Engineering extracellular electron transfer pathways of electroactive microorganisms by synthetic biology for energy and chemicals production.

The excessive consumption of fossil fuels causes massive emission of CO2, leading to climate deterioration and environmental pollution. The development of substitutes and sustainable energy sources to replace fossil fuels has become a worldwide priority. Bio-electrochemical systems (BESs), employing redox reactions of electroactive microorganisms (EAMs) on electrodes to achieve a meritorious combination of biocatalysis and electrocatalysis, provide a green and sustainable alternative approach for bioremediation, CO2 fixation, and energy and chemicals production. EAMs, including exoelectrogens and electrotrophs, perform extracellular electron transfer (EET) (i.e., outward and inward EET), respectively, to exchange energy with the environment, whose rate determines the efficiency and performance of BESs. Therefore, we review the synthetic biology strategies developed in the last decade for engineering EAMs to enhance the EET rate in cell-electrode interfaces for facilitating the production of electricity energy and value-added chemicals, which include (1) progress in genetic manipulation and editing tools to achieve the efficient regulation of gene expression, knockout, and knockdown of EAMs; (2) synthetic biological engineering strategies to enhance the outward EET of exoelectrogens to anodes for electricity power production and anodic electro-fermentation (AEF) for chemicals production, including (i) broadening and strengthening substrate utilization, (ii) increasing the intracellular releasable reducing equivalents, (iii) optimizing c-type cytochrome (c-Cyts) expression and maturation, (iv) enhancing conductive nanowire biosynthesis and modification, (v) promoting electron shuttle biosynthesis, secretion, and immobilization, (vi) engineering global regulators to promote EET rate, (vii) facilitating biofilm formation, and (viii) constructing cell-material hybrids; (3) the mechanisms of inward EET, CO2 fixation pathway, and engineering strategies for improving the inward EET of electrotrophic cells for CO2 reduction and chemical production, including (i) programming metabolic pathways of electrotrophs, (ii) rewiring bioelectrical circuits for enhancing inward EET, and (iii) constructing microbial (photo)electrosynthesis by cell-material hybridization; (4) perspectives on future challenges and opportunities for engineering EET to develop highly efficient BESs for sustainable energy and chemical production. We expect that this review will provide a theoretical basis for the future development of BESs in energy harvesting, CO2 fixation, and chemical synthesis.

Engineering extracellular polymer substrates biosynthesis and carbon felt-carbon nanotube hybrid electrode to promote biofilm electroactivity and bioelectricity production.

Organic-rich thin stillage is a significant by-product of the liquor brewing industry, and its direct release into the environment can cause severe water pollution. Microbial fuel cells (MFCs) offer the possibility for converting organic matters in thin stillage into clean electricity. However, limited biofilm formation and conductivity are crucial bottlenecks in restricting the power harvest of MFCs. Here, to efficiently harvest electricity power from thin stillage of liquor industry, we adopted a modular engineering strategy to increase biofilm formation and conductivity of Shewanella oneidensis via enhancing the component biosynthesis of extracellular polymer substrates (EPS) matrix, regulating intracellular c-di-GMP level, and constructing of artificial hybrid system. The results showed that the constructed CNTs@CF-EnBF2 hybrid system with low charge-transfer resistance enabled a maximum output power density of 576.77 mW/m2 in lactate-fed MFCs. Also, to evaluate the capability of harvesting electricity from actual wastewater, the CNTs@CF-EnBF2 system was employed to treat actual thin stillage, obtaining a maximum output power density of 495.86 mW/m2, 3.3-fold higher than the wild-type strain. Our research suggested that engineering and regulating EPS biosynthesis effectively promoted bioelectricity harvest, providing a green and sustainable treatment strategy for thin stillage.

In-vivo programmable acoustic manipulation of genetically engineered bacteria.

