Dual-signal output fluorescent aptasensor based on DNA programmability and gold nanoflowers for multiple mycotoxins detection.

Herein, a dual-signal output fluorescent aptamer sensor was constructed for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA) using the specific recognition ability of aptamers and the programmability of DNA. A functional capture probe (cDNA) was designed with the black hole quenching motif BHQ1 labeled at the 5' end and biotin (bio) labeled at the 3' end. The fluorescent dye Cy3-labeled aflatoxin B1 aptamer (AFB1-Apt) and the carboxyfluorescein FAM-labeled ochratoxin A aptamer (OTA-Apt) were used as two fluorescent probes. The cDNA is anchored to the quenching material gold nanoflowers (AuNFs) by the action of streptavidin (SA) and biotin. Its ends can be complementarily paired with two fluorescent probe bases to form a double-stranded structure. The fluorescence of Cy3 was quenched by AuNFs, and the fluorescence of FAM was quenched by BHQ1 through the fluorescence energy resonance transfer (FRET) effect, forming a fluorescence quenching system. Due to the high affinity of the target and the aptamer, the structure of the aptamer probe changes and detaches from the sensor when AFB1 and OTA are present, resulting in enhanced fluorescence. Under optimal conditions, the linear range of AFB1 was 0.1-100 ng/mL (R2 = 0.996), the limit of detection (LOD) was as low as 0.014 ng/mL, and the limit of quantification (LOQ) was 0.046 ng/mL. The linear range of OTA was 0.1-100 ng/mL (R2 = 0.995), the limit of detection (LOD) was as low as 0.027 ng/mL, and the limit of quantification (LOQ) was 0.089 ng/mL. The sensor had high accuracy in detecting both AFB1 and OTA in real sample analysis. The results of the t test show that there is no significant difference between the results of this study and the high-performance liquid phase (HPLC) method, indicating that the prepared sensor can be used as a potential platform for multiple mycotoxins detection.

A highly sensitive competitive aptasensor for AFB1 detection based on an exonuclease-assisted target recycling amplification strategy.

Aflatoxin B1 (AFB1) is a typical mycotoxin found in agricultural products, and poses a huge threat to both humans and animals. Accurate and rapid measurement of AFB1 is essential for environmental analysis and food safety. Based on molecular docking simulation design and exonuclease-assisted target recycling amplification, we designed a competitive fluorescence aptasensor to detect AFB1 rapidly and sensitively. According to the molecular docking simulations, a complementary strand (cDNA) was designed by searching for potential binding sites of the aptamer, which had the lowest binding energy. Magnetic beads modified with biotin-Apt were used as the capture probe, while FAM-labeled cDNA acted as the reporter probe. By using EXO I for target recycling amplification, this aptasensor was highly sensitive and selective for AFB1. The detection limit of the suggested aptasensor under optimal conditions was 0.36 ng mL-1 (S/N = 3) in the range of 1-1000 ng mL-1 (R2 = 0.991). The developed aptasensor was successfully used to analyze AFB1 in oil samples.

An electrochemical biosensor for the detection of aflatoxin B1 based on the specific aptamer and HCR biological magnification.

Aflatoxin B1 (AFB1) is a highly toxic mycotoxin, which causes severe acute or cumulative poisoning. Therefore, it is important to develop sensitive and selective detection methods for AFB1 for the safety of food and medicinal herbs. Herein, we have developed a "signal-on" electrochemical aptasensor based on the high specificity of the aptamer and hybridization chain reaction (HCR) biological amplification for AFB1 detection. In this work, thiol-modified complementary DNA (cDNA) immobilized on the surface of a gold electrode (GE) served as an initiator DNA. When AFB1 was present, it competed with the cDNA for binding to the aptamers, which resulted in the detaching of aptamers from the cDNA-aptamer duplexes. Then, the single-stranded cDNA acted as an initiator to trigger the HCR signal amplification. Therefore, long double-stranded DNA (dsDNA) products were produced, which could load large amounts of methylene blue (MB) molecules to generate a distinct electrochemical signal. Under the optimized conditions, the proposed electrochemical aptasensor achieved the ultrasensitive detection of AFB1 with a linear detection range of 0.01-100 pg mL-1, and a limit of detection (LOD) down to 2.84 fg mL-1. Furthermore, the electrochemical aptasensor was successfully applied for detecting AFB1 in corn and two kinds of traditional Chinese medicine samples, indicating the potential value for AFB1 detection in practical samples.

