides a simple and effective approach to improve lipid production, and makes us understand the mechanism of lipid accumulation and decomposition in Schizochytrium sp., offering new guidance for the exploitation of oleaginous microorganisms.

Advanced Strategies for the Synthesis of Terpenoids in Yarrowia lipolytica.

Terpenoids are an important class of secondary metabolites that play an important role in food, agriculture, and other fields. Microorganisms are rapidly emerging as a promising source for the production of terpenoids. As an oleaginous yeast, Yarrowia lipolytica contains a high lipid content which indicates that it must produce high amounts of acetyl-CoA, a necessary precursor for the biosynthesis of terpenoids. Y. lipolytica has a complete eukaryotic mevalonic acid (MVA) pathway but it has not yet seen commercial use due to its low productivity. Several metabolic engineering strategies have been developed to improve the terpenoids production of Y. lipolytica, including developing the orthogonal pathway for terpenoid synthesis, increasing the catalytic efficiency of terpenoids synthases, enhancing the supply of acetyl-CoA and NADPH, expressing rate-limiting genes, and modifying the branched pathway. Moreover, most of the acetyl-CoA is used to produce lipid, so it is an effective strategy to strike a balance of precursor distribution by rewiring the lipid biosynthesis pathway. Lastly, the latest developed non-homologous end-joining strategy for improving terpenoid production is introduced. This review summarizes the status and metabolic engineering strategies of terpenoids biosynthesis in Y. lipolytica and proposes new insights to move the field forward.

Biotechnological production of lipid and terpenoid from thraustochytrids.

As fungus-like protists, thraustochytrids have been increasingly studied for their faster growth rates and high lipid content. In the 1990s, thraustochytrids were used as docosahexaenoic acid (DHA) producers for the first time. Thraustochytrids genera, such as Thraustochytrium, Schizochytrium, and Aurantiochytrium have been developed and patented as industrial strains for DHA production. The high DHA yield is attributed to its unique and efficient polyketide-like synthase (PKS) pathway. Moreover, thraustochytrids possess a completed mevalonate (MVA) pathway, so it can be used as host for terpenoid production. In order to improve strain performance, the metabolic engineering strategies have been applied to promote or disrupt intracellular metabolic pathways, such as genetic engineering and addition of chemical activators. However, it is difficult to realize industrialization only by improving strain performance. Various operation strategies were developed to enlarge the production quantities from the laboratory-scale, including two-stage cultivation strategies, scale-up technologies and bioreactor design. Moreover, an economical and effective downstream process is also an important consideration for the industrial application of thraustochytrids. Downstream costs accounts for 20-60% of the overall process costs, which represents an attractive target for increasing the cost-competitiveness of thraustochytrids, including how to improve the efficiency of lipid extraction and the further application of biomass residues. This review aims to overview the whole lipid biotechnology of thraustochytrids to provide the background information for researchers.

[Clinical significance of insulin-like growth factor-1 receptor in platinum-based chemotherapy for non-small cell lung cancer].

UNLABELLED: OBJECTIVE; To explore the influence of the expression of insulin-like growth factor-1 receptor(IGF-1R)in end-stage non-small cell lung cancer. METHOD: The expression of IGF-1R was detected in 39 paraffin-embedded chemotherapy-naive non-small cell lung cancer tumor samples with immunohistochemical method,and the relationship between the outcomes of platinum-based chemotherapy and IGF-1R expression was analyzed. RESULTS: IGF-1R expression was detected in 21 cases (53.8%). The IGF-1R expression status shown no correlation with tumor pathological status,tumor differentiation status,history of smoking,as well as smoking index. Better outcomes of platinum-based chemotherapy were observed in patients with negative IGF-1R expression. CONCLUSION: IGF-1R expression may be used to predict the effectiveness of the first-line platinum-based chemotherapy for end-stage non-small cell lung cancer.

[Treatment and prognosis of 77 cases of small cell lung cancer].

OBJECTIVE: To investigate the clinical treatment modality and prognosis of small cell lung cancer(SCLC). METHOD: We retrospectively analyzed the clinical data of 77 SCLC patients who were admitted to our department after 2002. RESULTS: The disease was limited in 43 patients and extensive in 34 patients. For patients with limited SCLC, the 1-year, 2-year, and 5-year survival rate was 80%, 56%, and 21%, respectively. Four patients who had undergone surgical resection were all alive. Among patients who underwent adjuvant chemotherapy followed by radiotherapy, salvage chemotherapy, and salvage chemotherapy followed by radiotherapy, the median of survival period was 51 months, 12 months, and 28 months, respectively. For patients with extensive SCLC, the 1-year and 2-year survival rate was 56% and 25%, respectively. The median of survival period was 14.3 months. Stage was an independent factor in multifactor COX regression. Monofactor COX regression showed that radiotherapy and resection were factors correlated with survival. Brain metastasis had no impact on survival. CONCLUSIONS: Chemotherapy followed by radiotherapy is preferred for limited SCLC, while surgical resection remains questionable for early-stage patients. For extensive SCLC, multi-line chemotherapy may be helpful to improve the overall survival. Stage is an independent factor for predicting the prognosis.

