Survey of Cooperative Advanced Driver Assistance Systems: From a Holistic and Systemic Vision.

The design of cooperative advanced driver assistance systems (C-ADAS) involves a holistic and systemic vision that considers the bidirectional interaction among three main elements: the driver, the vehicle, and the surrounding environment. The evolution of these systems reflects this need. In this work, we present a survey of C-ADAS and describe a conceptual architecture that includes the driver, vehicle, and environment and their bidirectional interactions. We address the remote operation of this C-ADAS based on the Internet of vehicles (IoV) paradigm, as well as the involved enabling technologies. We describe the state of the art and the research challenges present in the development of C-ADAS. Finally, to quantify the performance of C-ADAS, we describe the principal evaluation mechanisms and performance metrics employed in these systems.

SCHC over LoRaWAN Efficiency: Evaluation and Experimental Performance of Packet Fragmentation.

Low Power Wide Area Networks (LPWAN) are expected to enable the massive connectivity of small and constrained devices to the Internet of Things. Due to the restricted nature of both end devices and network links, LPWAN technologies employ network stacks where there is no interoperable network layer as a general case; instead, application data are usually placed directly into technology-specific two-layer frames. Besides not being able to run standard IP-based protocols at the end device, the lack of an IP layer also causes LPWAN segments to operate in an isolated manner, requiring middleboxes to interface non-IP LPWAN technologies with the IP world. The IETF has standardized a compression and fragmentation scheme, called Static Context Header Compression and Fragmentation (SCHC), which can compress and fragment IPv6 and UDP headers for LPWAN in a way that enables IP-based communications on the constrained end device. This article presents a model to determine the channel occupation efficiency based on the transmission times of SCHC messages in the upstream channel of a LoRaWAN™ link using the ACK-on-Error mode of standard SCHC. The model is compared against experimental data obtained from the transmission of packets that are fragmented using a SCHC over LoRaWAN implementation. This modeling provides a relationship between the channel occupancy efficiency, the spreading factor of LoRa™, and the probability of an error of a SCHC message. The results show that the model correctly predicts the efficiency in channel occupation for all spreading factors. Furthermore, the SCHC ACK-on-Error mode implementation for the upstream channel has been made fully available for further use by the research community.

Direct-to-Satellite IoT Slotted Aloha Systems with Multiple Satellites and Unequal Erasure Probabilities.

Direct-to-satellite Internet of Things (IoT) solutions have attracted a lot of attention from industry and academia recently, as promising alternatives for large scale coverage of a massive number of IoT devices. In this work, we considered that a cluster of IoT devices was under the coverage of a constellation of low-Earth orbit (LEO) satellites, while slotted Aloha was used as a medium access control technique. Then, we analyzed the throughput and packet loss rate while considering potentially different erasure probabilities at each of the visible satellites within the constellation. We show that different combinations of erasure probabilities at the LEO satellites and the IoT traffic load can lead to considerable differences in the system's performance. Next, we introduce an intelligent traffic load distribution (ITLD) strategy, which, by choosing between a non-uniform allocation and the uniform traffic load distribution, guarantees a high overall system throughput, by allocating more appropriate amounts of traffic load at different positions (i.e., different sets of erasure probabilities) of the LEO constellation with respect to the IoT cluster. Finally, the results show that ITLD, a mechanism with low implementation complexity, allows the system to be much more scalable, intelligently exploiting the potential of the different positions of the satellite constellation.

Impact of Safety Message Generation Rules on the Awareness of Vulnerable Road Users.

