Corrigendum: Effects of concurrent training sequence on VO2max and lower limb strength performance: a systematic review and meta analysis.

[This corrects the article DOI: 10.3389/fphys.2023.1072679.].

Effects of concurrent training sequence on VO2max and lower limb strength performance: A systematic review and meta-analysis.

The aim of this study is to compare the effects of concurrent strength and endurance training sequences on VO2max and lower limb strength performance to provide scientific guidance for training practice. We searched PubMed, EBSCO, Web of Science (WOS), Wanfang, and China National Knowledge Infrastructure (CNKI) databases up to December 2022. The included articles were randomized controlled trials that allowed us to compare the strength-endurance (S-E) sequence and endurance-strength (E-S) sequence on VO2max, maximum knee extension strength, maximum knee flexion strength, and lower limb power. The Cochrane bias risk tool was used to evaluate the methodological quality of the included literature, and Stata 12.0 was used for the heterogeneity test, subgroup analysis, draw forest map, sensitivity analysis, and publication bias evaluation. The results have been presented as standardized mean differences (SMDs) between treatments with 95% confidence intervals and calculations performed using random effects models. Significance was accepted when p < 0.05. The studies included 19 randomized controlled trials (285 males and 197 females), 242 subjects in S-E sequence, and 240 subjects in E-S sequence in the analyses. No difference changes between S-E and E-S sequences has been observed on VO2max in the overall analysis (SMD = 0.02, 95% CI: -0.21-0.25, p = 0.859). The S-E sequence shows a greater increase in lower limb strength performance than does the E-S sequence (SMD = 0.19, 95% CI: 0.02-0.37, p = 0.032), which was manifested in the elderly (p = 0.039) and women (p = 0.017); in training periods >8 weeks (p = 0.002) and training frequencies twice a week (p = 0.003); and with maximum knee flexion (p = 0.040) and knee extension strength (p = 0.026), while no difference was found in lower limb power (p = 0.523). In conclusion, the effect of VO2max will not change with different concurrent training sequences. The S-E sequence improves lower limb strength more significantly, mainly in the improvement of knee flexion and knee extension. This advantage is more related to factors such as age, gender, training period, and training frequency.

Suppression of Continuous Wave Interference in Loran-C Signal Based on Sparse Optimization Using Tunable Q-Factor Wavelet Transform and Discrete Cosine Transform.

Loran-C is the most essential backup and supplementary system for the global navigation satellite system (GNSS). Continuous wave interference (CWI) is one of the main interferences in the Loran-C system, which will cause errors in the measurement of the time of arrival, thereby affecting positioning performance. The traditional adaptive notch filter method needs to know the frequency of CWI when removing it, and the number is limited. This paper presents a method based on sparseness to suppress the CWI in the Loran-C signal. According to the different morphological characteristics of the Loran-C signal and the CWI, we construct dictionaries suitable for the two components, respectively. We use the tunable Q-factor wavelet transform and the discrete cosine transform to make the two components obtain a good sparse representation in their respective dictionaries. Then, the two components are separated using the morphological component analysis theory. We illustrate this method using both synthetic data and actual data. A huge advantage of the proposed method is that there is no need to know the frequencies of the CWI for it can better cope with frequency changes of the CWI in the actual environments. Compared with the adaptive notch filter method, the results of the proposed method show that our approach is more effective and convenient.

[Molecular design, pharmacology and toxicology optimization of antimicrobial peptide from Hydrophis cyanocinctus, Hc-CATH].

Based on the cathelicidin family antimicrobial peptide Hc-CATH derived from sea snake, the Hc-16 and Hc-15 of 16 and 15 amino acid residues, were designed. By using CCK8, minimal inhibitory concentration, ELISA and bio-layer interferometry assays, their cytotoxicity, antibacterial activity, anti-inflammatory activity, and LPS neutralization activity was examined. Compared with Hc-15, Hc-16 had lower cytotoxicity and better broad-spectrum antibacterial activity against pathogens including clinically resistant bacteria, with the minimum inhibitory concentration of only 4.69 µg/mL. Hc-16 inhibited the expression of inflammatory cytokines of TNF-α and IL-6 induced by LPS, so as to significantly reduce the inflammatory response induced by infection. In addition, structure-activity relationship studies have shown that the phenylalanine at the C- and N-terminals of Hc-16 played a crucial role in its antibacterial and anti-inflammatory activity. Altogether, the designed Hc-16 has an excellent prospect to be developed into a novel antibiotic.

