Toward mosquito control with Chlamydomonas : expression of Cry genes from Bacillus thuringiensis israelensis in the chloroplast of Chlamydomonas reinhardti

Although crop plants that express Cry genes have been developed and used in the field to help control insect pests, genes that are toxic to mosquito larvae have not been exploited successfully for mosquito control. Chlamydomonas reinhardtii is a feasible platform for Bacillus thuringiensis israelensis (Bti) toxin expression and deployment as a mosquito larvicide. C. reinhardtii chloroplast genome engineering is well established and ideal for this application. The Bti Cry proteins Cry4Ba (128 kDa), Cry11Aa (72 kDa), and a truncated Cry4Aa (Cry4A700; 74 kDa) were recoded and expressed from the chloroplast genome of C. reinhardtii. Since these proteins can be toxic to heterologous hosts, the inducible Cyc6:Nac2-psbD expression system was used. The codon-optimized Cry genes were outfitted with a modified psbD 5’ region, and integrated into the chloroplast genome of the Ind41_18 strain; homoplasmicity of the transformants was verified by PCR. Western blots of total cell protein showed the accumulation of all three proteins under induction (i.e., minus Cu2+) conditions, with relative protein expression in the order of Cry4Aa700 >> Cry11Aa > Cry4Ba. A live-cell bioassay demonstrated the toxicity of the Cry4Aa700 and Cry11Aa transformants against larvae of Aedes aegypti and Culex quinquefasciatus. Surprisingly, the growth rates of the transformants under inducing and noninducing conditions were very similar, suggesting that these Cry gene constructs were not very toxic to these cells (Ind41_18 host strain under minus-Cu2+ conditions). Finally, RT-PCR analysis of the mRNAs suggested that Cry11Aa expression may be limited at the post-translational level. Constitutive expression of Cry toxins is essential for field application of Bti-Chlamydomonas. The same synthetic genes that were inducibly expressed in the Ind41_18 strain (above) were transformed into a wild-type strain of C. reinhardtii, but only Cry4Ba and Cry11Aa transformants reached homoplasmicity. Western blotting results confirmed the accumulation of Cry11Aa in those transformants, and indicated that it was at least as high as in the inducible system. The Cry11Aa wild-type strain was also toxic to A. aegypti larvae in a live cell bioassay. These results lay the foundation for obtaining Bti-Chlamydomonas strains that are highly toxic to mosquito larvae, and safe for non-target organisms. Taken together with the environmental and genetic controls on this organism and its chloroplast genes should make it an attractive biocontrol agent.Plant Biolog

Coexistence of UAVs and Terrestrial Users in Millimeter-Wave Urban Networks

5G millimeter-wave (mmWave) cellular networks are in the early phase of commercial deployments and present a unique opportunity for robust, high-data-rate communication to unmanned aerial vehicles (UAVs). A fundamental question is whether and how mmWave networks designed for terrestrial users should be modified to serve UAVs. The paper invokes realistic cell layouts, antenna patterns, and channel models trained from extensive ray tracing data to assess the performance of various network alternatives. Importantly, the study considers the addition of dedicated uptilted rooftop-mounted cells for aerial coverage, as well as novel spectrum sharing modes between terrestrial and aerial network operators. The effect of power control and of multiuser multiple-input multiple-output are also studied

Millimeter-Wave UAV Coveragein Urban Environments

With growing interest in mmWave connectivity for UAVs, a basic question is whether networks intended for terrestrial users can provide sufficient aerial coverage as well. To assess this possibility, the paper proposes a novel evaluation methodology using generative models trained on detailed ray tracing data. These models capture complex propagation characteristics and can be readily combined with antenna and beamforming assumptions. Extensive simulation using these models indicate that standard (street-level and downtilted) base stations at typical microcellular densities can indeed provide satisfactory UAV coverage. Interestingly, the coverage is possible via a conjunction of antenna sidelobes and strong reflections. With sparser deployments, the coverage is only guaranteed at progressively higher altitudes. Additional dedicated (rooftop-mounted and uptilted) base stations strengthen the coverage provided that their density is comparable to that of the standard deployment, and would be instrumental for sparse deployments of the latter