Acoustic tweezers can control target movement through the momentum interaction between an acoustic wave and an object. This technology has advantages over optical tweezers for in-vivo cell manipulation due to its high tissue penetrability and strong acoustic radiation force. However, normal cells are difficult to acoustically manipulate because of their small size and the similarity between their acoustic impedance and that of the medium. In this study, we use the heterologous expression of gene clusters to generate genetically engineered bacteria that can produce numerous sub-micron gas vesicles in the bacterial cytoplasm. We show that the presence of the gas vesicles significantly enhances the acoustic sensitivity of the engineering bacteria, which can be manipulated by ultrasound. We find that by employing phased-array-based acoustic tweezers, the engineering bacteria can be trapped into clusters and manipulated in vitro and in vivo via electronically steered acoustic beams, enabling the counter flow or on-demand flow of these bacteria in the vasculature of live mice. Furthermore, we demonstrate that the aggregation efficiency of engineering bacteria in a tumour is improved by utilizing this technology. This study provides a platform for the in-vivo manipulation of live cells, which will promote the progress of cell-based biomedical applications.

Performance Assessment of Self-Healing Polymer-Modified Bitumens by Evaluating the Suitability of Current Failure Definition, Failure Criterion, and Fatigue-Restoration Criteria.

Fatigue cracking is a common form of flexible pavement distress, which generally starts and spreads through bitumen. To address this issue, self-healing elastomer (SHE) modified bitumens were elaborated to assess whether these novel materials can overcome the neat asphalt (NA) fatigue performance and whether the current failure definition, failure criterion, and fatigue-restoration criteria can fit their performance. All bitumens were subjected to short-term and long-term aging. Linear amplitude sweep (LAS) test, LAS with rest period (LASH), and simplified viscoelastic-continuum-damage (S-VECD) model were utilized to appraise the behavior of the mentioned bitumens. The results showed that maximum stored pseudo-strain energy (PSE) and tau (τ) × N (number of loading cycles) failure definitions exhibited high efficiency to accommodate the fatigue life of NA and SHE-modified bitumens. Both failure criteria identified that SHE-modified bitumen (containing 1% of SHE) showed the highest increment of fatigue performance (67.1%) concerning NA. The failure criterion based on total released PSE, in terms of the area under the released PSE curve, was the only failure concept with high efficiency (R2 up to 0.999) to predict asphalt binder fatigue life. As well, the current framework to evaluate bitumen self-restoration failed to fully accommodate asphalt binder behavior, because bitumen with higher restoration could not exhibit greater fatigue performance. Consequently, a new procedure to assess this property including fatigue behavior was proposed, showing consistent results, and confirming that SHE-modified bitumen (containing 1% of SHE) exhibited the highest increment of fatigue performance (154.02%) after application of the rest period. Hence, the optimum SHE content in NA was 1%. Furthermore, it was found that a greater number of loading cycles to failure (Nf) did not ensure better fatigue performance and stored PSE influenced the bitumen fatigue behavior.

Engineering Shewanella oneidensis to efficiently harvest electricity power by co-utilizing glucose and lactate in thin stillage of liquor industry.

Thin stillage, rich in glucose and lactate, can seriously pollute water resources when directly discharged into the natural environment. Microbial fuel cells (MFC), as a green and sustainable technology, could utilize exoelectrogens to break down organics in wastewater and harvest electricity. Nevertheless, Shewanella oneidensis MR-1, cannot utilize thin stillage for efficient power generation. Here, to enable S. oneidensis to co-utilize glucose and lactate from thin stillage, an engineered S. oneidensis G7∆RSL1 was first created by constructing glucose metabolism pathway, promoting glucose and lactate co-utilization, and enhancing biofilm formation. Then, to enhance biofilm conductivity, we constructed a 3D self-assembled G7∆RSL1-rGO/CNT biohybrid with maximum power density of 560.4 mW m-2 and 373.7 mW m-2 in artificial and actual thin stillage, respectively, the highest among the reported genetically engineered S. oneidensis with thin stillage as carbon source. This study provides a new strategy to facilitate practical applications of MFC in wastewater remediation and efficient power recovery.