Electrochemical determination of hantavirus using gold nanoparticle-modified graphene as an electrode material and Cu-based metal-organic framework assisted signal generation.

An electrochemical biosensor was prepared for nucleic acid-based hantavirus detection using a Cu-based metal-organic framework (CuMOF) as a signal tag. The CuMOF was synthesized by the solvothermal method and then covalently bonded with signal DNA (sDNA) probes. The Au nanoparticles and reduced graphene oxide composite were deposited on the electrode surface by electroreduction as support substrate and was then functionalized with capture DNA (cDNA) probes by self-assembly. Through the complementary base pairing, the target DNA (tDNA) fragment of hantavirus hybridized with the cDNA and the sDNA in a sandwich-type format. The tDNA was detected according to the current signal of the CuMOF catalyzed reaction using o-phenylenediamine as redox substrate. The peak current of the biosensor at - 0.55 V increased linearly in proportion to the logarithmic value of the tDNA concentration from 10-15 to 10-9 mol/L, with a detection limit of 0.74 × 10-15 mol/L. Moreover, the proposed biosensor was successfully applied to detect hantavirus and was able to distinguish hantavirus from other arboviruses.

Hairpin-Bisulfite PCR.

Ligation of a hairpin oligonucleotide to genomic DNA prior to bisulfite conversion and PCR amplification physically links the two complementary DNA strands. This additional step in the conversion procedure overcomes the limitations of conventional bisulfite sequencing where information of the cytosine methylation status is only obtained from one of the two strands of an individual DNA molecule. Sequences derived from hairpin bisulfite PCR products reveal the dynamics of this epigenetic memory system on both strands of individual DNA molecules. The chapter describes a reliable step-by-step procedure to generate hairpin-linked DNA. It also provides a guide for efficient bisulfite conversion that is suitable for both conventional and hairpin bisulfite sequencing approaches.

Lipid-Encapsulated Silica Nanobowls as an Efficient and Versatile DNA Delivery System.

Nonmesoporous Janus silica nanobowls (NBs) are unique in that they possess two different nonporous surfaces per particle for loading biological molecules and can thus be designed with multifunctional properties. Although silica NBs have been successfully employed for both targeted therapeutic and diagnostic applications, their ability to deliver DNA has not yet been fully explored. The purpose of this study was to design and develop an in vitro transfection agent that would exploit the distinct characteristics of the silica NB. First, we determined that the NB surface can be linked to either supercoiled cDNA plasmids or vectorless, linear cDNA constructs. Additionally, the linearized cDNA can be functionalized and chemisorbed on NBs to obtain a controlled release. Second, the successful transfection of cells studied was dependent on lipid coating of the NB (LNBs). Although both NBs and LNBs were capable of undergoing endocytosis, NBs appeared to remain within vesicles as shown by transmission electron microscopy (TEM). Third, fluorescence microscopy and Western blotting assays revealed that transfection of four different cell lines and acutely isolated rat sensory neurons with LNBs loaded with either linear or supercoiled cDNA constructs coding for the fluorescent protein, clover and tdTomato, resulted in protein expression. Fourth, two separate opioid receptor-ion channel signaling pathways were functionally reconstituted in HEK cells transfected with LNBs loaded with three separate cDNA constructs. Overall, these results lay the foundation for the use and further development of LNBs as in vitro transfection agents.

Magnetically controlled colorimetric aptasensor for chlorpyrifos based on copper-based metal-organic framework nanoparticles with peroxidase mimetic property.

