Intelligent De Novo Design of Novel Antimicrobial Peptides against Antibiotic-Resistant Bacteria Strains.

Because of the growing number of clinical antibiotic resistance cases in recent years, novel antimicrobial peptides (AMPs) may be ideal for next-generation antibiotics. This study trained a Wasserstein generative adversarial network with gradient penalty (WGAN-GP) based on known AMPs to generate novel AMP candidates. The quality of the GAN-designed peptides was evaluated in silico, and eight of them, named GAN-pep 1-8, were selected by an AMP Artificial Intelligence (AI) classifier and synthesized for further experiments. Disc diffusion testing and minimum inhibitory concentration (MIC) determinations were used to identify the antibacterial effects of the synthesized GAN-designed peptides. Seven of the eight synthesized GAN-designed peptides displayed antibacterial activity. Additionally, GAN-pep 3 and GAN-pep 8 presented a broad spectrum of antibacterial effects and were effective against antibiotic-resistant bacteria strains, such as methicillin-resistant Staphylococcus aureus and carbapenem-resistant Pseudomonas aeruginosa. GAN-pep 3, the most promising GAN-designed peptide candidate, had low MICs against all the tested bacteria. In brief, our approach shows an efficient way to discover AMPs effective against general and antibiotic-resistant bacteria strains. In addition, such a strategy also allows other novel functional peptides to be quickly designed, identified, and synthesized for validation on the wet bench.

The thermal degradation and soil recovery of thermal treatment of field-weathered decabrominated diphenyl ether-contaminated soil.

A farm at Taoyuan in Taiwan was highly contaminated with decabrominated diphenyl ether (BDE-209), a widely used commercial brominated flame retardant and persistent in the environment, more than 10 years. Since crops are able to absorb and accumulate BDE-209 from soils in our previous research, posing a hazardous risk for humans, it is essential to develop a practical method of soil treatment. Thermal treatment was studied among different approaches. In our previous study (Ko et al., 2022), we found that heating to 450 °C for 30 min achieved a complete removal of BDE-209 in soil. However, the high temperature significantly decreased the original soil organic matter (SOM) from 2.47% to 0.27%, altering the soil texture, damaging microbial biomass, and thus affecting the revegetation after the thermal treatment. Sugarcane bagasse, a common agricultural residue, served as an amendment to restore soil fertility. Current results indicate that 2.5% bagasse can improve the SOM in soil by up to 2.73% and restore its bacterial composition, making the plant growth conditions similar to those of the untreated contaminated soil. In light of the high removal efficiency provided by the 450°C-thermal treatment and the high recovery efficiency of sugarcane bagasse, the strategy presented in this study serves to be a promising method for sustainable remediation.

The Puf-A Protein Is Required for Primordial Germ Cell Development.

Puf-A, a nucleolar Puf domain protein, is required for ribosome biogenesis. A study of Puf-A in zebrafish has shown that Puf-A is highly expressed in primordial germ cells (PGCs) and participates in PGC development. However, it remains unclear how Puf-A governs PGC development in mammals. Here, we generated transgenic mice carrying inducible Puf-A shRNA and obtained double heterozygous mice with Puf-A shRNA and Oct4-EGFP to examine the behavior of PGCs. It was found that the knockdown of Puf-A led to the loss of a considerable number of PGCs and a slowdown of the movement of the remaining PGCs. Puf-A and NPM1 colocalized in clusters in the nuclei of the PGCs. The silencing of Puf-A resulted in the translocation of NPM1 from nucleolus to nucleoplasm and the hyperactivation of p53 in the PGCs. The PGCs in Puf-A knockdown embryos showed a significant increase in subpopulations of PGCs at G1 arrest and apoptosis. Moreover, the expression of essential genes associated with PGC maintenance was decreased in the Puf-A knockdown PGCs. Our study showed that Puf-A governed PGC development by regulating the growth, survival, and maintenance of PGCs. We also observed the alterations of NPM1 and p53 upon Puf-A knockdown to be consistent with the previous study in cancer cells, which might explain the molecular mechanism for the role of Puf-A in PGC development.

Thermal treatment of decabrominated diphenyl ether in its highly contaminated soil in Taiwan.