Induction of mitophagy in C2C12 cells by electrical pulse stimulation involves increasing the level of the mitochondrial receptor FUNDC1 through the AMPK-ULK1 pathway.

The mitochondrial receptor protein FUN14 domain-containing-1 (FUNDC1) can induce mitophagy under hypoxic conditions, as well as playing important roles in normal metabolism and intracellular homeostasis. Exercise not only elevates mitochondrial biosynthesis, but also exerts a significant impact on mitochondrial fission, integration and mitophagy. However, it is still not clear whether FUNDC1 plays a regulatory role in this context. Electrical pulse stimulation (EPS) of cultured myotubes is widely used as an in vitro model of muscle contraction. We simulated the contraction of C2C12 myotubes by EPS (15 V, 1 Hz, 2 ms, 1 h) to examine the role of FUNDC1 in mitophagy. EPS was found to induce mitophagy by activating the AMPK-ULK1 pathway to an even greater extent than AICAR and FUNDC1 is involved in the associated mitophagy. However, when AMPK is inhibited, other pathways may regulate mitophagy. Our findings indicate that mitophagy helps maintain the normal functions of mitochondria. EPS of C2C12 myotubes results in contraction, induction of mitophagy and potential activation of the AMPK-ULK1 pathway that promotes the expression of FUNDC1.

[Effects of exercises with different durations and intensities on mitochondrial autophagy and FUNDC1 expression in rat skeletal muscles].

The aim of the present study was to investigate the effects of exercises with different durations and intensities on mitochondrial autophagy and FUNDC1 in rat skeletal muscles. Sixty male Sprague-Dawley rats were randomly divided into 2- and 4-week control groups (Con), moderate-intensity exercise groups (M-ex groups, treadmill exercise, 16 m/min, 1 h/d, 6 d/week), and high-intensity exercise groups (Hi-ex groups, treadmill exercise, 35 m/min, 20 min/d, 6 d/week). The bilateral soleus muscles were separated after the intervention, and paraffin sections were prepared for transmission electron microscopy. ELISA method was used to detect the content of citrate synthase (CS). The co-localizations of microtubule-associated protein 1 light chain 3 (LC3)/cytochrome c oxidase IV (COX-IV), FUNDC1/COX-IV and LC3/FUNDC1 were observed by immunofluorescent staining in frozen sections. The skeletal muscle mitochondria were extracted, and the expression of autophagy-related proteins, including AMPKα, p-AMPKα, Unc-51 like kinase 1 (ULK1), FUNDC1, LC3 and p62, were detected by Western blot. The results showed that exercise increased mitochondrial function, i.e. peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α), COX-I protein expression levels and CS content. There was no difference of mitochondrial function parameters between 2-week M-ex and 2-week Hi-ex groups, while mitochondrial function of 4-weeks Hi-ex group was significantly lower than that of 4-week M-ex group. Under the same exercise intensity, mitochondrial autophagy activation in skeletal muscle of 4-week exercise was higher than that in 2-week exercise group; Under the same duration of exercise, mitochondrial autophagy activation of Hi-ex group was higher than that in M-ex group. Both 2- and 4-week exercise intervention increased LC3/COX-IV, COX-IV/FUNDC1, and FUNDC1/LC3 co-localizations. Exercise increased LC3-II/LC3-I ratio, down-regulated p62 protein expression level, up-regulated FUNDC1, ULK1 protein expression levels and AMPKα phosphorylation, and the changes of these proteins in 4-week Hi-ex group were significantly greater than those in 4-week M-ex group. These results suggest exercise induces mitochondrial autophagy in skeletal muscles, and the activity of autophagy is related to the duration and intensity of exercise. The induction mechanism of exercise may involve the mediation of FUNDC1 expression through AMPK-ULK1 pathway.

Design of a Sea Snake Antimicrobial Peptide Derivative with Therapeutic Potential against Drug-Resistant Bacterial Infection.