Cellular Wireless Networks in the Upper Mid-Band

The upper mid-band -- roughly from 7 to 24 GHz -- has attracted considerable recent interest for new cellular services. This frequency range has vastly more spectrum than the highly congested bands below 7 GHz while offering more favorable propagation and coverage than the millimeter wave (mmWave) frequencies. Realizing the full potential of these bands, however, will require fundamental changes to the design of cellular systems. Most importantly, spectrum will likely need to be shared with incumbents including communication satellites, military RADAR, and radio astronomy. Also, due to the wide bandwidth, directional nature of transmission, and intermittent occupancy of incumbents, cellular systems will need to be agile to sense and intelligently use large spatial and bandwidth degrees of freedom. This paper attempts to provide an initial assessment of the feasibility and potential gains of wideband cellular systems operating in the upper mid-band. The study includes: (1) a system study to assess potential gains of multi-band systems in a representative dense urban environment; (2) propagation calculations to assess potential cross interference between satellites and terrestrial cellular services; and (3) design and evaluation of a compact multi-band antenna array structure. Leveraging these preliminary results, we identify potential future research directions to realize next-generation systems in these frequencies.Comment: 11 page

Analysis of Dual Connectivity Gain in Terms of Delay and Throughput

Dual connectivity is very important for gradual transition from LTE to 5G. Release 12 of the 3GPP specification introduced DC scheme, but what advantage we can get from DC is not evaluated yet. In this paper, we simulate the DC schemes (LTE LTE DC, LTE-mmWave DC, mmWave - mmWave DC) using newly modified network simulator (NS3) for 5G and evaluate the DC gains in terms of throughput and delay. We demonstrate the DC would increase the throughput under the fast switching scheme and decrease the end-to-end delay a lot.N

A Dual-Connection based Handover Scheme for Ultra-Dense Millimeter-Wave Cellular Networks

Mobile users in an ultra-dense millimeter-wave cellular network experience handover events more frequently than in conventional networks, which results in increased service interruption time and performance degradation due to blockages. Multi-connectivity has been proposed to resolve this, and it also extends the coverage of millimeter-wave communications. In this paper, we propose a dual-connection based handover scheme for mobile UEs in an environment where they are connected simultaneously with two millimeter-wave cells to overcome frequent handover problems. This scheme allows a mobile UE to choose its serving link between the two mmWave connections according to the measured SINRs and then the corresponding base stations may forward duplicate packets to the UE. We compare our dual-connection based scheme with a conventional single-connection based scheme through ns-3 simulation. The simulation results show that the proposed scheme significantly reduces handover rate and delay. Therefore, we argue that the dual-connection based scheme helps mobile users achieve performance goals they require in ultra-dense cellular environments.N

Performance Evaluation of Channel Bonding in Dense Scenario

IEEE 802.11ac standard has extended channel bonding to improve network throughput. However, in an increasingly dense network environment, 802.11ac has not achieved desired performance. To solve the problem, the latest standard 802.11ax which aims to improve the average user performance has been proposed. In addition, TGax presents a scenario that reflects the dense network environment. In this paper, we analyze the performance of using channel bonding in dense scenario through ns-3 simulations. We also find how use of RTS/CTS affects spatial reuse in the scenario.N

Overcoming Poor Transgene Expression in the Wild-Type Chlamydomonas Chloroplast: Creation of Highly Mosquitocidal Strains of Chlamydomonas reinhardtii

High-level expression of transgenes in the chloroplast of wild-type Chlamydomonas reinhardtii (C. reinhardtii) remains challenging for many genes (e.g., the cry toxin genes from Bacillus thuringiensis israelensis). The bottleneck is presumed to be post-transcriptional and mediated by the 5′ element and the coding region. Using 5′ elements from highly expressed photosynthesis genes such as atpA did not improve the outcome with cry11A regardless of the promoter. However, when we employed the 5′ UTR from mature rps4 mRNA with clean fusions to promoters, production of the rCry11A protein became largely promoter-dependent. The best results were obtained with the native 16S rrn promoter (−91 to −1). When it was fused to the mature 5′ rps4 UTR, rCry11A protein levels were ~50% higher than was obtained with the inducible system, or ~0.6% of total protein. This level was sufficient to visualize the 73-kDa rCry11A protein on Coomassie-stained gels of total algal protein. In addition, analysis of the expression of these transgenes by RT-PCR indicated that RNA levels roughly correlated with protein production. Live cell bioassays using the best strains as food for 3rd instar Aedes aegypti larvae showed that most larvae were killed even when the cell concentration was as low as 2 × 104 cells/mL. Finally, the results indicate that these highly toxic strains are also quite stable, and thus represent a key milestone in using C. reinhardtii for mosquito control