Polybrominated diphenyl ethers (PBDEs) were commonly used flame retardants in the world, while some of PBDEs have been listed as persistent organic pollutants (POPs). Decabrominated diphenyl ether (BDE-209) was the most commercially used PBDEs. A farm near the factory located in Northern Taiwan was highly contaminated with BDE-209. Since PBDEs in the contaminated soils can be uptake by crops shown in our previous studies and could be potentially consumed by humans, it is very important to establish a feasible treatment method for PBDE remediation in this contaminated farm. Thermal treatment of PBDEs in soil was studied. The initial concentration of BDE-209 in contaminated soil was 1.472 mg/kg. A series of thermal experiments under different operating conditions including various temperature (105, 150, 200, 250, 300, 350, 400 and 450 °C), holding time (10, 20 and 30 min), heating rate (5, 10, 20 and 40 °C/min), and soil amount (10, 100, 1000 and 2000 g) were investigated. The optimal heating conditions for thermal treatment of contaminated soil were heating at 450 °C for 30 min with a heating rate of 10 °C/min. Under this condition, the removal of BDE-209 in the different weights of contaminated soil was tested. The soils in the contaminated farm were tested to further evaluate the feasibility of remediating the on-site PBDE contaminated soil through thermal treatment, suggesting that the holding time was extended to 2 h for the field-scale contaminated soil. The results showed that BDE-209 had been removed to below the detection limit in on-site soil. This investigation is the first study using thermal treatment to remediate soils really contaminated with PBDEs.

Efficient hexabromocyclododecane-biodegrading microorganisms isolated in Taiwan.

The potential toxicity of hexabromocyclododecane (HBCD), its persistence in the environment, and its high bioaccumulation characteristics pose a need to remediate HBCD in the environment. Bacillus cereus and B. subtilis species complexes we isolated from Taiwan soil are capable of degrading HBCD. B. cereus can degrade HBCD with a half-life only 0.911 days. The highest efficiency of HBCD degradation by B. cereus was achieved at pH 7.0, 35 °C, and 0.10 ppm HBCD. The removal mechanism of HBCD by B. cereus is debromination and its pathway was proposed. The addition of surfactant Tween 60 improved HBCD removal but the addition of CaO2, slow-releasing oxygen, did not. These findings can facilitate the bioremediation of HBCD in the environment.

Cloning and characterisation of a prophenoloxidase from the haemocytes of mud crab Scylla serrata.

A prophenoloxidase (proPO) cDNA was cloned from the haemocytes of mud crab Scylla serrata using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has a full length of 2663bp, with an open reading frame of 2019bp, a 124-bp 5'-untranslated region, and a 520-bp 3'-untranslated region containing a poly A signal. It encodes a protein of 673 amino acids with a predicted molecular weight of 77.5kDa and with an estimated pI of 5.96. It contains two putative tyrosinase copper-binding motifs with six histidine residues (copper A, 185, 189, 211, and copper B, 346, 350, 386). The proPO has thiol-ester-like motif (GCGWPQHM), which showed similar structural features of proPOs from other decapod crustaceans. It also contains five possible glycosylation sites, and a conserved C-terminal region common to all known proPOs. Sequence comparison showed that the proPO-deduced amino acid of mud crab S. serrata has an overall similarity of 78%, 57%, 56%, 51-55%, 54%, 53%, 52%, 52%, and 52% to that of Dungeness crab Cancer magister, American lobster Homarus americanus, European lobster Homarus gammarus, kuruma prawn Marsupenaeus japonicus, crayfish Pacifastacus leniusculus, white shrimp Litopenaeus vannamei, tiger shrimp Penaeus monodon, green tiger shrimp Penaeus semisulcatus, and giant freshwater prawn Macrobrachium rosenbergii, respectively. The proPO was strongly expressed in haemocytes, but not in heart, eyestalk, gill, muscle, ovary, hepatopancreas, stomach, and intestine. The proPO transcript of mud crab S. serrata increased significantly in 12 and 24h post-lipopolysaccharide (LPS) injection, but returned to the original values in 72h post injection.

Expression and characterisation of tiger shrimp Penaeus monodon penaeidin (mo-penaeidin) in various tissues, during early embryonic development and moulting stages.

A penaeidin family, mo-penaeidin was cloned from the haemocytes of tiger shrimp Penaeus monodon using genomic polymerase chain reaction (PCR) by gene specific primers. Analysis of nucleotide sequence revealed that this mo-penaeidin consists of 1348 bp containing one intron (680 bp) and two exons (210 and 458 bp). It has an open reading frame (ORF) of 222 p, which encodes a protein of 74 amino acids including a signal peptide of 19 amino acids. The calculated molecular mass of the mature protein (55 amino acids) is 6.059 kDa with an estimated pI of 9.3. The deduced amino acid sequence of mo-penaeidin has similarity to that of penaeidin from Fenneropenaeus chinensis (73%), Farfantepenaeus paulensis (66%), Litopenaeus schmitti (53-67%), L. stylirostris (50-67%), L. setiferus (50-62%), L. vannamei (44-66%), and Marsupenaeus japonicus (33%), respectively. Phylogenetic tree analysis indicated that penaeidin (including mo-penaeidin, penaeidin, and penaeidin 5, 2, 3k, 3c1) of P. monodon is distinct from penaeidin 1, penaeidin 2, penaeidin 3 and penaeidin 4 of other penaeid shrimps. The mo-penaeidin mRNA was detected in various tissues including ovary and mandibular organ. The mo-penaeidin mRNA was present in one cell to postlarva stage with higher level at nauplius I (9h post hatching) and higher expression during the intermoult stage indicating an early innate immunity and different immunity at moulting stage.