Infections caused by drug-resistant pathogens are a worldwide challenge for public health. Antimicrobial peptides (AMPs) are regarded as promising antibiotic alternatives for the treatment of drug-resistant infections. In the present study, a series of small peptides were designed based on our previously reported sea snake AMP Hc-CATH. From them, the lead peptide HC1-D2, a truncated peptide entirely substituted by d-amino acids, was selected. HC1-D2 exhibited significantly improved stability and antibiofilm and anti-inflammatory activities. Meanwhile, HC1-D2 retained potent, broad-spectrum, and rapid antimicrobial properties against bacteria and fungi, especially drug-resistant bacteria. Moreover, HC1-D2 showed low propensity to induce bacterial resistance and low cytotoxicity and hemolytic activity. Notably, HC1-D2 showed potent in vivo anti-infective ability in mouse peritonitis models infected by both standard and drug-resistant bacteria. It significantly decreased the bacterial counts in the abdominal cavity and spleen of mice and apparently increased the survival rates of the mice. Acting through the MAPKs inflammatory pathway, HC1-D2 selectively induced the production of chemokine and the subsequent immune cell recruitment to the infection site, while inhibiting the production of pro-inflammatory cytokines with undesirable toxicities. These much improved properties make HC1-D2 a promising candidate for the development of novel peptide anti-infective agents against drug-resistant infections.

A small cytotoxic peptide from frog elicits potent antitumor immunity to prevent local tumor growth and metastases.

Aim: Anticancer immunochemotherapy represents an attractive paradigm to improve therapeutic responses and reduce side effects. Results & methodology: Here, we show that a naturally occurring host defense peptide, HN-1 inhibited multiple malignant cells proliferation and tumor growth in a xenografted human breast tumor model. Acting through MAPK/NF-κB pathways, HN-1 induced a caspase-independent mitochondrial apoptosis, as indicated by a p53-dependent increase of Bax/Bcl-2 ratio and the nuclear translocation of apoptosis inducing factor. Besides, HN-1 augmented CD4+/CD8+ T cells in 4T1 mammary carcinoma model, by enhancing the serum levels of cancer immunity-associated effectors. Meanwhile, HN-1 decreased the angiogenesis and infiltration of the tumor-associated macrophages. Conclusion: HN-1 induces caspase-independent cancer cells apoptosis and boosts cancer-resolving immunity without inducing potentially harmful pro-inflammatory responses.

Diversity, immunoregulatory action and structure-activity relationship of green sea turtle cathelicidins.

Cathelicidins are a family of gene-encoded immune effectors in vertebrate innate immunity. Here, we reported the diversity and biological activity of cathelicidins in green sea turtle, a marine reptile species known for long lifespan and disease resistance. Four novel cathelicidins (Cm-CATH1-4) were identified from green sea turtle. All of them, especially Cm-CATH2, exhibited potent, broad-spectrum and rapid bactericidal and anti-biofilm activities by inducing the disruption of cell membrane integrity. Additionally, Cm-CATH2 effectively induced the macrophages/monocytes and neutrophils trafficking to the infection site, and inhibited the LPS-induced production of inflammatory cytokines, by blocking TLR4/MD2 complex and the downstream signaling pathway activation. In mouse peritonitis and pneumonia models, Cm-CATH2 exhibited evident protection against drug-resistant bacterial infections. Taken together, the diverse structures and functions of Cm-CATHs indicated their pleiotropic role in innate immunity of green sea turtle, and the potent antimicrobial, anti-biofilm and immunomodulatory properties make them ideal candidates for the development of novel anti-infective drugs.

Combination of Human Umbilical Vein Endothelial Cell Vaccine and Docetaxel Generates Synergistic Anti-Breast Cancer Effects.

Background: A human umbilical vein endothelial cell (HUVEC) vaccine is a promising anti-angiogenesis therapy, but the modest therapeutic antitumor efficacy restricts its clinical use. Preclinical evidence supports the combination of antiangiogenic agents and chemotherapy for cancer treatment. Materials and Methods: In the present study, docetaxel (DOC) was combined with HUVEC vaccine to develop a HUVEC-DOC treatment regime. This study was designed to investigate the synergistic anti-breast cancer effects and mechanisms of the HUVEC-DOC treatment. Results: Compared with either agent monotherapy, HUVEC-DOC treatment exhibited more favorable anti-EMT-6 breast cancer effects in vivo. CD31 immunohistochemical analysis of the excised tumors showed notable decreases in vessel density after HUVEC-DOC administration, while T cells isolated from mice immunized with HUVEC-DOC showed increased cytotoxicity against HUVECs. Furthermore, the quantity of interferon gamma released from HUVEC-DOC-administered mice was significantly higher than the other three groups, and enhanced CD8+ T cell infiltration was observed more frequently in tumors excised from HUVEC-DOC-treated mice. Finally, the percentage of regulatory T cells was significantly decreased after HUVEC-DOC immunization. Conclusions: All the data verified that combining DOC with a HUVEC vaccine could generate synergistic anti-breast cancer activity, which might have the potential for combination treatment of human breast cancer.

