A catalase inhibitor: Targeting the NADPH-binding site for castration-resistant prostate cancer therapy.

Catalase (CAT) is an important antioxidant enzyme that breaks down H2O2 into water and oxygen. Inhibitor-modulating CAT activity in cancer cells is emerging as a potential anticancer strategy. However, the discovery of CAT inhibitors towards the heme active center located at the bottom of long and narrow channel has made little progress. Therefore, targeting new binding site is of great importance for the development of efficient CAT inhibitors. Here, the first NADPH-binding site inhibitor of CAT, BT-Br, was designed and synthesized successfully. The cocrystal structure of BT-Br-bound CAT complex was determined with a resolution of 2.2 Å (PDB ID:8HID), which showed clearly that BT-Br bound at the NADPH-binding site. Furthermore, BT-Br was demonstrated to induce ferroptosis in castration-resistant prostate cancer (CRPC) DU145 cells and eventually reduce CRPC tumors in vivo effectively. The work indicates that CAT has potential as a novel target for CRPC therapy based on ferroptosis inducing.

Discovery of Macrocycle-Based HPK1 Inhibitors for T-Cell-Based Immunotherapy.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell activation, and targeting HPK1 is considered a promising strategy for improving responses to antitumor immune therapies. The biggest challenge of HPK1 inhibitor design is to achieve a higher selectivity to GLK, an HPK1 homology protein as a positive regulator of T-cell activation. Herein, we report the design of a series of macrocycle-based HPK1 inhibitors via a conformational constraint strategy. The identified candidate compound 5i exhibited HPK1 inhibition with an IC50 value of 0.8 nM and 101.3-fold selectivity against GLK. Compound 5i also displayed good oral bioavailability (F = 27-49%) in mice and beagles and favorable metabolic stability (T1/2 > 186.4 min) in human liver microsomes. More importantly, compound 5i demonstrated a clear synergistic effect with anti-PD-1 in both MC38 (MSI) and CT26 (MSS) syngeneic tumor mouse models. These results showed that compound 5i has a great potential in immunotherapy.

Distribution- and Metabolism-Based Drug Discovery: A Potassium-Competitive Acid Blocker as a Proof of Concept.

Conventional methods of drug design require compromise in the form of side effects to achieve sufficient efficacy because targeting drugs to specific organs remains challenging. Thus, new strategies to design organ-specific drugs that induce little toxicity are needed. Based on characteristic tissue niche-mediated drug distribution (TNMDD) and patterns of drug metabolism into specific intermediates, we propose a strategy of distribution- and metabolism-based drug design (DMBDD); through a physicochemical property-driven distribution optimization cooperated with a well-designed metabolism pathway, SH-337, a candidate potassium-competitive acid blocker (P-CAB), was designed. SH-337 showed specific distribution in the stomach in the long term and was rapidly cleared from the systemic compartment. Therefore, SH-337 exerted a comparable pharmacological effect but a 3.3-fold higher no observed adverse effect level (NOAEL) compared with FDA-approved vonoprazan. This study contributes a proof-of-concept demonstration of DMBDD and provides a new perspective for the development of highly efficient, organ-specific drugs with low toxicity.

Discovery of 3-pyrazolyl-substituted pyrazolo[1,5-a]pyrimidine derivatives as potent TRK inhibitors to overcome clinically acquired resistance.

Several secondary tropomyosin receptor kinase (TRK) mutations located in the solvent front, xDFG, and gatekeeper regions, are a common cause of clinical resistance. Mutations in the xDFG motif in particular limit sensitivity to second-generation TRK inhibitors, which represent an unmet clinical need. We designed a series of 3-pyrazolyl-substituted pyrazolo[1,5-a]pyrimidine derivatives toward these secondary mutations using ring-opening and scaffold-hopping strategies. Compound 5n was the most potent, with IC50 values of 2.3 nM, 0.4 nM, and 0.5 nM against TRKAG667C, TRKAF589L, and TRKAG595R, compared to selitrectinib with IC50 values of 12.6 nM, 5.8 nM, and 7.6 nM, respectively (approximately 5.4, 14.5, and 15.2-fold increases). Furthermore, 5n displayed favorable pharmacokinetic properties and satisfactory antitumor efficacy (tumor growth inhibition of 97% at 30 mg/kg and 73% at 100 mg/kg) in TRKAWT and TRKAG667C xenograft mouse models. Collectively, 5n is a promising TRK inhibitor lead compound for overcoming clinically acquired resistance to second-generation inhibitors, particularly for resistant tumors harboring the TRKAG667C mutation in the xDFG motif.