Molecular cloning and characterisation of a thioester-containing alpha2-macroglobulin (alpha2-M) from the haemocytes of mud crab Scylla serrata.

Molecular approaches were used to clone thioester-containing alpha2-macroglobulin (alpha2-M) genes in the haemocytes of mud crab Scylla serrata. The full length sequence of alpha2-M was determined by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA clone consists of 5491bp with an open reading frame (ORF) of 4986bp encoding a protein of 1662 amino acids with 22 residues signal sequence. The calculated molecular mass of the mature protein is 184.2kDa with an estimated pI of 8.41. The S. serrata alpha2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate alpha2-Ms. Sequence comparison showed that alpha2-M deduced amino acid sequence of S. serrata has an overall similarity of 68% and 48% to that of kuruma shrimp Marsupenaeus japonicus and American horseshoe crab Limulus polyphemus, respectively. Phylogentic analysis revealed that S. serrata alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of S. serrata increased significantly in 24h- and 48h-post lipopolysaccharide (LPS) injection.

Molecular cloning of myostatin gene and characterization of tissue-specific and developmental stage-specific expression of the gene in orange spotted grouper, Epinephelus coioides.

In this article we report the molecular cloning and characterization of a nonmammalian myostatin (growth and differentiation factor-8, MSTN) homolog from the orange spotted grouper (Epinephelus coioides) by polymerase chain reaction (PCR) cloning. The grouper MSTN gene consists of two introns [Intron I (363 bp) and Intron II (811 bp)] flanked by three exons [Exon I (379 bp), Exon II (371 bp) and Exon III (381 bp)]. A full-length cDNA clone (2608 bp) of the MSTN gene (GenBank DQ493889, nucleotide sequence in the coding region identical to GeneBank AY856860) was also isolated. This cDNA encodes a polypeptide of 376 amino acid residues that showed 25% to 96% homology with MSTNs of molluscan, teleostean, avian, and mammalian species. Phylogenetic analysis of the grouper MSTN polypeptide confirmed the evolutionary relationships of this MSTN with other known MSTNs. Results of reverse transcription (RT)-PCR analysis of the total RNA extracted from different tissues revealed that MSTN gene is expressed not only in the skeletal muscle, but also in other tissues. MSTN mRNA was also detected in different embryonic developmental and larval stages. Because the tissue-specific expression of MSTN gene in grouper is different from that in mammals, it might suggest that MSTN gene may possess additional functions other than regulating muscle growth in fish.

Molecular cloning and characterisation of a proteinase inhibitor, alpha 2-macroglobulin (alpha2-M) from the haemocytes of tiger shrimp Penaeus monodon.

An alpha 2-macroglobulin (alpha2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA consists of 4876 bp with an open reading frame (ORF) of 4494 bp, a 52 bp 5'-untranslated region, and a 327 bp 3'-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence. The predicted molecular mass of the mature protein (1480 amino acids) is 167.7 kDa with an estimated pI of 5.30. The P. monodon alpha2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate alpha2-Ms. Sequence comparison showed that alpha2-M deduced amino acid sequence of P. monodon has an overall similarity of 85, 52 and 49% to that of kuruma shrimp Marsupenaeus japonicus, American horseshoe crab Limulus polyphemus and mud crab Scylla serrata, respectively. Alignment of the deduced amino acid sequence to other species alpha2-M showed that the overall structure is evolutionarily conserved and phylogenetic analysis revealed that P. monodon alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of P. monodon increased significantly in 12, 24 and 48 h post-peptidoglycan (PG) injection, but returned to the original values in 72 h post-PG injection.

Cloning and characterisation of a serine proteinase from the haemocytes of mud crab Scylla serrata.

A serine proteinase (SP) cDNA was cloned from the haemocytes of mud crab Scylla serrata using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1,131 bp encoding a protein of 376 amino acids. The calculated molecular mass of the SP mature protein is 39.54 kDa with an estimated pI of 5.37. The C-terminal half of S. serrata SP is composed of a trypsin-like domain, with a sequence similar to that of other invertebrate and vertebrate SP domain. The typical catalytic triad of SP required for functional activity (His150, Asp217 and Ser331) was conserved in the polypeptide sequence. Sequence comparison showed that SP deduced amino acid has an overall similarity of 55%, 51% and 50% to SP deduced amino acid from spiny lobster Panulirus argus, horseshoe crab Tachypleus tridentatus and crayfish Pacifastaus leniusculus, respectively. The SP was strongly expressed in haemocytes, but was weakly expressed in heart, eyestalk and antennules. The SP transcript decreased significantly for the S. serrata following 3 days exposure to pH 9.5. However, the SP transcript increased significantly 24 h post-zymosan injection.