Roles of polymorphic cathelicidins in innate immunity of soft-shell turtle, Pelodiscus sinensis.

Cathelicidins are a class of gene-encoded immune effectors in vertebrate innate immune system. Though being extensively studied in mammals, little is known about the roles of cathelicidins in turtles, the water-dwelling vertebrates in the order Testudines. In the present study, six novel cathelicidins (Ps-CATH1-6) with different tissue and inducible expression patterns were characterized from the turtle of economic importance, Chinese soft-shell turtle (Pelodiscus sinensis). Although the structures of Ps-CATH1-6 precursors were identical with most of the other known cathelicidins, the mature peptides of Ps-CATH1-6 showed low sequence similarity with the other cathelicidins. Functional studies indicated that some of them either directly kill pathogens via inducing the permeabilization in bacterial membrane (Ps-CATH4, 6), or boost infection-resolving immunity by selectively inhibiting pro-inflammatory responses (Ps-CATH2, 4, 6) through MAPKs and NF-κB pathways. Ps-CATH2, 4, and 6, which assume the hallmark amphipathic α-helical conformations as most of the other host defense peptides, exhibit evident in vivo protection by significantly reducing the bacterial loads in bacterial infected turtles. Collectively, the discovery of novel Ps-CATHs with pleiotropic structures and functions helps elucidating the roles of cathelicidins in the Chinese softshell turtle innate immunity, and better understanding the survival strategy of Chinese softshell turtle in harsh habitat.

Express Sequence Tag Analysis - Identification of Anseriformes Trypsin Genes from Full-Length cDNA Library of the Duck (Anas platyrhynchos) and Characterization of Their Structure and Function.

Trypsins are key proteins important in animal protein digestion by breaking down the peptide bonds on the carboxyl side of lysine and arginine residues, hence it has been used widely in various biotechnological processes. In the current study, a full-length cDNA library with capacity of 5·10(5) CFU/ml from the duck (Anas platyrhynchos) was constructed. Using express sequence tag (EST) sequencing, genes coding two trypsins were identified and two full-length trypsin cDNAs were then obtained by rapid-amplification of cDNA end (RACE)-PCR. Using Blast, they were classified into the trypsin I and II subfamilies, but both encoded a signal peptide, an activation peptide, and a 223-a.a. mature protein located in the C-terminus. The two deduced mature proteins were designated as trypsin-IAP and trypsin-IIAP, and their theoretical isoelectric points (pI) and molecular weights (MW) were 7.99/23466.4 Da and 4.65/24066.0 Da, respectively. Molecular characterizations of genes were further performed by detailed bioinformatics analysis. Phylogenetic analysis revealed that trypsin-IIAP has an evolution pattern distinct from trypsin-IAP, suggesting its evolutionary advantage. Then the duck trypsin-IIAP was expressed in an Escherichia coli system, and its kinetic parameters were measured. The three dimensional structures of trypsin-IAP and trypsin-IIAP were predicted by homology modeling, and the conserved residues required for functionality were identified. Two loops controlling the specificity of the trypsin and the substrate-binding pocket represented in the model are almost identical in primary sequences and backbone tertiary structures of the trypsin families.

Identification and Characterization of Novel Antioxidant Peptides Involved in Redox Homeostasis of Frog, Limnonectes fragilis.