Graphene quantum dots induce cascadic apoptosis via interaction with proteins associated with anti-oxidation after endocytosis by Trypanosoma brucei.

Trypanosoma brucei, the pathogen causing African sleeping sickness (trypanosomiasis) in humans, causes debilitating diseases in many regions of the world, but mainly in African countries with tropical and subtropical climates. Enormous efforts have been devoted to controlling trypanosomiasis, including expanding vector control programs, searching for novel anti-trypanosomial agents, and developing vaccines, but with limited success. In this study, we systematically investigated the effect of graphene quantum dots (GQDs) on trypanosomal parasites and their underlying mechanisms. Ultrasmall-sized GQDs can be efficiently endocytosed by T. brucei and with no toxicity to mammalian-derived cells, triggering a cascade of apoptotic reactions, including mitochondrial disorder, intracellular reactive oxygen species (ROS) elevation, Ca2+ accumulation, DNA fragmentation, adenosine triphosphate (ATP) synthesis impairment, and cell cycle arrest. All of these were caused by the direct interaction between GQDs and the proteins associated with cell apoptosis and anti-oxidation responses, such as trypanothione reductase (TryR), a key protein in anti-oxidation. GQDs specifically inhibited the enzymatic activity of TryR, leading to a reduction in the antioxidant capacity and, ultimately, parasite apoptotic death. These data, for the first time, provide a basis for the exploration of GQDs in the development of anti-trypanosomials.

Sonodynamic Therapy With Concentric Ultrasound Imaging Array for Precision Theranostics for Atherosclerotic Plaque.

Atherosclerotic cardiovascular disease is a major cause of human disability and mortality. Our previous study demonstrated the safety and efficacy of sonodynamic therapy (SDT) on atherosclerotic plaques. However, traditional single-element therapeutic transducer has single acoustic field, and positioning therapeutic and imaging transducers in the same position is difficult during ultrasound imaging-guided SDT. Continuously changing the position of transducers to intervene lesions in different positions is required, increasing the difficulty of treatment. Thus, an SDT device with precise theranostics is required. Therefore, we designed and fabricated a "concentric ultrasound transducer for theranostics" (CUST-T), comprising a central 8-MHz linear array transducer for ultrasound imaging, and a peripheral 1-MHz hollow two-dimensional (2-D) planar array transducer for generating phased-array focused ultrasound (PAFUS). The CUST-T exhibited high imaging resolution at a distance of up to 20 mm from the transducer and could generate a personalized complex PAFUS acoustic field to match various lesions. In vitro biomedical results showed that PAFUS-SDT induced RAW264.7-derived foam cell apoptosis leading to a targeting field apoptotic rate 4.36-6.24 times that of the nontargeting field and the significant apoptotic region was consistent with the PAFUS acoustic field. In vivo, PAFUS-SDT guided by ultrasound imaging significantly increased the lumen area ( ) and collagen level ( ), whereas the wall thickness ( ) and lipid content ( ) of rabbit femoral artery were reduced. In conclusion, CUST-T provided image guidance sufficient for accurate SDT for atherosclerotic plaques in peripheral arteries and could be applied in clinical practice.

Engineering Shewanella carassii, a newly isolated exoelectrogen from activated sludge, to enhance methyl orange degradation and bioelectricity harvest.

