Nervous necrosis virus (NNV), an aquatic RNA virus belonging to Betanodavirus, infects a variety of marine and freshwater fishes, leading to massive mortality of cultured larvae and juveniles and substantial economic losses. The enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is widely recognized as a central component in the innate immune response to cytosolic DNA derived from different pathogens. However, little is known about the response of cGAS to aquatic RNA viruses. This study found that Epinephelus coioides cGAS (EccGAS) overexpression inhibited NNV replication, whereas EccGAS silencing promoted NNV replication. The anti-NNV activity of EccGAS was involved in interferon (IFN) signaling activation including tumor necrosis factor receptor-associated factor family member-associated NF-kappa-B activator-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor 3 (IRF3) nuclear translocation, and the subsequent induction of IFNc and ISGs. Interestingly, NNV employed its capsid protein (CP) or Protein A (ProA) to negatively or positively modulate EccGAS-mediated IFN signaling by simultaneously targeting EccGAS. CP interacted with EccGAS via the arm-P, S-P, and SD structural domains and promoted its polyubiquitination with K48 and K63 linkages in an EcUBE3C (the ubiquitin ligase)-dependent manner, ultimately leading to EccGAS degradation. Conversely, ProA bound to EccGAS and inhibited its ubiquitination and degradation. In regulating EccGAS protein content, CP\'s inhibitory action was more pronounced than ProA\'s protective effect, allowing successful NNV replication. These novel findings suggest that NNV CP and ProA dynamically modulate the EccGAS-mediated IFN signaling pathway to facilitate the immune escape of NNV. Our findings shed light on a novel mechanism of virus-host interaction and provide a theoretical basis for the prevention and control of NNV.IMPORTANCEAs a well-known DNA sensor, cGAS is a pivotal component in innate anti-viral immunity to anti-DNA viruses. Although there is growing evidence regarding the function of cGAS in the resistance to RNA viruses, the mechanisms by which cGAS participates in RNA virus-induced immune responses in fish and how aquatic viruses evade cGAS-mediated immune surveillance remain elusive. Here, we investigated the detailed mechanism by which EccGAS positively regulates the anti-NNV response. Furthermore, NNV CP and ProA interacted with EccGAS, regulating its protein levels through ubiquitin-proteasome pathways, to dynamically modulate the EccGAS-mediated IFN signaling pathway and facilitate viral evasion. Notably, NNV CP was identified to promote the ubiquitination of EccGAS via ubiquitin ligase EcUBE3C. These findings unveil a novel strategy for aquatic RNA viruses to evade cGAS-mediated innate immunity, enhancing our understanding of virus-host interactions.

Protein A (ProA) high-performance liquid chromatography (HPLC) is a common analytical procedure for measuring monoclonal antibody (mAb) titers due to its high specificity and efficiency. Accurate and reliable results of this procedure are imperative, as the quantitation of the total mAb present for in-process samples directly impacts downstream purification steps related to the removal of process-related impurities. This study aimed to improve a platform ProA HPLC analytical procedure which was previously developed using traditional approaches and was not always reliable. By retrospectively applying Analytical Quality by Design (AQbD) principles and statistical assessments of performance, a bias in the calibration standard due to protein-adsorption to common sample vial materials was identified. The inclusion of Tween® 20 into the mobile phase used as sample diluent was optimized to ensure procedure performance and improve analytical range. The resulting procedure robustness was evaluated using Design of Experiment (DoE) approaches and performance was verified against Analytical Target Profile (ATP) criteria as recommended by regulatory agencies. The resulting linearity displayed R2 values of 1.00 with intercept biases of 1.2 % (analyst 1) and 0.8 % (analyst 2), accuracy across all levels was reported at 99.2 % recovery, and intermediate precision was reported as 3.0 % RSD. Application of this new platform procedure has since reduced development timelines for new mAb products by 50 % and allowed for accurate titer determination to support >5 early phase product-specific process decisions without requiring extensive analytical procedure development. This work demonstrates the utility and relative ease of adopting AQbD concepts, even for established procedures, and supporting them with a lifecycle approach to managing procedure performance.

To analyze the role played by the clinical pharmacist and its impact in antibiotic stewardship facing suspected allergy to beta-lactam antibiotics.
We performed 2 different independent bibliographic searches. A total of 35 articles were found, and the final number included in the study was 12. We analyzed the articles and collected variables of efficacy, safety, and applicability of evaluation tools applied to patients with suspected allergy to beta-lactams. Also, the variation in the consumption and prescription profile of alternative antibiotics was analyzed.
The selected studies analyzed questionnaires, allergy delabeling, intradermal tests, and oral challenge tests performed by pharmacists. Significant differences in the efficacy endpoint were found in 4 studies in favor of pharmaceutical intervention. In the study of Kwiatkowski et al., cefazolin use increased in surgical patients after pharmacist intervention (65% vs 28%; P < .01). In a quasi-experimental study, the mean defined daily dose of aztreonam and the mean days of therapy per 1000 patients/day decreased (21.23 vs 9.05, P <.01) and (8.79-4.24, P = .016), pre- and post-intervention, respectively, increasing antibiotic de-escalations (P = < .01). In another quasi-experimental study, the prescription of restricted use antibiotics decreased (42.5% vs 17.9%, P < .01)and the use of pre-surgical prophylactic antibiotics alternative to cefazolin (81.9% vs 55.9%, P < .01)in another study. Other study showed that the mean time per interview was 5.2 min per patient. No adverse events were reported in any study.
The pharmacist intervention in the evaluation of the patient with suspected allergy to beta-lactams is effective, safe, and feasible to implement on daily clinical practice. The standardization of protocols to clarify the history of allergies and development of evaluation tools represent simple screenings to perform delabeling or refer to the Immunoallergology service, improving penicilins use and reducing the need for second-line antibiotics. More studies are needed to standardize the desensitization tests made by pharmacists. However, despite these results, the involvement and leadership of the pharmacist in this area is limited and constitutes a future challenge for the profession.