Exploring the kinase-inhibitor fragment interaction space facilitates the discovery of kinase inhibitor overcoming resistance by mutations.

Protein kinases play crucial roles in many cellular signaling processes, making them become important targets for drug discovery. But drug resistance mediated by mutation puts a barrier to the therapeutic effect of kinase inhibitors. Fragment-based drug discovery has been successfully applied to overcome such resistance. However, the complicate kinase-inhibitor fragment interaction and fragment-to-lead process seriously limit the efficiency of kinase inhibitor discovery against resistance caused by mutation. Here, we constructed a comprehensive web platform KinaFrag for the fragment-based kinase inhibitor discovery to overcome resistance. The kinase-inhibitor fragment space was investigated from 7783 crystal kinase-inhibitor fragment complexes, and the structural requirements of kinase subpockets were analyzed. The core fragment-based virtual screening workflow towards specific subpockets was developed to generate new kinase inhibitors. A series of tropomyosin receptor kinase (TRK) inhibitors were designed, and the most potent compound YT9 exhibits up to 70-fold activity improvement than marketed drugs larotrectinib and selitrectinib against G595R, G667C and F589L mutations of TRKA. YT9 shows promising antiproliferative against tumor cells in vitro and effectively inhibits tumor growth in vivo for wild type TRK and TRK mutants. Our results illustrate the great potential of KinaFrag in the kinase inhibitor discovery to combat resistance mediated by mutation. KinaFrag is freely available at http://chemyang.ccnu.edu.cn/ccb/database/KinaFrag/.

Discovery of Next-Generation Tropomyosin Receptor Kinase Inhibitors for Combating Multiple Resistance Associated with Protein Mutation.

Tropomyosin receptor kinase (TRK) inhibition is an effective therapeutic approach for treatment of a variety of cancers. Despite the use of first-generation TRK inhibitor (TRKI) larotrectinib (1) resulting in significant therapeutic response in patients, acquired resistance develops invariably. The emergence of secondary mutations occurring at the solvent-front, xDFG, and gatekeeper regions of TRK represents a common mechanism for acquired resistance. However, xDFG mutations remain insensitive to second-generation macrocyclic TRKIs selitrectinib (3) and repotrectinib (4) designed to overcome the resistance mediated by solvent-front and gatekeeper mutations. Here, we report the structure-based drug design and discovery of a next-generation TRKI. The structure-activity relationship studies culminated in the identification of a promising drug candidate 8 that showed excellent in vitro potency on a panel of TRK mutants, especially TRKAG667C in the xDFG motif, and improved in vivo efficacy than 1 and 3 in TRK wild-type and mutant fusion-driven tumor xenograft models, respectively.

Insights into SARS-CoV-2: Medicinal Chemistry Approaches to Combat Its Structural and Functional Biology.

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still a pandemic around the world. Currently, specific antiviral drugs to control the epidemic remain deficient. Understanding the details of SARS-CoV-2 structural biology is extremely important for development of antiviral agents that will enable regulation of its life cycle. This review focuses on the structural biology and medicinal chemistry of various key proteins (Spike, ACE2, TMPRSS2, RdRp and Mpro) in the life cycle of SARS-CoV-2, as well as their inhibitors/drug candidates. Representative broad-spectrum antiviral drugs, especially those against the homologous virus SARS-CoV, are summarized with the expectation they will drive the development of effective, broad-spectrum inhibitors against coronaviruses. We are hopeful that this review will be a useful aid for discovery of novel, potent anti-SARS-CoV-2 drugs with excellent therapeutic results in the near future.

Grignard Reagent Utilization Enables a Practical and Scalable Construction of 3-Substituted 5-Chloro-1,6-naphthyridin-4-one Derivatives.