The fabrication of a magnetically controlled colorimetric aptasensor for chlorpyrifos is reported. The aptasensor was fabricated by the attachment of the colorimetric labels onto the magnetic carrier due to the hybridization reaction between the complementary DNA and aptamer. Chlorpyrifos detection was realized by monitoring the color changes of the TMB/H2O2 solution before and after incubation of the aptasensor with chlorpyrifos via exposure to external magnetic force. The color change was monitored at 650 nm by UV-Vis spectrophotometer. Under the optimal conditions, this magnetically controlled Cu-MOF-based aptasensor showed a detection limit of 4.4 ng/mL with a linear range of 0-1250 ng/mL. The colorimetric aptasensor displayed high selectivity for chlorpyrifos toward other interfering pesticides. The aptasensor was successfully applied for the spiked test of chlorpyrifos in fruits and vegetable samples with good recovery, which were in agreement with data obtained by GC-MS analysis. This magnetically controlled Cu-MOF-based sensing strategy not only leads to development of efficient and facile phase separation, but also expands the MOF's target scope from H2O2 or glucose to pesticides. Graphical abstract.

A colorimetric aptamer-based method for detection of cadmium using the enhanced peroxidase-like activity of Au-MoS2 nanocomposites.

In this work, a colorimetric aptamer-based method for detection of cadmium using gold nanoparticles modified MoS2 nanocomposites as enzyme mimic is established. In short, biotinylated Cd2+ aptamers are immobilized by biotin-avidin binding on the bottoms of the microplate, the complementary strands of Cd2+ aptamers are connected to the Au-MoS2 nanocomposites which have the function of enhanced peroxidase-like activity. The csDNA-Au-MoS2 signal probe and target Cd2+ compete for binding Cd2+ aptamer, the color change can be observed by addition of chromogenic substrate, thereby realizing visual detection of Cd2+. The absorbance of the solution at 450 nm has a clear linear relationship with the Cd2+ concentration. The linear range is 1-500 ng/mL, and the limit of detection is 0.7 ng/mL. The assay was used to test white wine samples, the results are consistent with those of atomic absorption spectrometry; which prove that this method can be used for detection of Cd2+ in real samples.

Strand-specific detection of overlapping transcripts via purification involving denaturation of biotinylated cDNA.

Reverse transcription-PCR (RT-PCR) is the most widely employed technique for gene expression analysis owing to its high sensitivity, easy reproducibility and fast output. It has been conceived that priming RT reactions with gene-specific primers generates cDNA only from the specific RNA. However, several reports have revealed that cDNA is synthesized even without addition of exogenous primers in RT reactions. Owing to such self-priming activity, the signals from specific strands cannot be accurately detected and can confound the expression analysis, especially in context of overlapping bidirectional transcripts. Here, we demonstrate that purification of biotin-tagged cDNA in conjunction with alkaline denaturation can obviate the problem of background priming and enable accurate strand-specific detection of overlapping transcripts.

Molecular cloning, expression and mimicking antiviral activity analysis of retinoic acid-inducible gene-I in duck (Anas platyrhynchos).

Intracellular double-stranded RNA (dsRNA) is a chief sign of replication for many viruses. Pattern recognition receptors(PRRs) of the innate immune system detected the dsRNA and initiate the antiviral responses. Retinoic acid-inducible gene I (RIG-I), a member of PRRs, plays an essential regulatory role in dsRNA-induced signalling. In this study, the full-length complementary DNA (cDNA) of duck RIG-I (duRIG-I) was cloned using the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). The cDNA of duRIG-I contained 97-bp 5'UTR, 141-bp 3'-UTR and 2802 bp complete open-reading frame (ORF) encoding 933 amino acids. Multiple sequence alignments showed that duRIG-I shared high similarity with RIG-I from other vertebrates. Quantitative real-time PCR (qRT-PCR) analysis revealed that duRIG-I mRNA was expressed in all tested tissues, with high levels in the liver, heart, spleen, kidney and thymus, while lower in the duodenum. duRIG-I could be induced by treatment with poly(I:C). Further, overexpression of duRIG-I significantly activated the transcription of poly(I:C)-induced IFN-b, IRF7, TRIF, Mx, STAT1 and STAT2 mRNA, and duRIG-I knockdown showed the opposite results. Overall, our results suggested that duRIG-I could be an important receptor for mimicking antiviral state in duck, which warrant further studies to show the possible mechanism.