Electroactive microorganisms (EAMs) play important roles in biogeochemical redox processes and have been of great interest in the fields of energy recovery, waste treatment, and environmental remediation. However, the currently identified EAMs are difficult to be widely used in complex and diverse environments, due to the existence of poor electron transfer capability, weak environmental adaptability, and difficulty with engineering modifications, etc. Therefore, rapid and efficient screening of high performance EAMs from environments is an effective strategy to facilitate applications of microbial fuel cells (MFCs). In this study, to achieve efficient degradation of methyl orange (MO) by MFC and electricity harvest, a more efficient exoelectrogen Shewanella carassii-D5 that belongs to Shewanella spp. was first isolated from activated sludge by WO3 nanocluster probe technique. Physiological properties experiments confirmed that S. carassii-D5 is a Gram-negative strain with rounded colonies and smooth, slightly reddish surface, which could survive in media containing lactate at 30 °C. Moreover, we found that S. carassii-D5 exhibited remarkable MO degradation ability, which could degrade 66% of MO within 72 h, 1.7 times higher than that of Shewanella oneidensis MR-1. Electrochemical measurements showed that MFCs inoculated with S. carassii-D5 could generate a maximum power density of 704.6 mW/m2, which was 5.6 times higher than that of S. oneidensis MR-1. Further investigation of the extracellular electron transfer (EET) mechanism found that S. carassii-D5 strain had high level of c-type cytochromes and strong biofilm formation ability compared with S. oneidensis MR-1, thus facilitating direct EET. Therefore, to enhance indirect electron transfer and MO degradation capacity, a synthetic gene cluster ribADEHC encoding riboflavin synthesis pathway from Bacillus subtilis was heterologously expressed in S. carassii-D5, increasing riboflavin yield from 1.9 to 9.0 mg/g DCW with 1286.3 mW/m2 power density output in lactate fed-MFCs. Furthermore, results showed that the high EET rate endowed a faster degradation efficient of MO from 66% to 86% with a maximum power density of 192.3 mW/m2, which was 1.3 and 1.6 times higher than that of S. carassii-D5, respectively. Our research suggests that screening and engineering high-efficient EAMs from sludge is a feasible strategy in treating organic pollutants.

A Programmable Toolkit to Dynamically Signal Cells Using Peptide Strand Displacement.

The native extracellular matrix communicates and interacts with cells by dynamically displaying signals to control their behavior. Mimicking this dynamic environment in vitro is essential in order to unravel how cell-matrix interactions guide cell fate. Here, we present a synthetic platform for the temporal display of cell-adhesive signals using coiled-coil peptides. By designing an integrin-engaging coiled-coil pair to have a toehold (unpaired domain), we were able to use a peptide strand displacement reaction to remove the cell cue from the surface. This allowed us to test how the user-defined display of RGDS ligands at variable duration and periodicity of ligand exposure influence cell spreading degree and kinetics. Transient display of αVß3-selective ligands instructed fibroblast cells to reversibly spread and contract in response to changes in ligand exposure over multiple cycles, exhibiting a universal kinetic response. Also, cells that were triggered to spread and contract repeatedly exhibited greater enrichment of integrins in focal adhesions versus cells cultured on persistent RGDS-displaying surfaces. This dynamic platform will allow us to uncover the molecular code by which cells sense and respond to changes in their environment and will provide insights into ways to program cellular behavior.

SNP rs12794714 of CYP2R1 is associated with serum vitamin D levels and recurrent spontaneous abortion (RSA): a case-control study.

PURPOSE: Vitamin D (VD) deficiency seems to be associated with the risk of recurrent spontaneous abortion (RSA). Vitamin D receptor (VDR) and cytochrome P450 family 2 subfamily R member 1 (CYP2R1) are two genes which are vital for VD metabolism and actions. However, whether single-nucleotide polymorphisms (SNPs) in these genes are correlated with the risk of RSA are poorly understood. Therefore, we aimed to characterize the relationships among VDR SNPs, CYP2R1 SNPs and RSA. METHODS: This case-control study enrolled 75 RSA patients and 83 controls. Serum VD and some cytokines were detected with LC-MS/MS and flow cytometry, respectively. Genotyping for three SNPs of CYP2R1 (rs10741657, rs10766197 and rs12794714) and five SNPs of VDR (rs7975232, rs1544410, rs2189480, rs2228570 and rs2239179) was done with polymerase chain reaction (PCR) and high-throughput sequencing. All the data were analyzed with appropriate methods and in different models. RESULTS: The results revealed a significant correlation between the AG genotype of CYP2R1 rs12794714 and VD levels (OR 0.686; 95% CI 0.49-0.96; p = 0.028). Besides, the AG and GG genotypes of CYP2R1 rs12794714 were markedly related to the risk of RSA (OR 52.394, 59.497; 95% CI 2.683-1023.265, 3.110-1138.367; p = 0.009, 0.007, respectively). CONCLUSION: Our results indicate that CYP2R1 rs12794714 might be a risk factor for RSA. Hence, early screening of pregnant women for CYP2R1 rs12794714 is necessary to warrant proactive counseling and treatment against RSA.