A robust, practical, and scalable approach for the construction of 3-substituted 5-chloro-1,6-naphthyridin-4-one derivatives 13 via the addition of Grignard reagents to 4-amino-2-chloronicotinonitrile (15) was developed. Starting with various Grignard reagents, a wide range of 3-substituted 5-chloro-1,6-naphthyridin-4-one derivatives 13 were conveniently synthesized in moderate-to-good yields through addition-acidolysis-cyclocondensation. In addition, the robustness and applicability of this synthetic route was proven on a 100 g scale, which would enable convenient sample preparation in the preclinical development of 1,6-naphthyridin-4-one-based MET-targeting antitumor drug candidates.

Structure-activity relationship study of novel quinazoline-based 1,6-naphthyridinones as MET inhibitors with potent antitumor efficacy.

As a privileged scaffold, the quinazoline ring is widely used in the development of EGFR inhibitors, while few quinazoline-based MET inhibitors are reported. In our ongoing efforts to develop new MET-targeted anticancer drug candidates, a series of quinazoline-based 1,6-naphthyridinone derivatives were designed, synthesized, and evaluated for their biological activities. The preliminary SARs studies indicate that the quinazoline scaffold was also acceptable for the block A of class II MET inhibitors. The further pharmacokinetic studies led to the identification of the most promising compound 22a with favorable in vitro potency (MET, IC50 = 9.0 nM), human microsomal metabolic stability (t1/2 = 621.2 min) and oral bioavailability (F = 42%). Moreover, 22a displayed good in vivo antitumor efficacy (IR of 81% in 75 mg/kg) in MET-positive human glioblastoma U-87 MG xenograft model. These positive results indicated that 22a is a potential new MET-targeted antitumor drug lead, which is worthy of further development.

Efficient Arylation of 2,7-Naphthyridin-1(2H)-one with Diaryliodonium Salts and Discovery of a New Selective MET/AXL Kinase Inhibitor.

New 8-chloro-2-phenyl-2,7-naphthyridin-1(2H)-one building blocks bearing diverse substitutes on the 2-phenyl group were synthesized via an efficient diaryliodonium salt-based N-arylation strategy with the advantage of mild conditions, short reaction times, and high yields. A small combinatorial library of 8-amino substituted 2-phenyl-2,7-naphthyridin-1(2H)-one was further conveniently constructed based on the above chlorinated naphthyridinones and substituted aniline. Preliminary biochemical screening resulted in the discovery of the new 2,7-naphthyridone-based MET/AXL kinase inhibitors. More importantly, 17c (IC50,MET of 13.8 nM) or 17e (IC50,AXl of 17.2 nM) and 17i (IC50,AXl of 31.8 nM) can efficient selectively inhibit MET or AXL kinase, respectively, while commercial cabozantinib showed no selectivity. The further exploration of the 8-substituted 2-phenyl-2,7-naphthyridin-1(2H)-one combinatorial library would significantly accelerate the discovery of more potent and selective inhibitors against diverse kinases.

Exosomes: Potential Therapies for Disease via Regulating TLRs.

Exosomes are small membrane vesicles that retain various substances such as proteins, nucleic acids, and small RNAs. Exosomes play crucial roles in many physiological and pathological processes, including innate immunity. Innate immunity is an important process that protects the organism through activating pattern recognition receptors (PRRs), which then can induce inflammatory factors to resist pathogen invasion. Toll-like receptor (TLR) is one member of PRRs and is important in pathogen clearance and nervous disease development. Although exosomes and TLRs are two independent materials, abundant evidences imply exosomes can regulate innate immunity through integrating with TLRs. Herein, we review the most recent data regarding exosome regulation of TLR pathways. Specifically, exosome-containing materials can regulate TLR pathways through the interaction with TLRs. This is a new strategy regulating immunity to resist pathogens and therapy diseases, which provide a potential method to cure diseases.

Synthesis and biological evaluation of new MET inhibitors with 1,6-naphthyridinone scaffold.