Cloning, characterisation and expression profile of kisspeptin1 and the kisspeptin1 receptor in the hypothalamic-pituitary-ovarian axis of Chinese alligator Alligator sinensis during the reproductive cycle.

Kisspeptin1 (Kiss1), a product of the Kiss1 gene, plays an important role in the regulation of reproduction in vertebrates by activating the Kiss1 receptor (Kiss1R) and its coexpression with gonadotrophin-releasing hormone (GnRH) in GnRH neurons. The purpose of this study was to clone the Kiss1 and Kiss1R genes found in the brain of Alligator sinensis and to explore their relationship with reproduction. The full-length cDNA of Kiss1 is 816bp, the open reading frame (ORF) is 417bp and the gene encodes a 138-amino acid precursor protein. The full-length cDNA of Kiss1R is 2348bp, the ORF is 1086bp and the gene encodes a 361-amino acid protein. Quantitative polymerase chain reaction showed that, except for Kiss1R expression in the hypothalamus, the expression of Kiss1 and Kiss1Rduring the reproductive period of A. sinensis was higher than that in the hypothalamus, pituitary gland and ovary during the hibernation period. The changes in GnRH2 mRNA in the hypothalamus were similar to those of GnRH1 and peaked during the reproductive period. This study confirms the existence of Kiss1 and Kiss1R in A. sinensis and the findings strongly suggest that Kiss1 and Kiss1R may participate in the regulation of GnRH secretion in the hypothalamus of alligators during the reproductive period. Furthermore, this is the first report of the full-length cDNA sequences of Kiss1 and Kiss1R in reptiles.

Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights.

A full-length cDNA encoding digestive lipase (SmDL) was cloned from the pancreas of the smooth-hound (Mustelus mustelus). The obtained cDNA was 1350 bp long encoding 451 amino acids. The deduced amino acid sequence has high similarity with known pancreatic lipases. Catalytic triad and disulphide bond positions are also conserved. According to the established phylogeny, the SmDL was grouped with those of tuna and Sparidae lipases into one fish digestive lipase cluster. The recently purified enzyme shows no dependence for bile salts and colipase. For this, the residue-level interactions between lipase-colipase are yet to be clearly understood. The structural model of the SmDL was built, and several dissimilarities were noticed when analyzing the SmDL amino acids corresponding to those involved in HPL binding to colipase. Interestingly, the C-terminal domain of SmDL which holds the colipase shows a significant role for colipase interaction. This is apt to prevent the interaction between fish lipase and the pancreatic colipase which and can provide more explanation on the fact that the classical colipase is unable to activate the SmDL.

Ultraselective antibiotic sensing with complementary strand DNA assisted aptamer/MoS2 field-effect transistors.

Although aptamer has been demonstrated as an important probe for antibiotic determination, the selective sensing of different antibiotics is still a challenge due to their structure similarities and wide folding degrees of aptamer. Herein, a field-effect transistor using MoS2 nanosheet as the channel and an aptamer DNA (APT) with its configuration shaped by a complementary strand DNA (CS) is employed for kanamycin (KAN) determination. This probe structure contributes to an enhanced selectivity and reliability with reduced device-to-device variations. This MoS2/APT/CS sensor shows time-dependent performance in antibiotic sensing. Prolonged detection time (20 s-300 s) leads to an enhanced sensitivity (1.85-4.43 M-1) and a lower limit of detection (1.06-0.66 nM), while a shorter detection time leads to a broader linear working range. A new sensing mechanism relying on charge release from probe is proposed, which is based on the "replacement reaction" between KAN and APT-CS. This sensor exhibits an extremely high selectivity (selectivity coefficient of 12.8) to kanamycin over other antibiotics including streptomycin, tobramycin, amoxicillin, ciprofloxacin and chloramphenicol. This work demonstrates the merits of probe engineering in label-free antibiotic detection with FET sensor, which presents significant promises in sensitive and selective chemical and biological sensing.