In the face of cooperative intelligent transportation systems (C-ITS) advancements, the inclusion of vulnerable road users (VRU), i.e., pedestrians, cyclists, and motorcyclists, has just recently become a part of the discussion. Including VRU in C-ITS presents new challenges, most notably the trade-off between the increase in VRU safety and the aggravation in channel congestion resulting from VRU-generated messages. However, previous studies mainly focus on network-related metrics without giving much consideration to VRU safety-related metrics. In this context, we evaluated such a trade-off with a study of motion-based message generation rules for VRU transmissions. The rules were analyzed using theoretical and simulation-based evaluations. In addition to studying the message generation rules using channel load metrics, such as channel busy ratio (CBR) and packet delivery ratio (PDR), we introduced a new metric: the VRU Awareness Probability (VAP). VAP uses the exchange of messages from active VRU to measure the probability of VRU detection by nearby vehicles. Results show that fixed message-filtering mechanisms reduce the overall channel load, but they could negatively impact VRU detection. We established the importance of quantifying the VRU awareness and its inclusion in C-ITS analysis because of its direct impact on VRU safety. We also discussed approaches that include VRU context and dynamism to improve the definition of message generation rules.

The Role of the Flagellar Protein FlgJ in the Virulence of Brucella abortus.

Brucella abortus is a facultative intracellular pathogen that causes a zoonosis called brucellosis. This disease leads to abortion and infertility in cattle, and diverse complications in humans. B. abortus is a successful intracellular bacterium that has developed the ability to evade the host's immune system and it replicates in professional and non-professional phagocytic cells, persisting in the different tissues, and organs of its hosts. It has been described that Brucella expresses a polar flagellum under certain conditions, but its function is still unknown. In this study we evaluated the role of the FlgJ, a protein, presumably a peptidoglycan hydrolase involved in flagellum formation and in the virulence of B. abortus strain 2308. B. abortus 2308 ΔflgJ mutant and complemented strains were constructed to study the function of the FlgJ protein in the context of the virulence of this pathogen in in vitro and in vivo assays. The results showed that the elimination of the flgJ gene delays the growth rate of B. abortus in culture, reduces its intracellular survival capacity in professional and non-professional phagocytic cells, rendering it unable to escape from the endocytic route and not reaching the endoplasmic reticulum. It also negatively affects their persistence in BALB/c mice. Functionally, the B. abortus 2308 flgJ gene restored motility to an E. coli flgJ mutant gene. Furthermore, it was discovered that the production of FlgJ protein is associated with the bacterial adherence by B. abortus. Therefore, although the specific function of the polar flagellum for Brucella is unknown, the data indicates that the flagellar flgJ gene and its product are required for full virulence of B. abortus 2308, since its deletion significantly reduces the fitness of this pathogen in vitro and in vivo.

Immunization of mice with chimeric antigens displaying selected epitopes confers protection against intestinal colonization and renal damage caused by Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) cause diarrhea and dysentery, which may progress to hemolytic uremic syndrome (HUS). Vaccination has been proposed as a preventive approach against STEC infection; however, there is no vaccine for humans and those used in animals reduce but do not eliminate the intestinal colonization of STEC. The OmpT, Cah and Hes proteins are widely distributed among clinical STEC strains and are recognized by serum IgG and IgA in patients with HUS. Here, we develop a vaccine formulation based on two chimeric antigens containing epitopes of OmpT, Cah and Hes proteins against STEC strains. Intramuscular and intranasal immunization of mice with these chimeric antigens elicited systemic and local long-lasting humoral responses. However, the class of antibodies generated was dependent on the adjuvant and the route of administration. Moreover, while intramuscular immunization with the combination of the chimeric antigens conferred protection against colonization by STEC O157:H7, the intranasal conferred protection against renal damage caused by STEC O91:H21. This preclinical study supports the potential use of this formulation based on recombinant chimeric proteins as a preventive strategy against STEC infections.

Review and Evaluation of MAC Protocols for Satellite IoT Systems Using Nanosatellites.