Previous studies have proved that a novel antioxidant system composed of various antioxidant peptides (AOPs) exists in the skin of ranid frogs, keeping the redox homeostasis. However, only a small number of AOPs have been identified so far. Here, a total of 47 cDNA sequences encoding 21 different AOPs belonging to 11 families were cloned from the skin cDNA library of Limnonectes fragilis. Among them, fragilin-A1 (VKRRGQDCIHGFCSD) and fragilin-B1 (GQFNDKRWIPFG) were also purified from skin secretions. They were selected with odorranain-Q-Lf (APIRMWYMYRKLTDMEPKPVA), the newest sequence among all 21 AOPs, to evaluate the antioxidant activities by direct free radical scavenging and lipid peroxidation inhibition assay. Results demonstrated that all peptides possessed strong DPPH and ABTS(.+) scavenging activities, and effectively inhibited lipid peroxidation in linoleic acid emulsion system during a 7- day test. No cytotoxic and hemolytic activity against human erythrocytes was observed for the three AOPs. The homology modeling analysis revealed that they all adopt tertiary structures ideally suited for the key residues to come into contact with the radicals. Current results reveal the existence of antioxidant system constituted of AOPs in the skin of the L. fragilis, and furthermore provide excellent templates for the development of novel antioxidant agents.

[Construction of novel thioredoxin fusion protein expression system and the production of recombinant Lf-CATH2].

The objective of this study was to construct an improved thioredoxin fusion protein expression system, and express the cathelicidin-derived peptide, Lf-CATH2. The improved fusion vector Lf-CATH2-pET32α(-TS) was successfully constructed by firstly deleting the thrombin site and S tag from the pET-32α vector, then inserting the Lf-CATH2 plus a thrombin site instead. Afterwards, Lf-CATH2 was expressed in Escherichia coli as fusion protein. After the cleavage by thrombin, Lf-CATH2 was released and subsequently separated using affinity chromatography. The antimicrobial activity of purified Lf-CATH2 was also examined. The improved expression vector significantly increased enzyme cleavage efficiency by 37%, and Lf-CATH2 could be expressed in high yield and maintain the biological activity. This novel thioredoxin fusion protein expression system enables a quick production of high-yield bioactive cationic peptides like cathelicidins.

Identification and Characterization of the First Cathelicidin from Sea Snakes with Potent Antimicrobial and Anti-inflammatory Activity and Special Mechanism.

Cathelicidins are a family of gene-encoded peptide effectors of innate immunity found exclusively in vertebrates. They play pivotal roles in host immune defense against microbial invasions. Dozens of cathelicidins have been identified from several vertebrate species. However, no cathelicidin from marine reptiles has been characterized previously. Here we report the identification and characterization of a novel cathelicidin (Hc-CATH) from the sea snake Hydrophis cyanocinctus. Hc-CATH is composed of 30 amino acids, and the sequence is KFFKRLLKSVRRAVKKFRKKPRLIGLSTLL. Circular dichroism spectroscopy and structure modeling analysis indicated that Hc-CATH mainly assumes an amphipathic α-helical conformation in bacterial membrane-mimetic solutions. It possesses potent broad-spectrum and rapid antimicrobial activity. Meanwhile, it is highly stable and shows low cytotoxicity toward mammalian cells. The microbial killing activity of Hc-CATH is executed through the disruption of cell membrane and lysis of bacterial cells. In addition, Hc-CATH exhibited potent anti-inflammatory activity by inhibiting the LPS-induced production of nitric oxide (NO) and pro-inflammatory cytokines such as TNF-α, IL-1ß, and IL-6. Hc-CATH directly binds with LPS to neutralize its toxicity, and it also binds to Toll-like receptor 4 (TLR4/MD2 complex), which therefore inhibits the binding of LPS to TLR4/MD2 complex and the subsequent activation of LPS-induced inflammatory response pathways. Taken together, our study demonstrates that Hc-CATH, the first cathelicidin from sea snake discovered to have both antimicrobial and anti-inflammatory activity, is a potent candidate for the development of peptide antibiotics.

Cathelicidins from the bullfrog Rana catesbeiana provides novel template for peptide antibiotic design.

