Signal Peptide Research Articles

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin (α-LA) is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants. In this study, Bacillus subtilis RIK1285 harboring AprE signal peptide (SP) was selected as the original strain for the production of α-LA. It was found that α-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant. The original strain most likely only produced α-LA intracellular, but not extracellular. To improve the expression and secretion of α-LA in RIK1285, a library of 173 homologous SPs from the B. subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285. SP YjcN was determined to be the best signal peptide. Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide. In addition, different promoters (PaprE, P43, and Pglv) were compared and applied. The results indicated that the strain RIK1285-pBE-Pglv-YjcN-LALBA had the highest α-LA yield, reaching 122.04 μg/mL. This study demonstrates successful expression and secretion of human α-LA in B. subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

ATR2C ala2 from Arabidopsis-infecting downy mildew requires 4 TIR-NLR immune receptors for full recognition.

Arabidopsis Col-0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2Cala2 effector was unknown. To reveal ATR2Cala2, an F2 population was generated from a cross between Hpa-Cala2 and Hpa-Noks1. We identified ATR2Cala2 as a non-canonical RxLR-type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection. ATR2Cala2 is recognized in accession Col-0 but not in Ler-0 in which RPP2A and RPP2B are absent. In ATR2Emoy2 and ATR2Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2Cala2. Stable and transient expression of ATR2Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility. Two additional Col-0 TIR-NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa-Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2Cala2-recognizing accessions.

Structure–activity relationships of the intramolecular disulphide bonds in LEAP2, an antimicrobial peptide from Acrossocheilus fasciatus

BackgroundThe liver-expressed antimicrobial peptide 2 (LEAP2) plays a pivotal role in the host’s immune response against pathogenic microorganisms. Numerous such antimicrobial peptides have recently been shown to mitigate infection risk in fish, and studying those harboured by the economically important fish Acrossocheilus fasciatus is imperative for enhancing its immune responses against pathogenic microorganisms. In this study, we cloned and sequenced LEAP2 cDNA from A. fasciatus to examine its expression in immune tissues and investigate the structure–activity relationships of its intramolecular disulphide bonds.ResultsThe predicted amino acid sequence of A. fasciatus LEAP2 was found to include a signal peptide, pro-domain, and mature peptide. Sequence analysis indicated that A. fasciatus LEAP2 is a member of the fish LEAP2A cluster and is closely related to Cyprinus carpio LEAP2A. A. fasciatus LEAP2 transcripts were expressed in various tissues, with the head kidney exhibiting the highest mRNA levels. Upon exposure to Aeromonas hydrophila infection, LEAP2 expression was significantly upregulated in the liver, head kidney, and spleen. A mature peptide of A. fasciatus LEAP2, consisting of two disulphide bonds (Af-LEAP2-cys), and a linear form of the LEAP2 mature peptide (Af-LEAP2) were chemically synthesised. The circular dichroism spectroscopy result shows differences between the secondary structures of Af-LEAP2 and Af-LEAP2-cys, with a lower proportion of alpha helix and a higher proportion of random coil in Af-LEAP2. Af-LEAP2 exhibited potent antimicrobial activity against most tested bacteria, including Acinetobacter guillouiae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Staphylococcus warneri. In contrast, Af-LEAP2-cys demonstrated weak or no antibacterial activity against the tested bacteria. Af-LEAP2 had a disruptive effect on bacterial cell membrane integrity, whereas Af-LEAP2-cys did not exhibit this effect. Additionally, neither Af-LEAP2 nor Af-LEAP2-cys displayed any observable ability to hydrolyse the genomic DNA of P. aeruginosa.ConclusionsOur study provides clear evidence that linear LEAP2 exhibits better antibacterial activity than oxidised LEAP2, thereby confirming, for the first time, this phenomenon in fish.