Extending the internet of things (IoT) networks to remote areas under extreme conditions or for serving sometimes unpredictable mobile applications has increased the need for satellite technology to provide effective connectivity. However, existent medium access control (MAC) protocols deployed in commercial satellite networks were not designed to offer scalable solutions for the increasing number of devices predicted for IoT in the near future, nor do they consider other specific IoT characteristics. In particular, CubeSats-a low-cost solution for space technology-have the potential to become a wireless access network for the IoT, if additional requirements, including simplicity and low demands in processing, storage, and energy consumption are incorporated into MAC protocol design for satellite IoT systems. Here we review MAC protocols employed or proposed for satellite systems and evaluate their performance considering the IoT scenario along with the trend of using CubeSats for IoT connectivity. Criteria include channel load, throughput, energy efficiency, and complexity. We have found that Aloha-based protocols and interference cancellation-based protocols stand out on some of the performance metrics. However, the tradeoffs among communications performance, energy consumption, and complexity require improvements in future designs, for which we identify specific challenges and open research areas for MAC protocols deployed with next low-cost nanosatellite IoT systems.

Draft Genome Sequence of Chilean Antarctic Pseudomonas sp. Strain K2I15.

We announce the draft genome sequence of Pseudomonas sp. strain K2I15, isolated from the rhizosphere of Deschampsia antarctica Desv. The genome sequence had 6,645,031 bp with a G+C content of 60.4%. This genome provides insights into the niche adaptation, prophage carriage, and evolution of this specific Antarctic bacteria.

Draft Genome Sequence of Bacillus sp. Strain K2I17, Isolated from the Rhizosphere of Deschampsia antarctica Desv.

We present here the draft genome sequence of Bacillus sp. strain K2I17, which was isolated from the rhizosphere of Deschampsia antarctica Desv. The genomic sequence contained 6,113,341 bp. This genome provides insights into the possible new biomedical and biotechnical applications of this specific Antarctic bacterium.

Genetic Diversity and Virulence Determinants of Escherichia coli Strains Isolated from Patients with Crohn's Disease in Spain and Chile.

Adherent-invasive Escherichia coli (AIEC) strains are genetically variable and virulence factors for AIEC are non-specific. FimH is the most studied pathogenicity-related protein, and there have been few studies on other proteins, such as Serine Protease Autotransporters of Enterobacteriacea (SPATEs). The goal of this study is to characterize E. coli strains isolated from patients with Crohn's disease (CD) in Chile and Spain, and identify genetic differences between strains associated with virulence markers and clonality. We characterized virulence factors and genetic variability by pulse field electrophoresis (PFGE) in 50 E. coli strains isolated from Chilean and Spanish patients with CD, and also determined which of these strains presented an AIEC phenotype. Twenty-six E. coli strains from control patients were also included. PFGE patterns were heterogeneous and we also observed a highly diverse profile of virulence genes among all E. coli strains obtained from patients with CD, including those strains defined as AIEC. Two iron transporter genes chuA, and irp2, were detected in various combinations in 68-84% of CD strains. We found that the most significant individual E. coli genetic marker associated with CD E. coli strains was chuA. In addition, patho-adaptative fimH mutations were absent in some of the highly adherent and invasive strains. The fimH adhesin, the iron transporter irp2, and Class-2 SPATEs did not show a significant association with CD strains. The V27A fimH mutation was detected in the most CD strains. This study highlights the genetic variability of E. coli CD strains from two distinct geographic origins, most of them affiliated with the B2 or D E. coli phylogroups and also reveals that nearly 40% of Chilean and Spanish CD patients are colonized with E.coli with a characteristic AIEC phenotype.

Evaluation of protective effect of DNA vaccines encoding the BAB1_0263 and BAB1_0278 open reading frames of Brucella abortus in BALB/c mice.