A potent and novel MET inhibitor, 5-((4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)amino)-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-ones (8), was designed and synthesized via a scaffold-hopping strategy of a 2,7-naphthyridinone MET kinase inhibitor 7. Lead compound 8 had good potency (IC50 of 9.8 nM), but unfavorable pharmacokinetic profiles (F = 12%, CL = 5.0 L/h/kg). Systematic structural optimization of compound 8 resulted in 9g (MET, IC50 = of 9.8 nM) with a comparable MET potency to that of compound 2 and a favorable pharmacokinetic profile (F = 63%, CL = 0.12 L/h/kg). Further study of the derivatization of N(1) amine group of 9g led to the discovery of 23a with good MET potency (IC50 of 7.1 nM), promising VEGFR-2 selectivity (3226-fold), and a markedly drug-likeness improvement (F = 57.7%, CL = 0.02 L/h/kg). The excellent VEGFR-2 selectivity and favorable drug-likeness of 23g suggest that the 1,6-naphthyridine moiety could be used as a new scaffold for kinase inhibitor discovery.

2,7-naphthyridinone-based MET kinase inhibitors: A promising novel scaffold for antitumor drug development.

As part of our effort to develop new molecular targeted antitumor drug, a novel 2,7-naphthyridone-based MET kinase inhibitor, 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)- 3-fluorophenyl)amino)-2-(4-fluorophenyl)-2,7-naphthyridin-1(2H)-one (13f), was identified. Knowledge of the binding mode of BMS-777607 in MET led to the design of new inhibitors that utilize novel 2,7-naphthyridone scaffold to conformationally restrain the key pharmacophoric groups (block C). Detailed SAR studies resulted in the discovery of a new MET inhibitor 13f, displaying favorable in vitro potency and oral bioavailability. More importantly, 13f exhibited excellent in vivo efficacy (tumor growth inhibition/TGI of 114% and 95% in 50 mg/kg, respectively) both in the U-87 MG and HT-29 xenograft models. The favorable drug-likeness of 13f indicated that 2,7-naphthyridinone may be used a promising novel scaffold for antitumor drug development. The preclinical studies of 13f are under way.

Various Profiles of tet Genes Addition to tet(X) in Riemerella anatipestifer Isolates From Ducks in China.

To investigate tetracycline resistance and resistant genotype in Riemerella anatipestifer, the tetracycline susceptibility of 212 R. anatipestifer isolates from China between 2011 and 2017 was tested. The results showed that 192 of 212 (90.6%) R. anatipestifer isolates exhibited resistance to tetracycline (the MICs ranged from 4 to 256 µg/ml). The results of PCR detection showed that, 170 of 212 (80.2%) R. anatipestifer isolates possessed the tet(X) gene. Other genes, including tet(A), tet(M), tet(Q), tet(O), tet(B), and tet(O/W/32/O), were found at frequencies of 20.8, 4.7, 1.4, 0.9, 0.9, and 0.5%, respectively. However, tet(C), tet(E), tet(G), tet(K), and tet(W) were not detected in any isolate. In these tet gene positive strains, 31 (14.6%), 2 (0.9%), 5 (2.4%), 1 (0.5%), 3 (1.4%) were detected containing tet(A)/tet(X), tet(M)/tet(O), tet(M)/tet(X), tet(O)/tet(X), and tet(Q)/tet(X) simultaneously, respectively. One isolates, R131, unexpectedly contained three tet genes, i.e., tet(M), tet(O), and tet(X). Sequence analysis of the tet gene ORFs cloned from R. anatipestifer isolates confirmed that tet(A), tet(B), tet(M), tet(O), tet(Q) and an unusual mosaic tet gene tet(O/W/32/O) were present in R. anatipestifer. The MIC results of R. anatipestifer ATCC 11845 transconjugants carrying tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes exhibited tetracycline resistance with MIC values ranging from 4 to 64 µg/ml. Additionally, the tet(X) gene could transfer into susceptible strain via natural transformation (transformation frequencies of ~10-6). In conclusion, the tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes were found and conferred tetracycline resistance in R. anatipestifer isolates. Moreover, the tet(X) is the main mechanism of tetracycline resistance in R. anatipestifer isolates. To our knowledge, this is the first report of tet(A), tet(B), tet(M), tet(O), tet(Q), and mosaic gene tet(O/W/32/O) in R. anatipestifer.