Detection fimH Gene in The Urine of UTI Patients at The Puskesmas Banyuanyar, as a Marker for The Presence of Escherichia coli

Urinary Tract Infection (UTI) is a bacterial infection characterized by a urine culture showing bacteriuria with bacterial counts exceeding 100,000 per milliliter of urine. Polymerase Chain Reaction (PCR) is a method that utilizes in-vitro enzymatic reactions to amplify specific DNA targets. This process synthesizes new DNA strands complementary to the target sequences using enzymes and oligonucleotide primers within a thermocycler. The fimH gene is among the most important of the UPEC strains and is associated with severe urinary tract infections. The aim of this study was to identify the presence of the fimH gene in urine samples from UTI patients at the Puskesmas Banyuanyar in Surakarta using Polymerase Chain Reaction (PCR). The research employed the Quota Sampling Technique, collecting a total of five urine samples from individuals diagnosed with urinary tract infections. The results of the control isolates using pure Escherichia coli isolates showed that the fimH gene PCR amplified control with annealing temperature of 52°C. The analysis of clinical isolates of Escherichia coli from the urine of urinary tract infection patients revealed that the fimH gene was PCR amplified in all five samples.

All-optical matching systems based on the squarer for QPSK and 8PSK optical signals

Photonic firewall which can conduct the data identification and intrusion detection directly at the optical layer is a high-speed and low-energy network security equipment. For the photonic firewall, the all-optical matching system is the core part. This paper focuses on designing all-optical matching systems for high-order phase modulated optical signals. Based on the squarer, two all-optical matching systems are designed for Quadrature Phase Shift Keying (QPSK) and 8 Phase Shift Keying (8PSK) optical signals respectively. The systems mainly consist of squarers, multiplexers, AND gates and regenerators. The squarer is implemented based on the four-wave mixing (FWM) effect of highly nonlinear fiber (HNLF). The theoretical analysis and simulation verification are conducted for the proposed system. The performances of feasibility and noise impact are evaluated for the proposed system with the data rate of 100 Gbaud. The results show that the system can correctly identify and localize the target sequence with the length of 4 and 8 symbols in the data sequence with the length of 16 symbols. The impact of noise is evaluated by the extinction ratio (ER) and contrast ratio (CR). The ER of two all-optical matching systems reaches 0 dB when the optical signal-to-noise ratio (OSNR) of input optical signal is about 5.59 dB and 9.24 dB respectively. The CR of two all-optical matching systems reaches 0 dB when the OSNR of input optical signal is about 4 dB and 8.62 dB respectively.

Multimodal mechanisms of pathogenic variants in the signal peptide of FIX leading to hemophilia B

AbstractSignal peptide (SP) is essential for protein secretion, and pathogenic variants in the SP of factor IX (FIX) have been identified in hemophilia B (HB). However, the underlying mechanism for the genotype-phenotype correlation of these variants has not been well studied. Here, we systematically examined the effects of 13 pathogenic point variants in the SP of FIX using different approaches. Our results showed that these point variants lead to HB by missense variants and/or aberrant premessenger RNA (pre-mRNA) splicing. The missense variants in a hydrophobic core (h-region) mainly affected the cotranslational translocation function of the SP, and those in C-terminal containing cleavage site (c-region) caused FIX deficiency mainly by disturbing the cotranslational translocation and/or cleavage of the SP. Almost absolute aberrant pre-mRNA splicing was only observed in variants of c.82T>G, but a slight change of splicing patterns was found in variants of c.53G>T, c.77C>A, c.82T>C, and c.83G>A, indicating that these variants might have different degrees of impact on pre-mRNA splicing. Although two 6-nt deletion aberrant pre-mRNA splicing products caused FIX deficiency by disturbing the SP cleavage, they could produce some functional mature FIX, and vitamin K could increase the secretion of functional FIX. Taken together, our data indicated that pathogenic variants in the SP of FIX caused HB through diverse molecular mechanisms or even a mixture of several mechanisms, and vitamin K availability could be partially attributed to varying bleeding tendencies in patients carrying the same variant in the SP.

