Inhaled SARS-CoV-2 vaccine for single-dose dry powder aerosol immunization.

The COVID-19 pandemic has fostered major advances in vaccination technologies1-4; however, there are urgent needs for vaccines that induce mucosal immune responses and for single-dose, non-invasive administration4-6. Here we develop an inhalable, single-dose, dry powder aerosol SARS-CoV-2 vaccine that induces potent systemic and mucosal immune responses. The vaccine encapsulates assembled nanoparticles comprising proteinaceous cholera toxin B subunits displaying the SARS-CoV-2 RBD antigen within microcapsules of optimal aerodynamic size, and this unique nano-micro coupled structure supports efficient alveoli delivery, sustained antigen release and antigen-presenting cell uptake, which are favourable features for the induction of immune responses. Moreover, this vaccine induces strong production of IgG and IgA, as well as a local T cell response, collectively conferring effective protection against SARS-CoV-2 in mice, hamsters and nonhuman primates. Finally, we also demonstrate a mosaic iteration of the vaccine that co-displays ancestral and Omicron antigens, extending the breadth of antibody response against co-circulating strains and transmission of the Omicron variant. These findings support the use of this inhaled vaccine as a promising multivalent platform for fighting COVID-19 and other respiratory infectious diseases.

Effects of 90 dB pure tone exposure on auditory and cardio-cerebral system functions in macaque monkeys.

Excessive noise exposure presents significant health risks to humans, affecting not just the auditory system but also the cardiovascular and central nervous systems. This study focused on three male macaque monkeys as subjects. 90 dB sound pressure level (SPL) pure tone exposure (frequency: 500Hz, repetition rate: 40Hz, 1 min per day, continuously exposed for 5 days) was administered. Assessments were performed before exposure, during exposure, immediately after exposure, and at 7-, 14-, and 28-days post-exposure, employing auditory brainstem response (ABR) tests, electrocardiograms (ECG), and electroencephalograms (EEG). The study found that the average threshold for the Ⅴ wave in the right ear increased by around 30 dB SPL right after exposure (P < 0.01) compared to pre-exposure. This elevation returned to normal within 7 days. The ECG results indicated that one of the macaque monkeys exhibited an RS-type QRS wave, and inverted T waves from immediately after exposure to 14 days, which normalized at 28 days. The other two monkeys showed no significant changes in their ECG parameters. Changes in EEG parameters demonstrated that main brain regions exhibited significant activation at 40Hz during noise exposure. After noise exposure, the power spectral density (PSD) in main brain regions, particularly those represented by the temporal lobe, exhibited a decreasing trend across all frequency bands, with no clear recovery over time. In summary, exposure to 90 dB SPL noise results in impaired auditory systems, aberrant brain functionality, and abnormal electrocardiographic indicators, albeit with individual variations. It has implications for establishing noise protection standards, although the precise mechanisms require further exploration by integrating pathological and behavioral indicators.

Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines.

The technology for obtaining the high-efficiency expression of target proteins through site-specific recombination has made progress. However, using the CRISPR/Cas9 system for site-specific integration of long fragments and the expression of active proteins remains a challenge. This study optimized the linear DNA circularization system, eliminated the prokaryotic plasmid backbone on the traditional foreign gene vector, and generated a homologous arm-free circular donor template with a single guide RNA target site (sgRNA TS). This strategy significantly increased the co-transfection efficiency of the 1.6 kb template and Cas9 plasmid by 1.15-fold, and the average knock-in (KI) efficiency of the 4.7 kb long-fragment template for the two target gene sites increased by 1.3-fold. Subsequently, we used rhBCHE as a reporter gene to efficiently integrate the 5.4 kb fragment containing the gene of interest (GOI) into specific sites in the HEK293T cell line to detect the expression of the circular template at different target sites. Overall, this study further verifies that the length of the circular donor is more conducive to non-homologous integration, and more importantly, we provide a simple and optimized strategy for the construction of long-fragment site integration cell lines.