A novel resistance gene, lnu(H), conferring resistance to lincosamides in Riemerella anatipestifer CH-2.

The Gram-negative bacterium Riemerella anatipestifer CH-2 is resistant to lincosamides, having a lincomycin (LCM) minimum inhibitory concentration (MIC) of 128 µg/mL. The G148_1775 gene of R. anatipestifer CH-2, designated lnu(H), encodes a 260-amino acid protein with ≤41% identity to other reported lincosamide nucleotidylyltransferases. Escherichia coli RosettaTM (DE3) containing the pBAD24-lnu(H) plasmid showed four- and two-fold increases in the MICs of LCM and clindamycin (CLI), respectively. A kinetic assay of the purified Lnu(H) enzyme for LCM and CLI showed that the protein could inactive lincosamides. Mass spectrometry analysis demonstrated that the Lnu(H) enzyme catalysed adenylylation of lincosamides. In addition, an lnu(H) gene deletion strain exhibited 512- and 32-fold decreases in LCM and CLI MICs, respectively. The wild-type level of lincosamide resistance could be restored by complementation with a shuttle plasmid carrying the lnu(H) gene. The transformant R. anatipestifer ATCC 11845 [lnu(H)] acquired by natural transformation also exhibited high-level lincosamide resistance. Moreover, among 175 R. anatipestifer field isolates, 56 (32.0%) were positive for the lnu(H) gene by PCR. In conclusion, Lnu(H) is a novel lincosamide nucleotidylyltransferase that inactivates LCM and CLI by nucleotidylylation, thus conferring high-level lincosamide resistance to R. anatipestifer CH-2.

Contribution of RaeB, a Putative RND-Type Transporter to Aminoglycoside and Detergent Resistance in Riemerella anatipestifer.

Riemerella anatipestifer is an important pathogenic bacterium that infects ducks. It exhibits resistance to multiple classes of antibiotics. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens and they are poorly understood in R. anatipestifer. In this study, a gene encoding the B739_0873 protein in R. anatipestifer CH-1, which belongs to the resistance-nodulation-cell division (RND) efflux pump family, was identified. With respect to the substrate specificity of B739_0873, the antibiotic susceptibility testing showed that the B739_0873 knockout strain was more sensitive to aminoglycosides and detergents than the wild-type strain. The transcription of B739_0873 was up-regulated when R. anatipestifer CH-1 was exposed to sub-inhibitory levels of these substrates. From the gentamicin accumulation assay, we concluded that B739_0873 was coupled to the proton motive force to pump out gentamicin. Furthermore, site-directed mutagenesis demonstrated that Asp 400, Asp 401, Lys 929, Arg 959, and Thr 966 were the crucial function sites of B739_0873 in terms of its ability to extrude aminoglycosides and detergents. Finally, we provided evidence that B739_0873 is co-transcribed with B739_0872, and that both B739_0872 and B739_0873 are required for aminoglycoside and detergent resistance. In view of these results, we designate B739_0873 as RaeB (Riemerella anatipestifer efflux).

The suppression of apoptosis by α-herpesvirus.

Apoptosis, an important innate immune mechanism that eliminates pathogen-infected cells, is primarily triggered by two signalling pathways: the death receptor pathway and the mitochondria-mediated pathway. However, many viruses have evolved various strategies to suppress apoptosis by encoding anti-apoptotic factors or regulating apoptotic signalling pathways, which promote viral propagation and evasion of the host defence. During its life cycle, α-herpesvirus utilizes an elegant multifarious anti-apoptotic strategy to suppress programmed cell death. This progress article primarily focuses on the current understanding of the apoptosis-inhibition mechanisms of α-herpesvirus anti-apoptotic genes and their expression products and discusses future directions, including how the anti-apoptotic function of herpesvirus could be targeted therapeutically.

Type B Chloramphenicol Acetyltransferases Are Responsible for Chloramphenicol Resistance in Riemerella anatipestifer, China.