Enhanced Antimalarial Efficacy Obtained by Targeted Delivery of Artemisinin in Heparin-Coated Magnetic Hollow Mesoporous Nanoparticles.

Malaria is one of the deadliest infectious diseases threatening half of the world population. With the deterioration of the parasiticidal effect of the current antimalarials, novel approaches such as screening of more specific inhibitors and targeted delivery of drugs have been under intensive research. Herein, we prepare hollow mesoporous ferrite nanoparticles (HMFNs) of 200 nm with ferromagnetic properties using a one-pot hydrothermal reaction. A magnetically targeted drug-delivery system coloaded with artemisinin in the inner magnetite shell and heparin on the outer mesoporous shell (HMFN@ART@HEP) is developed. Specific targeting of the magnetic nanoparticles to the parasite-infected erythrocytes is achieved by the attraction between the HMFNs and hemozoin (paramagnetic), a vital metabolite of plasmodium in the erythrocytic stage. With the hemozoin production reaching the maximum during the schizont period of the parasite, HMFN@ART@HEPs are adsorbed to the infected red blood cells (iRBCs), which not only interferes with the release of merozoites but also significantly enhances the inhibitory efficacy due to the increased local concentration of artemisinin. Subsequently, the heparin coated on the surface of the nanoparticles can efficiently interfere with the invasion of freshly released merozoites to new RBCs through the specific interaction between the parasite-derived ligands and heparin, which further increases the inhibitory effect on malaria. As a cluster of heparin, heparin-coated nanoparticles provide stronger blocking capability than free heparin, resulting from multivalent interactions with surface receptors on merozoite. Thus, we have developed a HMFN-based delivery system with considerable antimalarial efficacy, which is a promising platform for treatment against malaria.

Correction: A multi-center randomized prospective study on the treatment of infant bronchiolitis with interferon α1b nebulization.

[This corrects the article DOI: 10.1371/journal.pone.0228391.].

A multi-center randomized prospective study on the treatment of infant bronchiolitis with interferon α1b nebulization.

BACKGROUND: The respiratory syncytial virus (RSV) is the main cause of bronchiolitis in infants and interferon (IFN) α is a commercial antiviral drug. The nebulization of IFN α1b could be a viable treatment method. In this study, the therapeutic effects and safety of IFN α1b delivery via nebulization in infant bronchiolitis were investigated in this multi-center prospective study. METHODS AND FINDINGS: Bronchiolitis patients admitted to 22 hospitals who met the inclusion criteria were enrolled and randomly allocated to four groups: control, IFN Intramuscular Injection, IFN Nebulization 1 (1 µg/kg), and IFN Nebulization 2 (2 µg/kg) groups. All patients were observed for 7 days. The therapeutic effects and safety of different IFN delivery doses and delivery modes were evaluated. Coughing severity change, as scored by the researchers and parents, between days 1 and 3 was significantly different between the IFN Nebulization 2 and control groups. Lowell wheezing score change between days 3 and 5 was significantly different between IFN Nebulization 1 and control groups. There were no significant differences among the four groups regarding the number of consecutive days with fever, three-concave sign, fatigue and sleepiness, and loss of appetite. There were no cases of severe complications, no recurrence of fever, and no regression of mental status. CONCLUSIONS: IFN-α1b could more effectively alleviate coughing and wheezing in bronchiolitis. IFN-α1b nebulization had significant advantages in shortening the duration of wheezing and alleviating coughing.