Profiling gene expression reveals insights into pulmonary response to aerosolized botulinum toxin type A exposure in mice.

Botulinum neurotoxin type A (BoNT/A) is traditional medicine and well known for its therapeutic use as an anesthetic and in cosmetic applications that work through the inhibition of acetylcholine exocytosis in neuronal cells. BoNT/A also has the potential to function as a biological weapon due to its high mortality rate and ease of dispersal. Emerging evidence suggests that BoNT/A exhibits biological effects on nonneuronal cells. In cytology experiments, BoNT/A induces global gene expression alterations. However, pulmonary effects from exposure to aerosolized BoNT/A have not been evaluated. This study investigated the global transcriptional profile of lung tissues after botulism inhalation. A mice model of inhaled botulism was established using intratracheal exposure to aerosolized BoNT/A and described through histological examination and flow cytometry. Transcriptomic analysis revealed that genes related to acute inflammatory responses were upregulated at 12-h postexposure. Increased expression of multiple anti-inflammatory marker genes and decreased expression of pro-inflammatory marker genes were observed at 48- to 72-h postexposure, underscoring a transcriptional shift toward a pro-reparative phenotype. Histological examination and cell proportions analysis mirrored these expression patterns. Accordingly, the orchestration of a quick phenotype transition prompted by BoNT/A may have the potential for promoting the resolution of the inflammatory lung. To our knowledge, this study represents the first research to investigate the pulmonary transcriptional responses of aerosolized BoNT/A exposure; the results may provide new insights in elucidating the molecular mechanism for pulmonary inhaled botulism and highlight the potential therapeutic application of BoNT/A in mitigating inflammatory conditions.

Pretreatment with Retro-2 protects cells from death caused by ricin toxin by retaining the capacity of protein synthesis.

The current study explores the detoxification effect of Retro-2 on ricin toxin (RT) cytotoxicity, as well as the mechanisms underlying such effects, to provide a basis for follow-up clinical applications of Retro-2. The mouse-derived mononuclear/macrophage cell line, RAW264.7, was used to evaluate the detoxification effect of Retro-2 on RT by detecting cell viability, capacity for protein synthesis and the expression of cytokines, as well as endoplasmic reticulum stress (ERS)-related mRNA. The results indicated that many cells died when challenged with concentrations of RT ≥50ng/mL. The protein synthesis capacity of cells decreased when challenged with 200ng/mL RT for 2hours. Furthermore, the synthesis and release of many cytokines decreased, while the expression of cytokines or ERS-related mRNA increased when challenged with 200ng/mL of RT for 12 or more hours. However, cell viability, capacity for protein synthesis and release levels of many cytokines were higher, while the expression levels of cytokine, or ERS-related mRNA, were lower in cells pretreated with 20µm Retro-2 and challenged with RT, compared with those that had not been pretreated with Retro-2. In conclusion, Retro-2 retained the capacity for protein synthesis inhibited by RT, alleviated ERS induced by RT and increased the viability of cells challenged with RT. Retro-2 shows the potential for clinical applications.

Coexistence of a novel KPC-2-encoding MDR plasmid and an NDM-1-encoding pNDM-HN380-like plasmid in a clinical isolate of Citrobacter freundii.