Profiling the eukaryotic soil microbiome with differential primers and an antifungal peptide nucleic acid probe (PNA): Implications for diversity assessment

The analysis of 18S rRNA gene amplicons is an important tool to characterize the diversity of the eukaryotic soil microbiome. Here we analyzed two primer sets (TAReuk, EKeuk) and the impact of a newly designed antifungal peptide nucleic acid to enhance the detection of protists in silico and with soil DNA extracted from croplands and a forest. The in silico analyses showed for TAReuk pronounced specificities for protist SAR supergroup and Metazoa, while EKeuk was particularly specific for Ascomycota and Basidiomycota. In silico, the PNA matched with the majority of Ascomycota (81.3 %) and Basidiomycota (65.4 %), but with <6 % of protists. The contrasting primer specificities were confirmed with soil DNA, but the proportion of protist amplicons was similar. In contrast to in silico, effects of the PNA were not as clear with soil DNA, even though it completely inhibited the amplification of the targeted fungal sequences. PNA effects were more pronounced with TAReuk, and results with cropland and forest soil DNA were not consistent, e.g., for cropland, PNA decreased the relative abundance of fungi but for forest it was the opposite, possibly because of different fungal diversity. The divergence between PNA in silico-predictions and results with soil DNA are likely an outcome of primer binding to <100 % complementary target sequences and a still limited DNA sequence databases for soil microbial eukaryotes. With TAReuk, the presence of PNA enhanced the detection of Conosa and, thus, could be a useful tool to study this group in the future.

Heterogeneous identity, stiffness and growth characterise the shoot apex of Arabidopsis stem cell mutants.

Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3, produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clavata mutant meristems display massive overgrowth, which is thought to be caused by stem cell overproliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here we reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clavata meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation is lost in mutants, in which cells simultaneously express distinct fate markers. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally distinguishable from wild-type stem cells. Thus all benchmarks show that clavata meristem cells are different from wild-type stem cells, suggesting that overgrowth is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains in the meristem.

AMELX Mutations and Genotype-Phenotype Correlation in X-Linked Amelogenesis Imperfecta.

AMELX mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness) and/or hypomaturation (reduced hardness) enamel defects. In this study, we conducted whole exome analyses to unravel the disease-causing mutations for six AI families. Splicing assays, immunoblotting, and quantitative RT-PCR were conducted to investigate the molecular and cellular effects of the mutations. Four AMELX pathogenic variants (NM_182680.1:c.2T>C; c.29T>C; c.77del; c.145-1G>A) and a whole gene deletion (NG_012494.2:g.307534_403773del) were identified. The affected individuals exhibited enamel malformations, ranging from thin, poorly mineralized enamel with a "snow-capped" appearance to severe hypoplastic defects with minimal enamel. The c.145-1G>A mutation caused a -1 frameshift (NP_001133.1:p.Val35Cysfs*5). Overexpression of c.2T>C and c.29T>C AMELX demonstrated that mutant amelogenin proteins failed to be secreted, causing elevated endoplasmic reticulum stress and potential cell apoptosis. This study reveals a genotype-phenotype relationship for AMELX-associated AI: While amorphic mutations, including large deletions and 5' truncations, of AMELX cause hypoplastic-hypomaturation enamel with snow-capped teeth (AI types IIB and IIC) due to a complete loss of gene function, neomorphic variants, including signal peptide defects and 3' truncations, lead to severe hypoplastic/aplastic enamel (AI type IE) probably caused by "toxic" cellular effects of the mutant proteins.