Riemerella anatipestifer causes serositis and septicaemia in domestic ducks, geese, and turkeys. Traditionally, the antibiotics were used to treat this disease. Currently, our understanding of R. anatipestifer susceptibility to chloramphenicol and the underlying resistance mechanism is limited. In this study, the cat gene was identified in 69/192 (36%) R. anatipestifer isolated from different regions in China, including R. anatipestifer CH-2 that has been sequenced in previous study. Sequence analysis suggested that there are two copies of cat gene in this strain. Only both two copies of the cat mutant strain showed a significant decrease in resistance to chloramphenicol, exhibiting 4 µg/ml in the minimum inhibitory concentration for this antibiotic, but not for the single cat gene deletion strains. Functional analysis of the cat gene via expression in Escherichia coli BL21 (DE3) cells and in vitro site-directed mutagenesis indicated that His79 is the main catalytic residue of CAT in R. anatipestifer. These results suggested that chloramphenicol resistance of R. anatipestifer CH-2 is mediated by the cat genes. Finally, homology analysis of types A and B CATs indicate that R. anatipestifer comprises type B3 CATs.

Outbreak of Avian Tuberculosis in Commercial Domestic Pekin Ducks ( Anas platyrhynchos domestica).

Avian tuberculosis is a contagious disease affecting various domestic and wild bird species, and is caused by Mycobacterium avium . It is reported extremely rarely in commercial poultry flocks and has not been reported in commercial domestic ducks to date, with domestic ducks reported to be moderately resistant to M. avium infection. Here, we report the outbreak of avian tuberculosis in commercial Pekin duck ( Anas platyrhynchos domestica) flocks. Postmortem and histopathologic findings included nodules presenting in the visceral organs of ducks, and granulomas with central caseous necrosis surrounded by infiltrating lymphocytes. The M. avium pathogen was isolated and further identified by Ziehl-Neelsen staining and PCR based on insert sequence IS901 and the 16S rRNA gene. We highlight that avian tuberculosis not only has economic significance for the duck industry, but also presents a potential zoonotic hazard to humans.

Comparative genomic analysis identifies structural features of CRISPR-Cas systems in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer infection is a contagious disease that has resulted in major economic losses in the duck industry worldwide. This study attempted to characterize CRISPR-Cas systems in the disease-causing agent, Riemerella anatipestifer (R. anatipestifer). The CRISPR-Cas system provides adaptive immunity against foreign genetic elements in prokaryotes and CRISPR-cas loci extensively exist in the genomes of archaea and bacteria. However, the structure characteristics of R. anatipestifer CRISPR-Cas systems remains to be elucidated due to the limited availability of genomic data. RESULTS: To identify the structure and components associated with CRISPR-Cas systems in R. anatipestifer, we performed comparative genomic analysis of CRISPR-Cas systems in 25 R. anatipestifer strains using high-throughput sequencing. The results showed that most of the R. anatipestifer strains (20/25) that were analyzed have two CRISPR loci (CRISPR1 and CRISPR2). CRISPR1 was shown to be flanked on one side by cas genes, while CRISPR2 was designated as an orphan. The other analyzed strains harbored only one locus, either CRISPR1 or CRISPR2. The length and content of consensus direct repeat sequences, as well as the length of spacer sequences associated with the two loci, differed from each other. Only three cas genes (cas1, cas2 and cas9) were located upstream of CRISPR1. CRISPR1 was also shown to be flanked by a 107 bp-long putative leader sequence and a 16 nt-long anti-repeat sequence. Combined with analysis of spacer organization similarity and phylogenetic tree of the R. anatipestifer strains, CRISPR arrays can be divided into different subgroups. The diversity of spacer organization was observed in the same subgroup. In general, spacer organization in CRISPR1 was more divergent than that in CRISPR2. Additionally, only 8 % of spacers (13/153) were homologous with phage or plasmid sequences. The cas operon flanking CRISPR1 was observed to be relatively conserved based on multiple sequence alignments of Cas amino acid sequences. The phylogenetic analysis associated with Cas9 showed Cas9 sequence from R. anatipestifer was closely related to that of Bacteroides fragilis and formed part of the subtype II-C subcluster. CONCLUSIONS: Our data revealed for the first time the structural features of R. anatipestifer CRISPR-Cas systems. The illumination of structural features of CRISPR-Cas system may assist in studying the specific mechanism associated with CRISPR-mediated adaptive immunity and other biological functions in R. anatipestifer.