The immunogenicity of two DNA vaccines encoding open reading frames (ORFs) of genomic island 3 (GI-3), specific for Brucella abortus and Brucella melitensis, has been examined. Intramuscular injection of plasmid DNA carrying the BAB1_0263 and BAB1_0278 genes (pVF263 and pVF278, respectively) into BALB/c mice elicited both humoral and cellular immune responses. Mice injected with pVF263 or pVF278 had a dominant immunoglobulin G2a (IgG2a) response. In addition, both DNA vaccines elicited a T-cell-proliferative response, but only pVF263 induced significant levels of interferon gamma (INF-γ) upon restimulation with recombinant 263 protein. Neither DNA vaccine induced interleukin (IL)-10, nor IL-4, upon stimulation with an appropriate recombinant protein or crude Brucella protein, suggesting the induction of a typical T-helper 1 (Th1)-dominated immune response. Furthermore, the pVF278 DNA vaccines induced protection in BALB/c mice against challenge with the virulent strain B. abortus 2308. Taken together, these data suggest that DNA vaccination offers an improved delivery strategy for the BAB1_0278 antigen, and provide the first evidence of a protective effect of this antigen.

Roles of genomic island 3 (GI-3) BAB1_0278 and BAB1_0263 open reading frames (ORFs) in the virulence of Brucella abortus in BALB/c mice.

The genomic island 3 (GI-3) shared by Brucella melitensis and Brucella abortus contains 29 genes encoding mostly unknown proteins. Within this island, the open reading frames (ORFs) BAB1_0278 and BAB1_0263 are present, BAB1_0278 encodes a hypothetical protein of 64 amino acids sharing a domain with the GcrA superfamily, whereas the amino acid sequence of BAB1_0263 showed 42% identity with an iron regulated Lsr2 protein. We obtained one deletion mutant for each one of these ORFs present within the B. abortus GI-3 named BA-278 and BA-263, respectively. Both mutants were evaluated with respect to their ability to invade and replicate in nonprofessional and professional phagocytes (HeLa and J774.A1 cells) and their virulence in mice. Both mutants invaded efficiently HeLa and J774. A1 cells, however, 48-h post-infection the BA-278 mutant showed a lower intracellular persistence. The deletion of the ORF BAB1_0278, also affected the persistence of B. abortus in the spleens of mice, unlike to the deletion of the ORF BAB1_0263. These results allow us to conclude that BAB1_0278 ORF contributes to virulence of Brucella, since it is necessary to establish an optimal infectious process.

Characterization of genomic island 3 and genetic variability of Chilean field strains of Brucella abortus.

One of the capabilities developed by bacteria is the ability to gain large fragments of DNA from other bacteria or to lose portions of their own genomes. Among these exchangeable fragments are the genomic islands (GIs). Nine GIs have been identified in Brucella, and genomic island 3 (GI-3) is shared by two pathogenic species, B. melitensis and B. abortus. GI-3 encodes mostly unknown proteins. One of the aims of this study was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. abortus from Chile to determine whether these isolates are clonally related. Furthermore, we focused on the characterization of GI-3, studying its organization and the genetic conservation of the GI-3 sequence using techniques such as tiling-path PCR (TP-PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR). Our results, after PFGE was performed on 69 field isolates of B. abortus from Chile, showed that the strains were genetically homogeneous. To increase the power of genetic discrimination among these strains, we used multiple locus variable-number tandem-repeat (VNTR) analysis with 16 loci (MLVA-16). The results obtained by MLVA-16 showed that the strains of B. abortus were genetically heterogeneous and that most of them clustered according to their geographic origin. Of the genetic loci studied, panel 2B was the one describing the highest diversity in the analysis, as well as locus Bruce19 in panel 2A. In relation to the study of GI-3, our experimental analysis by TP-PCR identified and confirmed that GI-3 is present in all wild strains of B. abortus, demonstrating the high stability of gene cluster GI-3 in Chilean field strains.

Vaccination with recombinant Semliki Forest virus particles expressing translation initiation factor 3 of Brucella abortus induces protective immunity in BALB/c mice.

Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression of heterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying recombinant RNA encoding the Brucella abortus translation initiation factor 3 (IF3). The recombinant SFV particles (SFV-IF3 particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challenge with B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies at day 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either recombinant IF3 (rIF3) or crude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, but not interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challenge with the virulent B. abortus strain 2308 (P<0.01). These findings indicate that an SFV-based vector carrying RNA encoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.