In utero exposure to persistent and nonpersistent endocrine-disrupting chemicals and anogenital distance. A systematic review of epidemiological studies†.

Anti-androgenic endocrine-disrupting chemicals (EDCs) can cross the placenta to modify early offspring sexual dimorphic markers. These changes are linked to anogenital distance (AGD), which is an androgen-sensitive anthropometric parameter used as a biomarker of perineal growth and caudal migration of the genital tubercle. This review aimed to summarize strength of evidence for associations of in utero exposure to EDCs with AGD and to identify gaps and limitations in the literature so as to inform future research. We performed an electronic search of English literature in September 2019 in medical literature analysis and retrieval system online (MEDLINE), Web of Science and Toxline. We included epidemiological studies that examined in utero exposure to persistent and nonpersistent EDCs and considered AGD in offspring as an outcome. Our review contained 16 investigations examining exposure to persistent EDCs (nine studies) and nonpersistent EDCs (seven studies). Some individual studies reported an inverse association between exposure to bisphenol A (BPA), dioxins, perfluoroalkyl substances, and organochlorides and AGD in both male and female offspring. Meta-analysis of three studies found a small reduction of AGD in female offspring exposed to BPA. The number of studies per chemical is small, and number of subjects examined is limited; so, replication of these results is needed. To achieve more specificity and better replication of results, future studies should establish the association of nonpersistent EDCs using multiple urine samples, evaluate the cumulative impact of exposure to a mixture of anti-androgenic chemicals, and offer adequate consideration of more maternal- and children-related confounding factors.

Pulmonary invasive fungal disease and bacterial pneumonia: a comparative study with high-resolution CT.

BACKGROUND: Early diagnosis of invasive fungal disease (IFD) is challenging. High-resolution computed tomography (CT) may improve IFD diagnosis; however, there are no definitive imaging signs for differentiating between bacterial pneumonia and IFD. METHODS: We retrospectively evaluated CT images of 208 patients with IFD (n = 102) or bacterial pneumonia (n = 106). We classified pulmonary opacities as consolidations, ground-glass opacities (GGOs), or nodules and recorded the presence of perinodular ground-glass halos, reversed halo sign (RSH), and cavitation (crescent-shaped or not). RESULTS: Consolidation appeared in 83.3% and 92.5% of patients with IFD and bacterial pneumonia, respectively. Multifocal non-segmental consolidation was more common in IFD (48%) than bacterial pneumonia (22.6%; P < 0.05). Segmental or subsegmental consolidation was more common in bacterial pneumonia (43.4%) than IFD (7.8%; P < 0.01). GGOs and nodules were more common in IFD than bacterial pneumonia (60.8% vs. 24.5% and 54.9% vs. 15.1%, respectively; each P < 0.05). Consolidation combined with GGO, nodules, or both GGO and nodules was more frequent in IFD than in bacterial pneumonia (each P < 0.05). Nodules with halo sign (n = 23) appeared in 22.5% and 3.8% of patients with IFD and bacterial pneumonia, respectively. Nodules with RSH appeared only in IFD, and those with cavitation appeared in 11.8% and 1.9% of patients with IFD and bacterial pneumonia, respectively. CONCLUSIONS: Consolidation plus GGO and nodules or consolidation plus nodules is suggestive for IFD. Segmental or subsegmental consolidations are more frequent in bacterial pneumonia than in IFD. Large nodules, as well as nodules with halo sign or both small and large nodules, are related to IFD.

Sensitive detection of low-abundance in-frame deletions in EGFR exon 19 using novel wild-type blockers in real-time PCR.