Identification of phosphorylated small ORF-encoded peptides in Hep3B cells by LC/MS/MS

Small ORF-encoded peptides (SEPs) are a class of low molecular weight proteins and peptides comprising <100 amino acids with important functions in various life activities. Although the sequence length is short, SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. In this work, we enriched phosphopeptides with IMAC and TiO2 materials and analyzed the phosphorylated SEPs in Hep3B cells. A total of 24 phosphorylated SEPs were identified, and 11 SEPs were coded by ncRNA. For the sequence analysis, we found that the general characteristics of phosphorylated SEPs are roughly the same as canonical proteins. Besides, two phosphorylation SEPs have the Stathmin family signature 2 motif, which can regulate the microtubule cytoskeleton. Some SEPs have domains or signal peptides, indicating their specific functions and subcellular locations. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of some SEPs. However, only one-fifth of the predicted phosphorylation sites were identified by LC/MS/MS, indicating that many SEP PTMs are hidden in the dark, waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation. SignificanceSmall ORF-encoded peptides (SEPs) are important in various life activities. Although the sequence length is short (<100AA), SEPs might also have post-translational modification (PTM). Phosphorylation is one of the most essential PTMs of proteins. We enriched phosphopeptides and analyzed the phosphorylated SEPs in Hep3B cells. That is the first time to explore the PTM of SPEs systematically. Kinase network analysis found a small number of kinases that may be a clue to the specific functions of SEPs. More SEP PTMs are hidden in the dark and waiting to be uncovered and verified. This study helps expand our understanding of SEP and provides information for further SEP function investigation.

CRHR1 antagonist alleviated depression-like behavior by downregulating p62 in a rat model of post-stroke depression

Post-stroke depression (PSD) is a complication of cerebrovascular disease, which can increase mortality after stroke. CRH is one of the main signaling peptides released after activation of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. It affects synaptic plasticity by regulating inflammation, oxidative stress and autophagy in the central nervous system. And the loss of spines exacerbates depression-like behavior. Therefore, synaptic deficits induced by CRH may be related to post-stroke depression. However, the underlying mechanism remains unclear. The Keap1-Nrf2 complex is one of the core components of the antioxidant response. As an autophagy associated protein, p62 participates in the Keap1-NrF2 pathway through its Keap1 interaction domain. Oxidative stress is involved in the feedback regulation between Keap1-Nrf2 pathway and p62.However, whether the relationship between CRH and the Keap1-Nrf2-p62 pathway is involved in PSD remains unknown. This study found that serum levels of CRH in 22 patients with PSD were higher than those in healthy subjects. We used MCAO combined with CUMS single-cage SD rats to establish an animal model of PSD. Animal experiments showed that CRHR1 antagonist prevented synaptic loss in the hippocampus of PSD rats and alleviated depression-like behavior. CRH induced p62 accumulation in the prefrontal cortex of PSD rats through CRHR1. CRHR1 antagonist inhibited Keap1-Nrf2-p62 pathway by attenuating oxidative stress. In addition, we found that abnormal accumulation of p62 induces PSD. It alleviates depression-like behavior by inhibiting the expression of p62 and promoting the clearance of p62 in PSD rats. These findings can help explore the pathogenesis of PSD and design targeted treatments for PSD.

Protein target similarity is positive predictor of in vitro antipathogenic activity: a drug repurposing strategy for Plasmodium falciparum

Drug discovery is an intricate and costly process. Repurposing existing drugs and active compounds offers a viable pathway to develop new therapies for various diseases. By leveraging publicly available biomedical information, it is possible to predict compounds’ activity and identify their potential targets across diverse organisms. In this study, we aimed to assess the antiplasmodial activity of compounds from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library using in vitro and bioinformatics approaches. We assessed the in vitro antiplasmodial activity of the compounds using blood-stage and liver-stage drug susceptibility assays. We used protein sequences of known targets of the ReFRAME compounds with high antiplasmodial activity (EC50 < 10 uM) to conduct a protein-pairwise search to identify similar Plasmodium falciparum 3D7 proteins (from PlasmoDB) using NCBI protein BLAST. We further assessed the association between the compounds' in vitro antiplasmodial activity and level of similarity between their known and predicted P. falciparum target proteins using simple linear regression analyses. BLAST analyses revealed 735 P. falciparum proteins that were similar to the 226 known protein targets associated with the ReFRAME compounds. Antiplasmodial activity of the compounds was positively associated with the degree of similarity between the compounds’ known targets and predicted P. falciparum protein targets (percentage identity, E value, and bit score), the number of the predicted P. falciparum targets, and their respective mutagenesis index and fitness scores (R2 between 0.066 and 0.92, P < 0.05). Compounds predicted to target essential P. falciparum proteins or those with a druggability index of 1 showed the highest antiplasmodial activity.

Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis.

Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 Å-resolution X-ray structure that revealed a highly α-helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.

Genotyping Zebrafish Point Mutant by Allele-Specific Blocking PCR.

Genotyping zebrafish carrying wild-type, heterozygous, or homozygous copies of a mutant allele is often required for investigating gene specific functions, and is routinely performed to differentiate point mutants. In this study, we describe a modified allele-specific PCR method using an additional blocking primer to promote target sequence amplification while suppressing sequences with single mismatch. Using the tp53m214k point mutant as an example, we show that wild-type, heterozygous, and homozygous zebrafish can be easily distinguished using this simple PCR method, which could be widely adapted for genotyping zebrafish with point mutations or small nucleotide insertions/deletions.

Efficient signal sequence of mRNA vaccines enhances the antigen expression to expand the immune protection against viral infection

The signal sequence played a crucial role in the efficacy of mRNA vaccines against virus pandemic by influencing antigen translation. However, limited research had been conducted to compare and analyze the specific mechanisms involved. In this study, a novel approach was introduced by substituting the signal sequence of the mRNA antigen to enhance its immune response. Computational simulations demonstrated that various signal peptides differed in their binding capacities with the signal recognition particle (SRP) 54 M subunit, which positively correlated with antigen translation efficiency. Our data revealed that the signal sequences of tPA and IL-6-modified receptor binding domain (RBD) mRNA vaccines sequentially led to higher antigen expression and elicited more robust humoral and cellular immune protection against the SARS-CoV-2 compared to the original signal sequence. By highlighting the importance of the signal sequence, this research provided a foundational and safe approach for ongoing modifications in signal sequence-antigen design, aiming to optimize the efficacy of mRNA vaccines.

Identification, functional characterization and immune response profiles of interleukin-10 in Nibea albiflora

Interleukin-10 (IL-10) is an immunosuppressive cytokine, which plays a vital role in regulating inflammation for inhibiting the generation and function of pro-inflammatory cytokines in vivo or in vitro. In the present study, the full length cDNA of IL-10 was characterized from Nibea albiflora (named as NaIL-10) of 1238 base pairs (bp), containing a 5′-UTR (untranslated region) of 350 bp, a 3′-UTR of 333 bp and an open reading frame (ORF) of 555 bp (Fig. 1A) to encode 184 amino acid residues with a signal peptide at the N-terminus. The sequence analysis showed that NaIL-10 possessed the typical IL-10 family symbolic motif and conversed cysteine residues, similar to its teleost orthologues. Real-time PCR indicated that NaIL-10 had wide distribution in different healthy tissues, with a relatively high expression in immune-related tissues (head kidney, spleen, kidney, liver and gill). Significantly, up-regulations of NaIL-10 after infection against Vibrio parahaemolyticus, Vibrio alginolyticus and Poly I:C were also observed. Subcellular localization manifested that NaIL-10 mainly distributed in the cytoplasm unevenly and aggregately, and there was also a small amount on the cell membrane, indicating that NaIL-10 was secreted to the extracellular space as the known IL-10 homologous molecules. It could co-locate with IL-10 Rα on the membrane of HEK293T cells for their potential interaction, and GST pull-down and Co-IP studies certified the specific and direct interaction between NaIL-10 and NaIL-10 Rα, confirming that an IL-10 ligand-receptor system existed in N.albiflora. The expression of pro-inflammatory cytokines, including TNF-α, IL-6, IL-1β, were dramatically inhibited in LPS-stimulated RAW264.7 macrophages pre-incubated with recombinant NaIL-10 protein, demonstrating its anti-inflammatory roles. Taken together, the results demonstrated the existence of IL-10 ligand-receptor system in N.albiflora for the first time, and indicated the suppressive function of NaIL-10 on pro-inflammatory cytokine expression in inflammatory response, which would be conducive to better comprehending the role of IL-10 in the immunomodulatory mechanisms of teleost.