OBJECTIVES: The objective of this study was to characterize the molecular mechanism of coproduction of KPC-2 and NDM-1 in Citrobacter freundii. METHODS: C. freundii strain 112298 was isolated from a human case of septic shock in a Chinese teaching hospital. The major carbapenemase and ESBL genes were detected by PCR. The MIC values were determined by using VITEK 2 and antimicrobial susceptibility was judged by CLSI standards. The resistance plasmid was transferred into Escherichia coli by electroporation, followed by plasmid DNA isolation from the electroporant, and then fully sequenced and compared with closely related plasmids. RESULTS: Strain 112298 produces KPC-2 and NDM-1, encoded by the novel non-typeable plasmid p112298-KPC and an IncX3-type plasmid p112298-NDM, respectively. In p112298-KPC, a Tn1722-based blaKPC-2-carrying transposon is associated with several additional resistance modules, constituting a single MDR region. Assembly of these resistance modules is likely mediated by homologous recombination between five copies of IS26 elements at different sites within the MDR region. p112298-NDM is a very close relation of pNDM-HN380. blaNDM-1 in p112298-NDM is carried by a Tn125 variant, which differs from the prototype Tn125 as observed in pNDM-BJ01 by disruption of an upstream copy of ISAba125 by IS5 and absence of a downstream copy of ISAba125. CONCLUSIONS: Production of KPC-2 and NDM-1 by p112298-KPC and p112298-NDM, respectively, makes C. freundii 112298 highly resistant to carbapenems and, moreover, these two plasmids still harbour genes for resistance to cephalosporins, chloramphenicol, chromate, fosfomycin, quaternary ammonium, rifampicin and sulphonamides.

Characterization of the gut butyrate-producing bacteria and lipid metabolism in African green monkey as a natural host of simian immunodeficiency virus infection.

OBJECTIVE: Natural hosts of simian immunodeficiency virus (SIV), such as the African green monkey (AGM), possess the ability to avoid acquired immune deficiency syndrome (AIDS) despite lifelong infection. The underlying mechanisms are not completely understood. This study aimed to characterize the gut microbiome and metabolite profiles of different nonhuman primates (NHPs) to provide potential insight into AIDS resistance. DESIGN AND METHODS: Fresh feces from Cynomolgus macaques (CMs), and Rhesus macaques (RMs), SIV- AGMs (AGM_N), and SIV+ AGMs (AGM_P) were collected and used for metagenomic sequencing and metabonomic analysis. RESULTS: Compared with CMs and RMs, significant decreases in the abundances of Streptococcus , Alistipes , Treponema , Bacteroides , and Methanobrevibacter ( P  < 0.01), and significant increases in the abundances of Clostridium , Eubacterium , Blautia , Roseburia , Faecalibacterium , and Dialister ( P  < 0.01) were detected in AGM_N. Compared with AGM_N, a trend toward increased abundances of Streptococcus and Roseburia were found in AGM_P. The levels of metabolites involved in lipid metabolism and butanoate metabolism significantly differed among AGM_P, AGM_N and CM ( P  < 0.05). CONCLUSIONS: Our data, for the first time, demonstrated distinguishing features in the abundances of butyrate-producing bacteria and lipid metabolism capacities between different NHP hosts of SIV infection. These findings may correlate with the different characteristics observed among these hosts in the maintenance of intestinal epithelial barrier integrity, regulation of inflammation, and provide insights into AIDS resistance in AGMs.

Engineered probiotic ameliorates ulcerative colitis by restoring gut microbiota and redox homeostasis.

Probiotics are potential treatments for ulcerative colitis (UC), but their efficacy is frequently compromised by gastrointestinal conditions that limit adhesion and activity. Here, we use machine learning and bioinformatics to confirm that patients with UC have decreased prevalence of Lactobacillus genus and increased oxidative stress, which correlate with inflammation severity. Accordingly, we developed a probiotic-based therapeutic that synergistically restores intestinal redox and microbiota homeostasis. Lactobacillus casei (Lac) were induced to form a pericellular film, providing a polysaccharide network for spatially confined crystallization of ultrasmall but highly active selenium dots (Se-Lac). Upon oral administration, the selenium dot-embedded pericellular film efficiently enhanced gastric acid resistance and intestinal mucoadhesion of Lac cells. At the lesion site, the selenium dots scavenged reactive oxygen species, while Lac modulated the gut microbiota. In multiple mouse models and non-human primates, this therapeutic effectively relieved inflammation and reduced colonic damage, thus showing promise as a UC treatment.

Exosome-loaded degradable polymeric microcapsules for the treatment of vitreoretinal diseases.