[Clinical characteristics of gastric cancer in the elderly patients].

OBJECTIVE: To investigate the clinical manifestations,treatment,and prognosis of gastric cancer in the elderly patients. METHOD: A total of 252 patients with gastric cancer who admitted to the Oncology Department of Peking Union Medical College Hospital were divided into elderly group (≥ 65 years) and non-elderly group (< 65 years) and the clinical characteristics of these two groups were analyzed and compared. RESULTS: The elderly accounted for 36.0% of all gastric cancer patients in our department. The proportion of male was significantly higher in elderly group than non-elderly group (male:female = 3.74:1, P=0.020). Abdominal satiety and pain were the most common symptoms,which were significantly lower in elderly group (43.3% vs. 61.7%, P=0.005). However,the frequency of weight loss was significantly higher in the elderly group (15.6% vs. 6.2%, P = 0.015). Significantly more elderly patients with gastric cancer were found the second tumors (12.2% vs. 2.5%, P=0.002). The most common tumor location was cardia (36.7%) in elderly group and antrum (34.6%) in non-elderly group. A small proportion (2.2%) of elderly patients had multi-original lesions, which was not found in non-elderly group. The overall rate of surgery and R0 resection rate were 77.8% and 70.9% respectively, which were similar in both groups. The overall rate of chemotherapy was 98%. The ratio was one third compared with younger patients who received three and more than three lines chemotherapy (3.3% vs. 9.3%), but did not reach statistical difference. More elderly patients chose FOLFOX / XELOX regimen (73.3%) compared with younger arm. The median survival time was 26.5 months in elderly group and 28.0 months in non-elderly group (P=0.835). Subgroup analysis showed that the median survival time of stage 4 gastric cancer was longer in elderly group than in non-elderly group (22.7 months and 16.1 months, respectively; P=0.057), which was marginally statistically significant. CONCLUSIONS: More old men may get gastric cancer. More elderly patients may present with weight loss. Cardia is the most common tumor location. The ratio of multi-original lesions and secondary tumors is higher for elderly patients. Elderly patients with good performance status can receive surgery and chemotherapy safely. The resection rate is similar between elderly and non-elderly patients. Elderly patients receive more two-drug combination regimens. The overall prognoses are similar between elderly patients and non-elderly patients.

Application of 18F-fluorodeoxyglucose positron emission tomography in diagnosis of malignant diseases.

OBJECTIVE: To testify the efficacy of 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) in the diagnosis of cancer. METHODS: A total of 170 patients with diagnosed cancer or suspicious cancer were enrolled in this study, and underwent 18F-FDG PET. The standard uptake value (SUV) and diameter for each abnormal region in PET images were analyzed. All data were analyzed by SPSS 11.5. RESULTS: PET scan identified a primary cancer in 45.8% (11/24) patients. The sensitivity and specificity of PET scan in differentiating malignant lesions from benign ones were 78.8% (52/66) and 77.1% (27/35) respectively. Twenty-nine out of 68 (42.6%) lesions were detected earlier by PET than by computed tomography. The SUV of primary cancer was significantly higher than that of metastatic lymph nodes (5.84 +/- 3.12 vs. 3.14 +/- 2.24, P<0.001). And SUV of primary lung cancer was also significantly higher than that of metastatic lung cancer (6.30 +/- 3.01 vs. 2.86 +/- 2.37, P<0.01). CONCLUSION: 18F-FDG PET plays a very important role in cancer diagnosis.

(RS)-3-Acetyl-2-methyl-4-(3-nitro-phen-yl)-1,4,5,6,7,8-hexa-hydro-quinolin-5-one.

In the title compound, C(18)H(18)N(2)O(4), the nitro group, a methyl group, the acetyl group and some atoms of the dihydro-quinolinone group are disordered over two sites with the ratio of occupancies fixed at 0.57:0.43. The relationship between the major and minor components of disorder is that of diastereomers. In the crystal structure, inter-molecular N-H⋯O, weak C-H⋯O and C-H⋯π inter-actions link the mol-ecules into two-dimensional layers running parallel to the (010) plane.