Impact of dietary chitosan oligosaccharide and its effects on coccidia challenge in broiler chickens.

1. Two experiments were conducted, the first to determine the optimum inclusion of chitosan oligosaccharide (COS) in broiler diets to support growth performance, digestive functions, intestinal morphology, and immune organs. The second experiment evaluated the immune-protective properties of COS on broiler chickens during coccidia challenge (CC).2. Experiment 1 investigated the effect of graded dietary concentration of COS in the diets of broiler chickens using eight cage replicates for each of the six diets. A corn-soybean meal-based diet was used as the basal diet and supplemented with 0.0, 0.5, 1.0, 1.5, 2.0, or 2.5 g of COS/kg feed to form the six treatments.3. The diet supplemented with 1.0 g COS/kg of feed provided the optimal inclusion level for broiler chickens regarding body weight (BW) gain, jejunal villus height, villus height to crypt depth ratio, and ileal energy digestibility at d 22 of age.4. Experiment 2 investigated the immune-protective properties of COS in broiler chickens during CC. A total of 224 male broiler chicks were randomly assigned to eight replicate cages in a 2 × 2 factorial arrangement of treatments with two COS concentrations (0 or 1 g of COS/kg of diet), with or without CC.5. On d 18 of age, birds in the CC group received twice the recommended coccidia vaccine dose of 30 doses/kg BW.6. Coccidia challenge reduced (P < 0.05) and dietary COS increased (P < 0.05) BW gain, and feed intake. Dietary COS mitigated (P < 0.05) the CC-induced effects on gain:feed. Dietary COS supplementation attenuated the CC-induced effects (P < 0.05) on the expression of occludin genes.7. In conclusion, dietary COS improved performance, and the immune-related beneficial impact of COS supplementation was associated with reduced expression of pro-inflammatory cytokine genes.

A versatile and accessible polymer coating for functionalizable zwitterionic quantum dots with high DNA grafting efficiency.

Efficient and versatile functionalization of poly(anhydride maleic-alt-isobutylene) (PIMA), with economical commercial reagents, results in the one-step/one-day production of a copper-free click chemistry-ready carboxybetaine-like coating for quantum dots (QDs). The QDs are bright and stable in aqueous media and easily grafted with DNA with >95% efficiency.

An aptamer assay for aflatoxin B1 detection using Mg2+ mediated free zone capillary electrophoresis coupled with laser induced fluorescence.

We described an aptamer based and Mg2+ mediated free zone capillary electrophoresis-laser induced fluorescence (CE-LIF) assay for aflatoxin B1 (AFB1) detection. This CE-LIF assay applied an anti-AFB1 aptamer with a single fluorescein (FAM) label at 5' end and a short complementary DNA (cDNA). In the absence of AFB1, the cDNA hybridized with the aptamer probe and formed a duplex DNA. The use of running buffer containing MgCl2 allowed good isolation of the duplex DNA from the single stranded DNA in CE. We found introducing a biotin label on the cDNA further improved the isolation. When AFB1 existed in sample solution, the aptamer probe bound with AFB1, dissociating from the duplex DNA. Thus, the duplex DNA peak decreased, while the aptamer probe peak increased during CE-LIF analysis. We achieved detection of AFB1 by measuring the aptamer probe peak. The length of cDNA, the ratio of aptamer to cDNA, and the concentration of MgCl2 in sample buffer and separation buffer had great effect on the aptamer based CE-LIF assay. Under optimized conditions, the detection limit of AFB1 was 0.2 nM, and the dynamic range was from 0.2 nM to 500 nM. Limit of quantitation was 0.5 nM. This CE-LIF assay enabled detection of AFB1 spiked in diluted human serum, diluted human urine, and corn flour samples. This assay exhibits potential for wide application as it integrates the rapidity, high sensitivity, low sample consumption of CE-LIF analysis and the strengths of aptamer.

Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches.

Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.

Graphene Oxide-Based Nanostructured DNA Sensor.

Quick detection of DNA sequence is vital for many fields, especially, early-stage diagnosis. Here, we develop a graphene oxide-based fluorescence quenching sensor to quickly and accurately detect small amounts of a single strand of DNA. In this paper, fluorescent magnetic nanoparticles (FMNPs) modified with target DNA sequence (DNA-t) were bound onto the modified graphene oxide acting as the fluorescence quenching element. FMNPs are made of iron oxide (Fe3O4) core and fluorescent silica (SiO2) shell. The average particle size of FMNPs was 74 ± 6 nm and the average thickness of the silica shell, estimated from TEM results, was 30 ± 4 nm. The photoluminescence and magnetic properties of FMNPs have been investigated. Target oligonucleotide (DNA-t) was conjugated onto FMNPs through glutaraldehyde crosslinking. Meanwhile, graphene oxide (GO) nanosheets were produced by a modified Hummers method. A complementary oligonucleotide (DNA-c) was designed to interact with GO. In the presence of GO-modified with DNA-c, the fluorescence intensity of FMNPs modified with DNA-t was quenched through a FRET quenching mechanism. Our study indicates that FMNPs can not only act as a FRET donor, but also enhance the sensor accuracy by magnetically separating the sensing system from free DNA and non-hybridized GO. Results indicate that this sensing system is ideal to detect small amounts of DNA-t with limitation detection at 0.12 µM.

Aptamer Structure Switch Fluorescence Anisotropy Assay for Small Molecules Using Streptavidin as an Effective Signal Amplifier Based on Proximity Effect.

Fluorescence polarization/anisotropy (FP/FA) approaches are appealing for targets sensing in homogeneous solution due to simplicity, reproducibility and sensitivity. Taking advantage of aptamers, aptamer structure switch FA methods are unique for small molecule detection based on the competition between aptamer-target binding and the hybridization of aptamer and complementary DNA (cDNA). However, usually small FA change is generated in these aptamer assays that only rely on size change caused by hybridization of an oligonucleotide because of the rapid local rotation of fluorophores and small mass change. Here we describe a simple and general aptamer structure switch FA assay for small molecules by employing a large-sized streptavidin (SA) as an effective signal amplifier based on proximity effect to reduce local rotation of fluorophore. In this design, the SA-labeled cDNA hybridizes with fluorescein (FAM)-labeled aptamer, drawing FAM close to SA and bringing a much higher FA value due to restricted local rotation of FAM. Small molecule-aptamer probe binding causes displacement of the SA-labeled cDNA and great decrease of FA. The closeness of SA to FAM in the duplex is key for this proposed strategy to produce large FA changes in target detection. Our method enabled to detect 60 pM aflatoxin B1 (AFB1), 1 nM ochratoxin A (OTA), and 0.5 µM adenosine triphosphate (ATP), respectively. This aptamer FA method combines the merits of aptamers and FA analysis, and it is promising in applications of detection of small molecules with good sensitivity.

Molecular and functional characterization of a short-type peptidoglycan recognition protein, PGRP-S in the amphibian Xenopus laevis.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the function of frog PGRPs. In this study, a short-type PGRP (termed Xl-PGRP-S) gene was identified in the African clawed frog, Xenopus laevis. The predicted protein of Xl-PGRP-S contains several structural features known in PGRPs, including a typical PGRP domain and two closely spaced conserved cysteines. Xl-PGRP-S gene was constitutively expressed in all tissues examined, with the highest expression level observed in muscle. As a typical PRR, Xl-PGRP-S is inducible after peptidoglycan (PGN) stimulation, and has an ability to bind PGN. In addition, Xl-PGRP-S has been proven to have Zn2+-dependent amidase activity and antibacterial activity against Edwardsiella tarda. The present study represents the first discovery on the function of frog PGRPs, thus contributing to a better understanding of the functional evolution of PGRPs in early tetrapods.