The therapeutic benefits of many cell types involve paracrine mechanisms. Inspired by the paracrine functions of exosomes and the sustained degradation properties of microcapsules, here we report the therapeutic benefits of exosome-loaded degradable poly(lactic-co-glycolic acid) microcapsules with micrometric pores for the treatment of vitreoretinal diseases. On intravitreal injection in a mouse model of retinal ischaemia-reperfusion injury, microcapsules encapsulating mouse mesenchymal-stem-cell-derived exosomes settled in the inferior vitreous cavity, released exosomes for over one month as they underwent degradation and led to the restoration of retinal thickness to nearly that of the healthy retina. In mice and non-human primates with primed mycobacterial uveitis, intravitreally injected microcapsules loaded with exosomes from monkey regulatory T cells resulted in a substantial reduction in the levels of inflammatory cells. The exosome-encapsulating microcapsules, which can be lyophilised, may offer alternative treatment options for vitreoretinal diseases.

Genome sequences of three live attenuated vaccine strains of Brucella species and implications for pathogenesis and differential diagnosis.

Live attenuated vaccines play essential roles in the prevention of brucellosis. Here, we report the draft genome sequences of three vaccine strains, Brucella melitensis M5-10, B. suis S2-30, and B. abortus 104M. Primary genome sequence analysis identified mutations, deletions, and insertions which have implications for attenuation and signatures for differential diagnosis.

Complete genome sequence of Brucella canis BCB018, a strain isolated from a human patient.

Brucella canis is considered a rare cause of human brucellosis because of difficulties in presumptive diagnosis and underestimation of the incidence. Here, we report the draft genome sequence of a Brucella canis isolate, BCB018, isolated from a human patient, providing precious resources for comparative genomics analysis of Brucella field strains.

Sequence difference of angiotensin-converting enzyme 2 between nonhuman primates affects its binding-affinity with SARS-CoV-2 S receptor binding domain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused many deaths and contributed to a tremendous public health concern worldwide since 2020. Angiotensin-converting enzyme 2 (ACE2) binds to the SARS-CoV-2 virus as a receptor. The challenge of different nonhuman primate (NHP) species by SARS-CoV-2 virus demonstrated different effects on virus replication and disease pathology. This study characterizes differences between host ACE2 sequences of three NHP species: Macaca mulatta, Macaca fascicularis, and Chlorocebus sabaeus. In addition, the binding affinity between the ACE2 ectodomain and the SARS-CoV-2 S receptor-binding domain (RBD) was analyzed. Variation of ACE2 sequence among NHP species and the binding affinity may account for different susceptibility and responses to SARS-CoV-2 infection.

Docosahexaenoic Acid Ester of Phloridzin Reduces Inflammation and Insulin Resistance via AMPK.

BACKGROUND: Docosahexaenoic acid-acylated phloridzin (PZ-DHA), a novel polyphenol fatty acid ester derivative, is synthesized through an acylation reaction of phloridzin (PZ) and docosahexaenoic acid (DHA). PZ-DHA is more stable than DHA and exhibits higher cellular uptake and bioavailability than PZ. OBJECTIVE: The study aims to investigate the effects of PZ-DHA on insulin resistance in the skeletal muscle and the related mechanisms; we used palmitic acid (PA)-treated C2C12 myotubes as an insulin resistance model. RESULTS: We found that PZ-DHA increased the activity of AMP-activated protein kinase (AMPK) and improved glucose uptake and mitochondrial function in an AMPK-dependent manner in untreated C2C12 myotubes. PZ-DHA treatment of the myotubes reversed PA-induced insulin resistance; this was indicated by increases in glucose uptake and the expression of membrane glucose transporter 4 (Glut4) and phosphorylated Akt. Moreover, PZ-DHA treatment reversed PA-induced inflammation and oxidative stress. These effects of PZ-DHA were mediated by AMPK. Furthermore, the increase in AMPK activity, improvement in insulin resistance, and decrease in inflammatory and oxidative responses after PZ-DHA treatment diminished upon co-treatment with a liver kinase B1 (LKB1) inhibitor, suggesting that PZ-DHA improved AMPK activity by regulating its upstream kinase, LKB1. CONCLUSION: The effects of PZ-DHA on insulin resistance in C2C12 myotubes may be mediated by the LKB1- AMPK signaling pathway. Hence, PZ-DHA is a promising therapeutic agent for insulin resistance in type 2 diabetes.