Assessment of the antimicrobial and immunomodulatory activity of QS-CATH, a promising therapeutic agent isolated from the Chinese spiny frogs (Quasipaa spinosa)

Cathelicidins are important antimicrobial peptides in various vertebrate species where they are crucial parts of the innate immune system. The current understanding of amphibian cathelicidins is limited, particularly with regard to their immunomodulatory effects. To address this knowledge gap, we produced the cDNA sequence of the cathelicidin gene from a skin transcriptome of the Chinese spiny frog Quasipaa spinosa. The amino acid sequence of the Quasipaa spinosa cathelicidin (QS-CATH) was predicted to consist of a signal peptide, a cathelin domain, and a mature peptide. Comparative analysis of the QS-CATH amino acid sequence with that of other amphibian cathelicidins revealed high variability in the functional mature peptide among amphibians, whereas the cathelin domain was conserved. The QS-CATH gene was expressed in several tissues, with the highest level of expression in the spleen. Upregulation of QS-CATH after Aeromonas hydrophila infection occurred in the kidney, gut, spleen, skin, and liver. Chemically synthesized QS-CATH exhibited pronounced antibacterial activity against Shigella flexneri, Staphylococcus warneri, Escherichia coli, Salmonella enterica, and Listeria monocytogenes. Furthermore, QS-CATH disrupted the cell membrane integrity of S. flexneri, as evidenced by a lactate dehydrogenase release assay, and it hydrolyzed the genomic DNA of S. flexneri. Additionally, QS-CATH elicited chemotaxis and modulated the expression of inflammatory cytokine genes in RAW264.7 mouse leukemic monocyte/macrophage cells. These findings confirm the antimicrobial effects of amphibian cathelicidin and its ability to influence immune cell function. This will expedite the potential utilization of amphibian antimicrobial peptides as therapeutic agents.

Comprehensive genome-wide analysis of wheat xylanase inhibitor protein (XIP) genes: unveiling their role in Fusarium head blight resistance and plant immune mechanisms.

In this comprehensive genome-wide study, we identified and classified 83 Xylanase Inhibitor Protein (XIP) genes in wheat, grouped into five distinct categories, to enhance understanding of wheat's resistance to Fusarium head blight (FHB), a significant fungal threat to global wheat production. Our analysis reveals the unique distribution of XIP genes across wheat chromosomes, particularly at terminal regions, suggesting their role in the evolutionary expansion of the gene family. Several XIP genes lack signal peptides, indicating potential alternative secretion pathways that could be pivotal in plant defense against FHB. The study also uncovers the sequence homology between XIPs and chitinases, hinting at a functional diversification within the XIP gene family. Additionally, the research explores the association of XIP genes with plant immune mechanisms, particularly their linkage with plant hormone signaling pathways like abscisic acid and jasmonic acid. XIP-7A3, in particular, demonstrates a significant increase in expression upon FHB infection, highlighting its potential as a key candidate gene for enhancing wheat's resistance to this disease. This research not only enriches our understanding of the XIP gene family in wheat but also provides a foundation for future investigations into their role in developing FHB-resistant wheat cultivars. The findings offer significant implications for wheat genomics and breeding, contributing to the development of more resilient crops against fungal diseases.

