Comparison of microbially induced calcium carbonate precipitation eligibility using sporosarcina pasteurii and bacillus licheniformis on two different sands

The use of biological means for ground improvement have become popular, which generally works through the process called microbially-induced calcium carbonate precipitation (MICP). Many studies indicate successful application of MICP based improvement with multiple bacteria and on several soils. Given the proven performance of MICP, this study aims to examine the MICP process by comparing the calcium carbonate precipitation ability of widely studied bacteria, i.e., Sporosarcina pasteurii and relatively under-recognized bacteria, i.e., Bacillus licheniformis to outline the formation success. For this purpose, two different sands were tested for observing precipitation behavior using a series of syringe tests. Furthermore, the effect of concentration and inclusion of calcium chloride for nutrition of bacteria, saturation with water, and hybrid use of two bacteria were investigated in some tests for diversification. X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive x-ray spectroscopy (EDS) were used for the interpretation of results. Results indicated that Sporosarcina pasteurii had performed superior over Bacillus licheniformis when achieving calcium carbonate precipitation in tests for both sands. In addition, many intriguing SEM images contributed to the literature of MICP monitoring, highlighting the effects of the variables investigated.     

Fluoranthene determination based on a rapid and sensitive syringe extraction and solid-phase fluorescence technique

A rapid and sensitive strategy was proposed for the detection of fluoranthene (FL), which is a polycyclic aromatic hydrocarbon (PAH), in water samples. In this work, syringe solid-phase extraction (SPE) combined with solid-phase fluorescence spectrometry was used to determine FL in PAHs polluted environmental samples. The fluorescence signals were directly monitored on the membrane surface after FL was enriched by syringe SPE. Under the optimal conditions, the proposed method showed a linear relationship in the concentration range 2–50 μg/L with a correlation coefficient (R²) of 0.998, and the limit of detection was 0.143 μg/L. The recoveries varied from 93.47% to 109.81% in the actual samples, with the relative standard deviations (n = 3) ranging from 2.06% to 6.32%. According to the results, the established method can be applied in the field of rapid detection as it is fast, simple, portable, and highly sensitive, and has strong anti-interference.     

Simple cost-effective larval injection method for dsRNA delivery to induce RNAi response in Helicoverpa armigera (Hübner)

Microinjection is considered as an effective method for dsRNA delivery in insects. It also facilitates the delivery of a precise quantity of dsRNA in the host insect, inducing an efficient RNAi response. However, the microinjection method needs prior optimization of several parameters like concentration of dsRNA, site of injection, developmental stage of insect etc. for inducing effective RNAi response. Moreover, sophisticated microinjection devices are largely expensive with high maintenance cost. The Old-World bollworm, Helicoverpa armigera (Hübner) is known to be a detrimental polyphagous pest with widespread infestations across the globe. In the present study, we demonstrate a low-cost and effective dsRNA delivery method for inducing RNAi response in H. armigera with the aid of basic insulin injection syringe and fabricated micropipette tip. In order to validate the RNAi response following dsRNA injection, we have selected three key genes from the chitin biosynthesis pathway of the insect. Besides these, argonaute 1 (ago1) was also used as an indicator gene for dsRNA-mediated RNAi induction. Delivery of dsRNA using injection with insulin syringe caused significant upregulation of the ago1 gene in the insect irrespective of any of the three target genes concerned viz. HaNAGK (3.9 fold; p < 0.001), HaAGM (6.3 fold; p < 0.001) and HaUAP (5.9 fold; p < 0.01) respectively, as compared to control injected with nuclease-free water. The dsRNA-injected insects showed aberrant developmental symptoms typical of impeded chitin synthesis, eventually leading to anomalous ecdysis with substantial mortality (up to 69.04%), as compared to controls. The described protocol reduces insect injury, enabling easy restraining of larva and quick execution of dsRNA injection with efficient RNAi response.     

一种昆虫呼吸代谢测定方法的改进

为了探索精准、简易的昆虫呼吸代谢测定办法,本文基于Sable小动物呼吸测量系统,比较了应用Sable呼吸测量系统配置的8通道气路转换器呼吸室和采用鲁尔接头注射器作为替代呼吸室测试昆虫的呼吸代谢。结果表明:应用测量系统自带呼吸室和应用替代呼吸室检测棉铃虫蛹O_2消耗量(前者为0.2425(±0.0143) mL/g·h,后者为0.2389(±0.0146) mL/g·h)和CO_2释放量(前者为0.1562(±0.0098) mL/g·h,后者为0.1639(±0.0092) mL/g·h),两种测试方法无显著差异。与采用系统配置8通道气路转换器和自带呼吸室每测试7个样本耗时2.30 h相比较,应用替代呼吸室测试21个样本仅耗时2.75 h,明显节省测试时间。应用替代呼吸室,从呼出二氧化碳动态亦可以区分黑纹粉蝶不同虫态或不同发育状态的呼吸代谢差异。通过对两种测试方法的分析,推荐应用鲁尔接头注射器作为替代呼吸室的改进方法进行昆虫呼吸代谢生理的研究。     

Cupric ions induce both an efflux of potassium and low molecular mass metabolites in Pseudomonas syringae

Abstract Cu²⁺ induced an efflux of potassium, inorganic phosphate, 260 nm-absorbing materials and ribose-containing molecules in Pseudomonas syringae . No detectable amounts of aspartic and glutamic acids leaked from the cells. It is proposed that the release of inorganic phosphate and other low molecular mass anionic metabolites probably played a role in re-equilibrating the internal charge balance after the exit of K⁺ ions.     

The role of the T-pilus in horizontal gene transfer and tumorigenesis

The T-pilus is a flexuous filamentous appendage that is essential for Agrobacterium tumefaciens virulence. T-pilus subunits are derived from a VirB2-processing reaction that generates cyclized polypeptide subunits. The T-pilus filament has a diameter of 10 nm and contains a lumen approximately 2 nm in diameter. Biogenesis of the T-pilus requires all 11 VirB proteins, but not the VirD4 protein, which is used in conjugal plasmid transfer. VirB4 and VirB11 are two ATPases that may form homohexameric rings within the transport apparatus, which is composed of VirB6-10 proteins.     

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).     

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.     

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.