Search

Research on reducing the movement of pests on wood products has led to several options for safer trade including heat treatment of wood to mitigate pests. In this study, pathogenic organisms commonly regulated in the trade of forest products were tested to determine the minimum heat dose (temperature and time) required to cause mortality. The mycelial stage of tree pathogens, Heterobasidion occidentale, Grosmannia clavigera, Bretziella fagacearum, Phytophthora cinnamomi, P. lateralis, P. ramorum and P. xmultiformis, which may be found in untreated wood products, were tested in vitro using the Humble water bath with parameters simulating the rate of heat applied to wood in a commercial kiln. RNA detection using reverse transcription real-time PCR was used to validate pathogen mortality following treatment for: P. ramorum, P. lateralis, P. cinnamomi, P. xmultiformis and G. clavigera. The lethal temperature for all pathogens ranged from 44 to 50°C for a 30-min treatment duration. Using this method to evaluate heat treatment for other forest product pests is recommended to accurately identify the minimum dose required to support phytosanitary trade. With more data potentially lower heat treatment applications may be recommended under specific conditions to produce more efficient and economical heat treatment schedules and reduce environmental impacts.

Dengue virus (DENV) is responsible for approximately 100 million cases of dengue fever annually, including severe forms such as hemorrhagic dengue and dengue shock syndrome. Despite intensive vaccine research and development spanning several decades, a universally accepted and approved vaccine against dengue fever has not yet been developed. The major challenge associated with the development of such a vaccine is that it should induce simultaneous and equal protection against the four DENV serotypes, because past infection with one serotype may greatly increase the severity of secondary infection with a distinct serotype, a phenomenon known as antibody-dependent enhancement (ADE). Using a lentiviral vector platform that is particularly suitable for the induction of cellular immune responses, we designed a tetravalent T-cell vaccine candidate against DENV (“LV-DEN”). This vaccine candidate has a strong CD8+ T-cell immunogenicity against the targeted non-structural DENV proteins, without inducing antibody response against surface antigens. Evaluation of its protective potential in the preclinical flavivirus infection model, i.e., mice knockout for the receptor to the type I IFN, demonstrated its significant protective effect against four distinct DENV serotypes, based on reduced weight loss, viremia, and viral loads in peripheral organs of the challenged mice. These results provide proof of concept for the use of lentiviral vectors for the development of efficient polyvalent T-cell vaccine candidates against all DENV serotypes.

Wastewater surveillance could provide a means of monitoring SARS-CoV-2 prevalence that does not rely on testing individuals. Here, the authors report results from England’s national wastewater surveillance program, use it to estimate prevalence, and compare estimates with those from population-based prevalence surveys.

ABSTRACT Integrons are mobile genetic elements that have played an important role in the dissemination of antibiotic resistance. Under stress, the integron can generate combinatorial variation in resistance cassette expression by cassette reshuffling, accelerating the evolution of resistance. However, the flexibility of the integron integrase site recognition motif hints at potential off-target effects of the integrase on the rest of the genome that may have important evolutionary consequences. Here, we test this hypothesis by selecting for increased-piperacillin-resistance populations of Pseudomonas aeruginosa with a mobile integron containing a difficult-to-mobilize β-lactamase cassette to minimize the potential for adaptive cassette reshuffling. We found that integron activity can decrease the overall survival rate but also improve the fitness of the surviving populations. Off-target inversions mediated by the integron accelerated plasmid adaptation by disrupting costly conjugative genes otherwise mutated in control populations lacking a functional integrase. Plasmids containing integron-mediated inversions were associated with lower plasmid costs and higher stability than plasmids carrying mutations albeit at the cost of a reduced conjugative ability. These findings highlight the potential for integrons to create structural variation that can drive bacterial evolution, and they provide an interesting example showing how antibiotic pressure can drive the loss of conjugative genes. IMPORTANCE Tackling the public health challenge created by antibiotic resistance requires understanding the mechanisms driving its evolution. Mobile integrons are widespread genetic platforms heavily involved in the spread of antibiotic resistance. Through the action of the integrase enzyme, integrons allow bacteria to capture, excise, and shuffle antibiotic resistance gene cassettes. This integrase enzyme is characterized by its ability to recognize a wide range of recombination sites, which allows it to easily capture diverse resistance cassettes but which may also lead to off-target reactions with the rest of the genome. Using experimental evolution, we tested the off-target impact of integron activity. We found that integrons increased the fitness of the surviving bacteria through extensive genomic rearrangements of the plasmids carrying the integrons, reducing their ability to spread horizontally. These results show that integrons not only accelerate resistance evolution but also can generate extensive structural variation, driving bacterial evolution beyond antibiotic resistance.