To analyze the role played by the clinical pharmacist and its impact in antibiotic stewardship facing suspected allergy to beta-lactam antibiotics.
We performed two different independent bibliographic searches. A total of 35 articles were found, and the final number included in the study was 12. We analysed the articles and collected variables of efficacy, safety and applicability of evaluation tools applied to patients with suspected allergy to beta-lactams. Also, the variation in the consumption and prescription profile of alternative antibiotics was analyzed.
The selected studies analysed questionnaires, allergy delabeling, intradermal tests and oral challenge tests performed by pharmacists. Significant differences in the efficacy endpoint were found in 4 studies in favour of pharmaceutical intervention. In the study of Kwiatkowski et al, cefazolin use increased in surgical patients after pharmacist intervention (65 vs. 28%; p < 0.01). In a quasi-experimental study, the mean defined daily dose of aztreonam and the mean days of therapy per 1000 patients/day decreased (21.23 vs 9.05, p <0.01) and (8.79-4.24, p = 0.016), pre and post-intervention, respectively, increasing antibiotic de-escalations (p ≤ 0.01). In another quasi-experimental study, the prescription of restricted-use antibiotics decreased (42.5% vs. 17.9%, p < 0.01) and the use of pre-surgical prophylactic antibiotics alternative to cefazolin (81.9% vs 55.9%, p<0.01) in another study. Other study showed that the mean time per interview was 5.2 minutes per patient. No adverse events were reported in any study.
The pharmacist intervention in the evaluation of the patient with suspected allergy to beta-lactams is effective, safe and feasible to implement on daily clinical practice. The standardization of protocols to clarify the history of allergies and development of evaluation tools represent simple screenings to perform delabelling or refer to the Immunoallergology service, improving penicilins use and reducing the need for second line antibiotics. More studies are needed to standardize the desensitization tests made by pharmacists. However, despite these results, the involvement and leadership of the pharmacist in this area is limited and constitutes a future challenge for the profession.

Free-living nematodes harbor and disseminate various soil-borne bacterial pathogens. Whether they function as vectors or environmental reservoirs for the aquatic L. pneumophila, the causative agent of Legionnaires\' disease, is unknown. A survey screening of biofilms of natural (swimming lakes) and technical (cooling towers) water habitats in Germany revealed that nematodes can act as potential reservoirs, vectors or grazers of L. pneumophila in cooling towers. Consequently, the nematode species Plectus similis and L. pneumophila were isolated from the same cooling tower biofilm and taken into a monoxenic culture. Using pharyngeal pumping assays, potential feeding relationships between P. similis and different L. pneumophila strains and mutants were examined and compared with Plectus sp., a species isolated from a L. pneumophila-positive thermal source biofilm. The assays showed that bacterial suspensions and supernatants of the L. pneumophila cooling tower isolate KV02 decreased pumping rate and feeding activity in nematodes. However, assays investigating the hypothesized negative impact of Legionella\'s major secretory protein ProA on pumping rate revealed opposite effects on nematodes, which points to a species-specific response to ProA. To extend the food chain by a further trophic level, Acanthamoebae castellanii infected with L. pneumphila KV02 were offered to nematodes. The pumping rates of P. similis increased when fed with L. pneumophila-infected A. castellanii, while Plectus sp. pumping rates were similar when fed either infected or non-infected A. castellanii. This study revealed that cooling towers are the main water bodies where L. pneumophila and free-living nematodes coexist and is the first step in elucidating the trophic links between coexisting taxa from that habitat. Investigating the Legionella-nematode-amoebae interactions underlined the importance of amoebae as reservoirs and transmission vehicles of the pathogen for nematode predators.