Reduction of choroidal neovascularization via cleavable VEGF antibodies conjugated to exosomes derived from regulatory T cells.

Choroidal neovascularization induced by age-related macular degeneration and retinal neovascularization induced by diabetic retinopathy-two leading causes of blindness-are often treated using antibodies targeting vascular endothelial growth factor (VEGF). Here we report a strong association between inflammation and high VEGF expression in aqueous humour samples from patients with choroidal or retinal neovascularization, and show that intravitreally injected exosomes derived from regulatory T cells and conjugated with an anti-VEGF antibody via a peptide linker that is cleavable by matrix metalloproteinases markedly suppressed ocular neovascularization in mouse and non-human primate models of choroidal neovascularization. The engineered exosomes, which selectively accumulate in the neovascularization lesions, could be adapted for other combination therapies of therapeutic antibodies and anti-inflammatory cargo.

Acute High Level Noise Exposure Can Cause Physiological Dysfunction in Macaque Monkeys: Insight on the Medical Protection for Special Working Environmental Personnel.

The high level noise caused by intense acoustic weapons and blasting is a common source of acute acoustic trauma faced by some special environmental personnel. Studies have shown that high level noise can cause auditory and non-auditory effects. However, there are few reports on the biological effects, especially the non-auditory effects of acute high level noise exposure in simulated special working environments, and the great differences between experimental animals and human beings make it difficult to extrapolate from research conclusions. In this study, macaque monkeys were used to detect the effects of acute high level noise exposure on hearing, cognition, and cardiovascular function. Auditory brainstem response, auditory P300, and electrocardiogram (ECG) of macaque monkeys were measured. Results showed that acute high level noise exposure caused permanent hearing threshold shifts; partial hearing loss which couldn't recover to normal levels in the detection period; pathological changes in T wave and QRS complexes; and large fluctuations in cognitive ability after exposure, which finally recovered to normal. These alterations may be a combination of effects caused by stress-induced neuroendocrine dysfunction and mechanical damage of auditory organs. To elaborate the exact mechanism, further studies are still needed. Meanwhile, positive measures should be taken to reduce the incidence of acute high level noise injury.

Time-course transcriptome analysis of lungs from mice exposed to ricin by intratracheal inoculation.

In this study, a ricin toxin (RT)-induced pulmonary intoxication model was established in mice by intratracheal-delivered RT at a dose of 2× LD50. Based on this model, the histopathological evaluation of the lungs at 24 h and 48 h post-exposure was executed, and the genome-wide transcriptome of the lungs at 4, 12, 24 and 48 h post-exposure was analyzed. Histopathological analysis showed that a large number of neutrophils infiltrated the lungs at 24 h post-exposure, and slight pulmonary edema and perivascular-peribronchiolar edema appeared in the lungs at 48 h. Transcriptome analysis showed that the expression of a large number of genes related to leukocyte migration and chemotaxis consistently increased in the lungs upon exposure to RT, and the expression of genes that participate in acute phase immune and/or inflammatory response, also increased within 12 h of exposure to RT, which could be confirmed by the measurement of cytokines, such as IL-1ß, TNF-α and IL-6, in bronchoalveolar lavage fluid. While the expression of genes related to cellular components of the extracellular matrix and cell membrane integrity consistently decreased in the lungs, and the expression of genes related to antioxidant activity also decreased within the first 12 h. There are 17 differentially expressed genes (DEGs) that participate in ribotoxic stress response, endoplasmic reticulum stress response or immune response in the lungs at 4 h post-exposure. The expression of these DEGs was upregulated, and the number of these DEGs accounted for about 59 % of all DEGs at 4 h. The 17 DEGs may play an important role in the occurrence and development of inflammation. Notably, Atf3, Egr1, Gdf15 and Osm, which are poorly studied, may be important targets for the subsequent research of RT-induced pulmonary intoxication. This study provides new information and insights for RT-induced pulmonary intoxication, and it can provide a reference for the subsequent study of the toxicological mechanism and therapeutic approaches for RT-induced pulmonary intoxication.