A new mRNA structure prediction based approach to identifying improved signal peptides for bone morphogenetic protein 2

BackgroundSignal peptide (SP) engineering has proven able to improve production of many proteins yet is a laborious process that still relies on trial and error. mRNA structure around the translational start site is important in translation initiation and has rarely been considered in this context, with recent improvements in in silico mRNA structure potentially rendering it a useful predictive tool for SP selection. Here we attempt to create a method to systematically screen candidate signal peptide sequences in silico based on both their nucleotide and amino acid sequences. Several recently released computational tools were used to predict signal peptide activity (SignalP), localization target (DeepLoc) and predicted mRNA structure (MXFold2). The method was tested with Bone Morphogenetic Protein 2 (BMP2), an osteogenic growth factor used clinically for bone regeneration. It was hoped more effective BMP2 SPs could improve BMP2-based gene therapies and reduce the cost of recombinant BMP2 production.ResultsAmino acid sequence analysis indicated 2,611 SPs from the TGF-β superfamily were predicted to function when attached to BMP2. mRNA structure prediction indicated structures at the translational start site were likely highly variable. The five sequences with the most accessible translational start sites, a codon optimized BMP2 SP variant and the well-established hIL2 SP sequence were taken forward to in vitro testing. The top five candidates showed non-significant improvements in BMP2 secretion in HEK293T cells. All showed reductions in secretion versus the native sequence in C2C12 cells, with several showing large and significant decreases. None of the tested sequences were able to increase alkaline phosphatase activity above background in C2C12s. The codon optimized control sequence and hIL2 SP showed reasonable activity in HEK293T but very poor activity in C2C12.ConclusionsThese results support the use of peptide sequence based in silico tools for basic predictions around signal peptide activity in a synthetic biology context. However, mRNA structure prediction requires improvement before it can produce reliable predictions for this application. The poor activity of the codon optimized BMP2 SP variant in C2C12 emphasizes the importance of codon choice, mRNA structure, and cellular context for SP activity.

Exploring class III cellobiose dehydrogenase: sequence analysis and optimized recombinant expression

BackgroundCellobiose dehydrogenase (CDH) is an extracellular fungal oxidoreductase with multiple functions in plant biomass degradation. Its primary function as an auxiliary enzyme of lytic polysaccharide monooxygenase (LPMO) facilitates the efficient depolymerization of cellulose, hemicelluloses and other carbohydrate-based polymers. The synergistic action of CDH and LPMO that supports biomass-degrading hydrolases holds significant promise to harness renewable resources for the production of biofuels, chemicals, and modified materials in an environmentally sustainable manner. While previous phylogenetic analyses have identified four distinct classes of CDHs, only class I and II have been biochemically characterized so far.ResultsFollowing a comprehensive database search aimed at identifying CDH sequences belonging to the so far uncharacterized class III for subsequent expression and biochemical characterization, we have curated an extensive compilation of putative CDH amino acid sequences. A sequence similarity network analysis was used to cluster them into the four distinct CDH classes. A total of 1237 sequences encoding putative class III CDHs were extracted from the network and used for phylogenetic analyses. The obtained phylogenetic tree was used to guide the selection of 11 cdhIII genes for recombinant expression in Komagataella phaffii. A small-scale expression screening procedure identified a promising cdhIII gene originating from the plant pathogen Fusarium solani (FsCDH), which was selected for expression optimization by signal peptide shuffling and subsequent production in a 5-L bioreactor. The purified FsCDH exhibits a UV-Vis spectrum and enzymatic activity similar to other characterized CDH classes.ConclusionThe successful production and functional characterization of FsCDH proved that class III CDHs are catalytical active enzymes resembling the key properties of class I and class II CDHs. A detailed biochemical characterization based on the established expression and purification strategy can provide new insights into the evolutionary process shaping CDHs and leading to their differentiation into the four distinct classes. The findings have the potential to broaden our understanding of the biocatalytic application of CDH and LPMO for the oxidative depolymerization of polysaccharides.