In 2012, The Spanish Societies of Infectious Diseases and Clinical Microbiology (SEIMC), Hospital Pharmacy (SEFH), and Preventive Medicine, Public Health and Healthcare Management (SEMPSGS) lead a consensus document including recommendations for the implementation of antimicrobial stewardship (AMS) programs (AMSP; PROA in Spanish) in acute care hospitals in Spain. While these recommendations were critical for the development of these programs in many centres, there is a need for guidance in the development of AMS activities for specific patient populations, syndromes or other specific aspects which were not included in the previous document or have developed significantly since then. The objective of this expert recommendation guidance document is to review the available information about these activities in these patient populations or circumstances, and to provide guidance recommendations about them. With this objective the SEIMC, SEFH, SEMPSPGS, the Spanish Society of Intensive Care Medicine (SEMICYUC) and the Spanish Pediatric Infectious Disease Society (SEIP) selected a panel of experts who chose the different aspects to include in the document. Because of the lack of high-level evidence in the implementation of the activities, the panel opted to perform a narrative review of the literature for the different topics for which recommendations were agreed by consensus. The document was open to public consultation for the members of these societies for their comments and suggestions, which were reviewed and considered by the panel.

BACKGROUND: One of the most important strategies of PROA in the Emergency Department (ED) is the accurate diagnosis of infection to avoid inappropriate prescription. Our objective is to evaluate patients who receive antibiotics despite not having objective data of infection.
METHODS: We carried out a cross-sectional study of patients admitted to the ED of the Hospital Universitario Fundación Alcorcón in which it was recommended to suspend the antibiotic through the PROA. Clinical and epidemiological characteristics and 30-day follow-up were analyzed to assess readmissions and mortality.
RESULTS: 145 patients were analyzed. It was recommended to suspend the antibiotic in 25. 44% of them had a diagnosis of urinary infection. The suspension recommendation was accepted by 88%. No patient died and one was readmitted.
CONCLUSIONS: An important percentage of patients are prescribed antibiotics despite not having infection criteria, the clinical evolution after suspension of antibiotics was favorable.

Ralstonia solanacearum RSc2741 has been predicted as a gamma-glutamyl phosphate reductase ProA catalyzing the second reaction of proline formation from glutamate. Here, we experimentally demonstrated that proA mutants were proline auxotrophs that failed to grow in a minimal medium, and supplementary proline, but not glutamate, fully restored the diminished growth, confirming that ProA is responsible for the biosynthesis of proline from glutamate in R. solanacearum. ProA was previously identified as one of the candidates regulating the expression of genes for type three secretion system (T3SS), one of the essential pathogenicity determinants of R. solanacearum. Supplementary proline significantly enhanced the T3SS expression both in vitro and in planta, indicating that proline is a novel inducer of the T3SS expression. Deletion of proA substantially impaired the T3SS expression both in vitro and in planta even under proline-supplemented conditions, indicating that ProA plays additional roles apart from proline biosynthesis in promoting the expression of the T3SS genes. It was further revealed that the involvement of ProA in the T3SS expression was mediated through the pathway of PrhG-HrpB. Both the proA mutants and the wild-type strain grew in the intercellular spaces of tobacco leaves, while their ability to invade and colonize tobacco xylem vessels was substantially impaired, which was about a 1-day delay for proA mutants to successfully invade xylem vessels and was about one order of magnitude less than the wild-type strain to proliferate to the maximum densities in xylem vessels. It thus resulted in substantially impaired virulence of proA mutants toward host tobacco plants. The impaired abilities of proA mutants to invade and colonize xylem vessels were not due to possible proline insufficiency in the rhizosphere soil or inside the plants. All taken together, these results extend novel insights into the understanding of the biological function of ProA and sophisticated regulation of the T3SS and pathogenicity in R. solanacearum.

The environmental bacterium Legionella pneumophila is an intracellular pathogen of various protozoan hosts and able to cause Legionnaires\' disease, a severe pneumonia in humans. By encoding a wide selection of virulence factors, the infectious agent possesses several strategies to manipulate its host cells and evade immune detection. In the present study, we demonstrate that the L. pneumophila zinc metalloprotease ProA functions as a modulator of flagellin-mediated TLR5 stimulation and subsequent activation of the pro-inflammatory NF-κB pathway. We found ProA to be capable of directly degrading immunogenic FlaA monomers but not the polymeric form of bacterial flagella. These results indicate a role of the protease in antagonizing immune stimulation, which was further substantiated in HEK-BlueTM hTLR5 Detection assays. Addition of purified proteins, bacterial suspensions of L. pneumophila mutant strains as well as supernatants of human lung tissue explant infection to this reporter cell line demonstrated that ProA specifically decreases the TLR5 response via FlaA degradation. Conclusively, the zinc metalloprotease ProA serves as a powerful regulator of exogenous flagellin and presumably creates an important advantage for L. pneumophila proliferation in mammalian hosts by promoting immune evasion.

The evolution of novel enzymes has fueled the diversification of life on earth for billions of years. Insights into events that set the stage for the evolution of a new enzyme can be obtained from ancestral reconstruction and laboratory evolution. Ancestral reconstruction can reveal the emergence of a promiscuous activity in a pre-existing protein and the impact of subsequent mutations that enhance a new activity. Laboratory evolution provides a more holistic view by revealing mutations elsewhere in the genome that indirectly enhance the level of a newly important enzymatic activity. This review will highlight recent studies that probe the early stages of the evolution of a new enzyme from these complementary points of view.