Epidermal growth factor receptor (EGFR) mutations are associated with response of tyrosine kinase inhibitors (TKIs) for patients with advanced non-small cell lung cancer (NSCLC). However, the existing methods for detection of samples having rare mutations(i.e. ~0.01%) have limits in terms of specificity, time consumption or cost. In the current study, novel wild-type blocking (WTB) oligonucleotides modified with phosphorothioate or inverted dT at the 5'-termini were designed to precisely detect 11 common deletion mutations in exon 19 of EGFR gene (E19del) using a WTB-PCR assay. And internal competitive leptin amplifications were further applied to enhance the specificity of the WTB-PCR system. Our results showed that WTB-PCR could completely block amplification of wild-type EGFR when 200 ng of DNA was used as template. Furthermore, the current WTB-PCR assay facilitated the detection of E19del mutations with a selectivity of 0.01% and sensitivity as low as a single copy. And, the results showed that the current WTB-PCR system exceeded detection limits afforded by the ARMS-PCR assay. In conclusion, the current WTB-PCR strategy represents a simple and cost-effective method to precisely detect various low-abundance deletion mutations.

Clinical efficacy of bevacizumab combined with gemcitabine and cisplatin combination chemotherapy in the treatment of advanced non-small cell lung cancer.

PURPOSE: To investigate the clinical efficacy of bevacizumab combined with gemcitabine and cisplatin (GP) combination chemotherapy in the treatment of advanced non-small cell lung cancer (NSCLC). METHODS: A total of 186 patients with advanced NSCLC who were admitted to the First Affiliated Hospital of Kunming Medical University from October 2013 to June 2016 were randomly divided into control group and observation group, with 93 cases in each group. Patients in the control group were treated with GP chemotherapy, while patients in observation group were treated with intravenous infusion of bevacizumab combined with GP chemotherapy. Treatment was administered for 3 courses, every 3 weeks. After treatment, clinical efficacy, tumor markers levels (CEA and CYFRA21-1), serum vascular endothelial growth factor (VEGF) levels and adverse reactions were compared between two groups. RESULTS: After treatment, the total effective rate and disease control rate in the control group were 40.86% and 70.97%, respectively, while the total effective rate and disease control rate in the observation group were 70.97% and 90.32% respectively, (p<0.05). After treatment, the levels of CEA, CYFRA21-1 and serum VEGF in both groups were significantly lower than those before treatment (p<0.05), and decreases were more significant in the observation group than in the control group (p<0.05). The overall 1-, 3- and 5-year survival rates were 52.69% (49 cases), 36.56% (34 cases) and 25.81% (24 cases) for the observation group, and 43.01% (40 cases), 27.96% (26 cases) and 15.05 % (14 cases) for the control group. Overall survival rate in the observation group was significantly higher than the one in the control group (p<0.05). CONCLUSION: The combination of bevacizumab plus GP chemotherapy for advanced NSCLC can improve serum tumor markers and clinical efficacy, thus prolonging the long-term survival of patients. It is worthy of clinical application.

Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR/Cas9 genomic editing technology.

IAA2 is a member of the Aux/IAA auxin responsive gene family in Arabidopsis thaliana. No iaa2 mutant has been reported until now, thus hindering its further mechanistic investigations. The normal genomic editing technology of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) uses only a single guide RNA (sgRNA) to target one site in a specific gene, and the gene knockout efficiency is not high. Instead, multiple sgRNAs can target multiple sites; therefore, the efficiency may be improved. In the present investigation, we used the golden-gate cloning strategy and two rounds of PCR reactions to combine three sgRNAs in the same entry vector. The final expression vector was obtained by LR reactions with the destination vector containing the Cas9 expression cassette. Four out of the six sgRNAs were effective, and we also obtained a lot of insertion and deletion mutations. Compared with one sgRNA approach, multiple sgRNAs displayed higher gene-knockout efficiency and produced more germ-line mutants. Thus, we established a more rapid and efficient method and generated five mutants for further studies of IAA2 functions.