Abstract COVID‐19 vaccines already in use or in clinical development may have reduced efficacy against emerging SARS‐CoV‐2 variants. In addition, although the neurotropism of SARS‐CoV‐2 is well established, the vaccine strategies currently developed have not taken into account protection of the central nervous system. Here, we generated a transgenic mouse strain expressing the human angiotensin‐converting enzyme 2, and displaying unprecedented brain permissiveness to SARS‐CoV‐2 replication, in addition to high permissiveness levels in the lung. Using this stringent transgenic model, we demonstrated that a non‐integrative lentiviral vector, encoding for the spike glycoprotein of the ancestral SARS‐CoV‐2, used in intramuscular prime and intranasal boost elicits sterilizing protection of lung and brain against both the ancestral virus, and the Gamma (P.1) variant of concern, which carries multiple vaccine escape mutations. Beyond induction of strong neutralizing antibodies, the mechanism underlying this broad protection spectrum involves a robust protective T‐cell immunity, unaffected by the recent mutations accumulated in the emerging SARS‐CoV‐2 variants.

Zika virus (ZIKV) is a mosquito-borne flavivirus that recently emerged in the South Pacific, Americas, and Caribbean islands, where the larger epidemics were documented. ZIKV infection in humans is responsible for neurological disorders and microcephaly. Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted as a hexameric lipoprotein particle. Intracellular NS1 exists as a dimer that is required for viral replication, whereas the secreted NS1 hexamer interacts with host factors, leading to pathophysiological conditions. In an effort to dispose of specific anti-ZIKV NS1 immune serum, Vero cells were transduced with a lentiviral vector containing the NS1 gene from an epidemic strain of ZIKV. We showed that stably transduced Vero/ZIKV NS1 cell clone was efficient in the secretion of recombinant NS1 oligomer. Immunization of adult rat with purified extracellular NS1 developed anti-ZIKV antibodies that specifically react with the NS1 dimer produced in human cells infected with African and Asian strains of ZIKV. The rat antibody against ZIKV NS1 dimer is a reliable biological tool that enables the immunological detection of secreted NS1 from host-cells infected with ZIKV.

Tackling the challenge created by antibiotic resistance requires understanding the mechanisms behind its evolution. Like any evolutionary process, the evolution of antimicrobial resistance (AMR) is driven by the underlying variation in a bacterial population and the selective pressures acting upon it. Importantly, both selection and variation will depend on the scale at which resistance evolution is considered (from evolution within a single patient to the host population level). While laboratory experiments have generated fundamental insights into the mechanisms underlying antibiotic resistance evolution, the technological advances in whole genome sequencing now allow us to probe antibiotic resistance evolution beyond the lab and directly record it in individual patients and host populations. Here we review the evolutionary forces driving antibiotic resistance at each of these scales, highlight gaps in our current understanding of AMR evolution, and discuss future steps toward evolution-guided interventions. [ABSTRACT FROM AUTHOR]

Ku et al. report a lentiviral (LV) vector system in which expression of vaccine antigens is driven by β2-microglobulin (β2m) promoter. Conducting comparative studies in mice and rats, they find that LV-based vaccine outperforms the ‘gold-standard’ Ad5 by inducing a polyfunctional and long-lived immune response.