Intratracheal inoculation of AHc vaccine induces protection against aerosolized botulinum neurotoxin A challenge in mice.

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is generally known to be the most poisonous of all biological toxins. In this study, we evaluate the protection conferred by intratracheal (i.t.) inoculation immunization with recombinant Hc subunit (AHc) vaccines against aerosolized BoNT/A intoxication. Three AHc vaccine formulations, i.e., conventional liquid, dry powder produced by spray freeze drying, and AHc dry powder reconstituted in water are prepared, and mice are immunized via i.t. inoculation or subcutaneous (s.c.) injection. Compared with s.c.-AHc-immunized mice, i.t.-AHc-immunized mice exhibit a slightly stronger protection against a challenge with 30,000× LD50 aerosolized BoNT/A. Of note, only i.t.-AHc induces a significantly higher level of toxin-neutralizing mucosal secretory IgA (SIgA) production in the bronchoalveolar lavage of mice. In conclusion, our study demonstrates that the immune protection conferred by the three formulations of AHc is comparable, while i.t. immunization of AHc is superior to s.c. immunization against aerosolized BoNT/A intoxication.

Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge.

OBJECTIVE: LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study. METHODS: A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography. RESULTS: Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 106 CFU of Y. pestis virulent strain 141. CONCLUSION: The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

Biosynthesis of Self-Assembled Proteinaceous Nanoparticles for Vaccination.

Recent years have seen enormous advances in nanovaccines for both prophylactic and therapeutic applications, but most of these technologies employ chemical or hybrid semi-biosynthetic production methods. Thus, production of nanovaccines has to date failed to exploit biology-only processes like complex sequential post-translational biochemical modifications and scalability, limiting the realization of the initial promise for offering major performance advantages and improved therapeutic outcomes over conventional vaccines. A Nano-B5 platform for in vivo production of fully protein-based, self-assembling, stable nanovaccines bearing diverse antigens including peptides and polysaccharides is presented here. Combined with the self-assembly capacities of pentamer domains from the bacterial AB5 toxin and unnatural trimer peptides, diverse nanovaccine structures can be produced in common Escherichia coli strains and in attenuated pathogenic strains. Notably, the chassis of these nanovaccines functions as an immunostimulant. After showing excellent lymph node targeting and immunoresponse elicitation and safety performance in both mouse and monkey models, the strong prophylactic effects of these nanovaccines against infection, as well as their efficient therapeutic effects against tumors are further demonstrated. Thus, the Nano-B5 platform can efficiently combine diverse modular components and antigen cargos to efficiently generate a potentially very large diversity of nanovaccine structures using many bacterial species.

Anaplasma platys-Like Infection in Goats, Beijing, China.

As one of the important tick-borne zoonotic pathogens, Anaplasma has both veterinary and public health significance. Here, we performed a survey of Anaplasma infection in the goats from a farm in Beijing, China, and found 44.6% (41/92) were infected with Anaplasma capra, and 22.8% (21/92) were infected with Anaplasma sp. This Anaplasma sp. bacterium was close to a recently emerging Anaplasma platys strain based on gltA and groEL gene phylogenetic analysis. As to further understand the characteristics of Anaplasma sp., we raised a couple of positive goats (n = 2) in the laboratory with tick-free settings. We observed inappetence, vomiting, high fever, and weakness of limbs in the goat's offspring (n = 3). In addition, the blood samples from all offspring were all positive of this Anaplasma spp. We did not see any intracellular morulae in neutrophils, monocytes, and erythrocytes, but we identified some in the platelets of the blood smears from the positive goats by light microscopy. We named it A. platys-like and suggested it may infect platelets and be transmitted vertically through the placenta of goats. These findings deserve further evaluation.