Cathelicidins, a class of gene-encoded effector molecules of vertebrate innate immunity, provide a first line of defense against microbial invasions. Although cathelicidins from mammals, birds, reptiles and fishes have been extensively studied, little is known about cathelicidins from amphibians. Here we report the identification and characterization of two cathelicidins (cathelicidin-RC1 and cathelicidin-RC2) from the bullfrog Rana catesbeiana. The cDNA sequences (677 and 700 bp, respectively) encoding the two peptides were successfully cloned from the constructed lung cDNA library of R. catesbeiana. And the deduced mature peptides are composed of 28 and 33 residues, respectively. Structural analysis indicated that cathelicidin-RC1 mainly assumes an amphipathic alpha-helical conformation, while cathelicidin-RC2 could not form stable amphipathic structure. Antimicrobial and bacterial killing kinetic analysis indicated that the synthetic cathelicidin-RC1 possesses potent, broad-spectrum and rapid antimicrobial potency, while cathelicidin-RC2 exhibited very weak antimicrobial activity. Besides, the antimicrobial activity of cathelicidin-RC1 is salt-independent and highly stable. Scanning electron microscopy (SEM) analysis indicated that cathelicidin-RC1 kills microorganisms through the disruption of microbial membrane. Moreover, cathelicidin-RC1 exhibited low cytotoxic activity against mammalian normal or tumor cell lines, and low hemolytic activity against human erythrocytes. The potent, broad-spectrum and rapid antimicrobial activity combined with the salt-independence, high stability, low cytotoxic and hemolytic activities make cathelicidin-RC1 an ideal template for the development of novel peptide antibiotics.

Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

Identification and polymorphism discovery of the cathelicidins, Lf-CATHs in ranid amphibian (Limnonectes fragilis).

To protect themselves against the invasion of microorganisms, amphibians, especially the Rana frogs, are possibly equipped with complex combinations of antimicrobial peptides (AMPs). The two major AMP families, ranid skin secretion AMPs and cathelicidins might together constitute the host innate immune system of amphibians. Cathelicidins are a group of cationic peptides found in leukocytes and epithelial cells, and they play a central role in the early innate immune defense found in virtually all species of mammals. However, they have rarely been reported from amphibians. Here, we report the identification and discovery of polymorphism cathelicidins in Limnonectes fragilis. The expression profile indicated high cathelicidin transcript levels in frog spleen, liver and kidney, but lower levels in lung, skin and stomach. According to the amphibian's unique proteolytic pattern, R125 and L121 of the prepropeptides are predicted to be the processing positions for protease to generate the mature peptides, Lf-CATH1 and -2, respectively. Both consist of 30 amino acid residues, of which two were cysteines positionally conserved among a few known amphibian cathelicidins. Homology modeling analysis revealed that Lf-CATH1 and -2 adopt a tertiary structure with a mostly α helix that is representative of small cationic cathelicidin family peptides. Recombinant Lf-CATH1 (rLf-CATH1) was produced in Escherichia coli. Synthetic Lf-CATH1 and -2 displayed potent antimicrobial activities in vitro against a broad spectrum of microorganisms, including standard and clinically isolated drug-resistant strains, while showing neglectable hemolysis and cytotoxicities.

Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

Structural and functional characterization of CATH_BRALE, the defense molecule in the ancient salmonoid, Brachymystax lenok.

Thick-lipped lenok, Brachymystax lenok is one of the ancient fish species in China and northeast Asia countries. Due to the overfishing, the population of lenok has been declined significantly. Cathelicidins are innate immune effectors that possess both bactericidal activities and immunomodulatory functions. This report identifies and characterizes the salmonoid cathelicidin (CATH_BRALE) from this ancient fish. It consists of open reading frame (ORF) of 886 bp encoding the putative peptide of 199 amino acids. Sequence alignment with other representative salmonid cathelicidins displayed two distinctive features of current lenok cathelicidin: high level of arginine, resulting in high positive charge and glycine residues, which is significantly different from most acknowledged types of cathelicidins; and the six-aminoacid tandem repeated sequence of RPGGGS detected in a variable number of copies among fish cathelicidins, suggesting the existence of a genetically unstable region similar to that found in some mammalian cathelicidins. Expression of CATH_BRALE is predominantly found in gill, with lower levels in the gastrointestinal tract and spleen. The homology modeled structure of CATH_BRALE exhibits structural features of antiparallel b-sheets flanked by a-helices that are representative of small cationic cathelicidin family peptides. CATH_BRALE possesses much stronger antimicrobial activity against gram-negative bacteria than that of the human ortholog, LL-37. The growth of two typical fish bacterial pathogens, gram-negative bacterium of Aeromonas salmonicida and Aeromonas hydrophila was substantially inhibited by synthetic CATH_BRALE, with both MICs as low as 9.38 mM.