Purpose: Accurate surveillance of the COVID-19 pandemic can be weakened by under-reporting of cases, particularly due to asymptomatic or pre-symptomatic infections, resulting in bias. Quantification of SARS-CoV-2 RNA in wastewater (WW) can be used to infer infection prevalence, but uncertainty in sensitivity and considerable variability has meant that accurate measurement remains elusive. Methods & Materials: Data from 44 sewage sites in England, covering 31% of the population, are used in this analysis where samples are available from July 2020 to present day. Samples include the raw SARS-CoV-2 gene copy number and associated meta-data. To establish the sensitivity and specificity of the WW data, we compare to population representative prevalence surveys available across England (the ONS Covid Infection Survey - CIS). The WW data were mapped to sub-regional data of the CIS and fitted using mathematical modelling. First, a phenomenological model was developed to model how infected individuals shed SARS-CoV-2 into WW and how the markers may degrade in time and compare this to the data. Second, we develop a model to estimate SARS-CoV-2 prevalence directly from WW data which is trained on the CIS data. Results: Data from 44 sewage sites in England, shows that SARS-CoV-2 prevalence is estimated to within 1.1% of estimates from representative prevalence surveys (with 95% confidence). Using machine learning and phenomenological models, differences between sampled sites, particularly the WW flow rate, influence prevalence estimation and require careful interpretation. SARS-CoV-2 signals in WW appear 4-5 days earlier in comparison to clinical testing data but are coincident with prevalence surveys suggesting that WW surveillance can be a leading indicator for asymptomatic viral infections. Conclusion: Wastewater-based epidemiology complements and strengthens traditional surveillance, with significant implications for public health. Using WW to quantify infection prevalence requires knowledge of additional meta-data and outbreak detection needs to account for unexplained aberrations in WW data to improve reliability

Background CAR-T cells immunotherapy is a breakthrough in the treatment of hematological malignancies such as acute lymphoblastic leukemia (ALL) and B-cell malignancies. However, CAR-T therapies face major hurdles such as the lack of tumor-specific antigen (TSA), and immunosuppressive tumor microenvironment sometimes caused by the tumorous expression of immune checkpoints (ICPs) such as HLA-G. Indeed, HLA-G is remarkable because it is both a potent ICP and a TSA. HLA-G tumor expression causes immune escape by impairing innate and adaptive immune responses and by inducing a suppressive microenvironment. Yet, to date, no immunotherapy targets it.Methods We have developed two anti-HLA-G third-generation CARs based on new anti-HLA-G monoclonal antibodies.Results Anti-HLA-G CAR-T cells were specific for immunosuppressive HLA-G isoforms. HLA-G-activated CAR-T cells polarized toward T helper 1, and became cytotoxic against HLA-G+ tumor cells. In vivo, anti-HLA-G CAR-T cells were able to control and eliminate HLA-G+ tumor cells. The interaction of tumor-HLA-G with interleukin (IL)T2-expressing T cells is known to result in effector T cell functional inhibition, but anti-HLA-G CAR-T cells were insensitive to this inhibition and still exerted their function even when expressing ILT2. Lastly, we show that anti-HLA-G CAR-T cells differentiated into long-term memory effector cells, and seemed not to lose function even after repeated stimulation by HLA-G-expressing tumor cells.Conclusion We report for the first time that HLA-G, which is both a TSA and an ICP, constitutes a valid target for CAR-T cell therapy to specifically target and eliminate both tumor cells and HLA-G+ suppressive cells.

Mobile integrons are widespread genetic platforms that allow bacteria to modulate the expression of antibiotic resistance cassettes by shuffling their position from a common promoter. Antibiotic stress induces the expression of an integrase that excises and integrates cassettes, and this unique recombination and expression system is thought to allow bacteria to ‘evolve on demand’ in response to antibiotic pressure. To test this hypothesis, we inserted a custom three-cassette integron into Pseudomonas aeruginosa and used experimental evolution to measure the impact of integrase activity on adaptation to gentamicin. Crucially, integrase activity accelerated evolution by increasing the expression of a gentamicin resistance cassette through duplications and by eliminating redundant cassettes. Importantly, we found no evidence of deleterious off-target effects of integrase activity. In summary, integrons accelerate resistance evolution by rapidly generating combinatorial variation in cassette composition while maintaining genomic integrity.

Fault zones exhibit 3D variable thickness, a feature that remains inadequately explored, particularly with regard to the impact on fluid flow. Upon analyzing an analytic solution, we examine 3D thermal‐hydraulic (TH) dynamical models through a benchmark experiment, which incorporates a fault zone with thickness variations corresponding to realistic orders of magnitude. The findings emphasize an area of interest where vigorous convection drives fluid flow, resulting in a temperature increase to 150°C at a shallow depth of 2.7 km in the thickest sections of the fault zone. Moreover, by considering various tectonic regimes (compressional, extensional, and strike‐slip) within 3D thermal‐hydraulic‐mechanical (THM) models and comparing them to the benchmark experiment, we observe variations in fluid pressure induced by poroelastic forces acting on fluid flow within the area of interest. These tectonic‐induced pressure changes influence the thermal distribution of the region and the intensity of temperature anomalies. Outcomes of this study emphasize the impact of poroelasticity‐driven forces on transfer processes and highlight the importance of addressing fault geometry as a crucial parameter in future investigations of fluid flow in fractured systems. Such research has relevant applications in geothermal energy, CO2 storage, and mineral deposits. Plain Language Summary: Exploring critical parameters affecting the fluid flow within fault zones in the Earth's crust is of fundamental scientific and economic interest. Among them, fault zone thickness and tectonic regimes are two parameters whose role remains unexplored. The results of this study show that fluid flow by convection is vigorous in the zone where fault thickness is largest. In this area of interest, the tectonic regimes impact convective dynamics and modify the thermal distribution of the system. These generic outcomes are discussed in relation to real‐cases scenarios and emphasize the fundamental role of considering a Geometric parameter (G), the fault zone thickness variation, as well as tectonic regimes, during the exploratory phase for geothermal energy, CO2 storage, and mineral exploration. Key Points: Based on a critical Rayleigh number analysis and 3D numerical modeling, we explore the effects of fault zone thickness variations on fluid flowIn line with the critical Rayleigh number analysis, our numerical results reveal that thermal convection is more efficient in areas where the fault zone thickness is largestThe poroelasticity‐driven force alters the convective dynamics within the zone of interest in the fault zone (i.e., the thickest part) [ABSTRACT FROM AUTHOR]

The lethal heat treatment dose (time and temperature) for the prepupal life stage of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), emerald ash borer (EAB), was determined through an in vitro application using a carefully calibrated heat treatment apparatus.The lethal and sublethal effects of heat on A. planipennis prepupae were assessed through a ramped heat delivery application, simulating industrial kilns and conventional heat chamber operations, for treatments combining target temperatures of 54 °C, 55 °C, and 56 °C, and exposure durations of 0 min (i.e., kiln temperature ramp only), 15 min, or 30 min. Prepupal EAB larvae did not survive exposure to 56 °C for 15 min or longer, or to 55 °C for 30 min. Sublethal effects were observed for all other treatments. Sublethal effects included delayed development and failure to complete the pupal and adult life stages. [ABSTRACT FROM AUTHOR]

Intracellular pathogens include all viruses, many bacteria and parasites capable of invading and surviving within host cells. Key to survival is the subversion of host cell pathways by the pathogen for the purpose of propagation and evading the immune system. The intracellular bacterium Shigella flexneri, the causative agent of bacillary dysentery, invades host cells in a vacuole that is subsequently ruptured to allow growth of the pathogen within the host cytoplasm. S. flexneri invasion has been classically described as a macropinocytosis-like process, however the underlying details and the role of macropinosomes in the intracellular bacterial lifestyle have remained elusive. We applied dynamic imaging and advanced large volume correlative light electron microscopy (CLEM) to study the highly transient events of S. flexneri's early invasion into host epithelial cells and elucidate some of its fundamental features. First, we demonstrate a clear distinction between two compartments formed during the first step of invasion: the bacterial containing vacuole and surrounding macropinosomes, often considered identical. Next, we report a functional link between macropinosomes and the process of vacuolar rupture, demonstrating that rupture timing is dependent on the availability of macropinosomes as well as the activity of the small GTPase Rab11 recruited directly to macropinosomes. We go on to reveal that the bacterial containing vacuole and macropinosomes come into direct contact at the onset of vacuolar rupture. Finally, we demonstrate that S. flexneri does not subvert pre-existing host endocytic vesicles during the invasion steps leading to vacuolar rupture, and propose that macropinosomes are the major compartment involved in these events. These results provide the basis for a new model of the early steps of S. flexneri epithelial cell invasion, establishing a different view of the enigmatic process of cytoplasmic access by invasive bacterial pathogens.