Coevolution between marine Aeromonas and phages reveals temporal trade-off patterns of phage resistance and host population fitness.

Coevolution of bacteria and phages is an important host and parasite dynamic in marine ecosystems, contributing to the understanding of bacterial community diversity. On the time scale, questions remain concerning what is the difference between phage resistance patterns in marine bacteria and how advantageous mutations gradually accumulate during coevolution. In this study, marine Aeromonas was co-cultured with its phage for 180 days and their genetic and phenotypic dynamics were measured every 30 days. We identified 11 phage resistance genes and classified them into three categories: lipopolysaccharide (LPS), outer membrane protein (OMP), and two-component system (TCS). LPS shortening and OMP mutations are two distinct modes of complete phage resistance, while TCS mutants mediate incomplete resistance by repressing the transcription of phage genes. The co-mutation of LPS and OMP was a major mode for bacterial resistance at a low cost. The mutations led to significant reductions in the growth and virulence of bacterial populations during the first 60 days of coevolution, with subsequent leveling off. Our findings reveal the marine bacterial community dynamics and evolutionary trade-offs of phage resistance during coevolution, thus granting further understanding of the interaction of marine microbes.

Design combinations of evolved phage and antibiotic for antibacterial guided by analyzing the phage resistance of poorly antimicrobial phage.

Although antibiotics are the primary method against bacterial infections, the rapid emergence of antibiotic resistance has forced interest in alternative antimicrobial strategies. Phage has been considered a new biological antimicrobial agent due to its high effectiveness in treating bacterial infections. However, the applications of phage therapy have been limited by the quick development of phage-resistant bacteria. Therefore, more effective phage treatment strategies need to be explored guided by characterizing phage-resistant mutants. In this study, Pseudomonas plecoglossicida phage vB_PpS_SYP was isolated from the sewage but exhibited weak antibacterial activity caused by phage-resistant bacteria. Phage-resistant mutants were isolated and their whole genomes were analyzed for differences. The results showed that mutations in glycosyltransferase family 1 (GT-1) and hypothetical outer membrane protein (homP) led to bacterial phage resistance. The GT-1 mutants had lower biofilm biomass and higher antibiotic sensitivity than wild-type strain. Phage SYP evolved a broader host range and improved antimicrobial efficacy to infect homP mutants. Therefore, we designed a strategy for combined antibiotic and evolved phage inhibition driven by the two phage-resistant mutants. The results showed that the combination was more effective against bacteria than either antibiotics or phage alone. Our findings presented a novel approach to utilizing poorly antimicrobial phages by characterizing their phage-resistant mutants, with the potential to be expanded to include phage therapy for a variety of pathogens. IMPORTANCE The rapid emergence of antibiotic resistance renews interest in phage therapy. However, the lack of efficient phages against bacteria and the emergence of phage resistance impaired the efficiency of phage therapy. In this study, the isolated Pseudomonas plecoglossicida phage exhibited poor antibacterial capacity and was not available for phage therapy. Analysis of phage-resistant mutants guided the design of antibacterial strategies for the combination of antibiotics with evolved phages. The combination has a good antibacterial effect compared to the original phage. Our findings facilitate ideas for the development of antimicrobial-incapable phage, which have the potential to be applied to the phage treatment of other pathogens.

Integration of transcriptome, miRNA and degradome sequencing reveals the early browning response in fresh-cut apple.

Surface browning negatively impacts the shelf-life of fresh-cut apple. Herein, we found that the browning of fresh-cut apple aggravated rapidly after 24 h post-cutting, then the transcriptomic and miRNA expression profiles of fresh-cut apple immediately after cutting (T0) and 24 h post-cutting (T24) were analyzed to explore the molecular mechanism of early browning response. A total of 3156 differentially expressed mRNAs (DEGs) and 23 differentially expressed miRNAs (DEmiRNAs) were identified in T24 vs T0. Most DEGs related to respiratory, energy, antioxidant, lipid and secondary metabolism were activated in the early stage of browning. There were 63 target genes of 10 DEmiRNAs validated by degradome sequencing and among them, mdm-miR156aa_L + 1_1 targets 12-oxophytodienoate reductase, ptc-miR6478_R-1 targets patatin-like protein, mdm-miR156aa_L + 1_1 and mdm-miR156aa_L + 1_2 co-target SPLs might participate in the early browning response through regulating antioxidant, lipid and secondary metabolism. Our results will be beneficial for the technological innovation of browning amelioration for fresh-cut apple.

Detection of Cassava Component in Sweet Potato Noodles by Real-Time Loop-mediated Isothermal Amplification (Real-time LAMP) Method.

Sweet potato (Ipomoea batatas) noodles are a traditional Chinese food with a high nutritional value; however, starch adulteration is a big concern. The objective of this study was to develop a reliable method for the rapid detection of cassava (Manihot esculenta) components in sweet potato noodles to protect consumers from commercial adulteration. Five specific Loop-mediated Isothermal Amplification (LAMP) primers targeting the internal transcribed spacer (ITS) of cassava were designed, genomic DNA was extracted, the LAMP reaction system was optimized, and the specificity of the primers was verified with genomic DNA of cassava, Ipomoea batatas, Zea mays, and Solanum tuberosum; the detection limit was determined with a serial dilution of adulterated sweet potato starch with cassava starch, and the real-time LAMP method for the detection of the cassava-derived ingredient in sweet potato noodles was established. The results showed that the real-time LAMP method can accurately and specifically detect the cassava component in sweet potato noodles with a detection limit of 1%. Furthermore, the LAMP assay was validated using commercial sweet potato noodle samples, and results showed that 57.7% of sweet potato noodle products (30/52) from retail markets were adulterated with cassava starch in China. This study provides a promising solution for facilitating the surveillance of the commercial adulteration of sweet potato noodles from retail markets.

Reduced expression of the Kinesin-Associated Protein 3 (KIFAP3) gene increases survival in sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a degenerative disorder of motor neurons that typically develops in the 6th decade and is uniformly fatal, usually within 5 years. To identify genetic variants associated with susceptibility and phenotypes in sporadic ALS, we performed a genome-wide SNP analysis in sporadic ALS cases and controls. A total of 288,357 SNPs were screened in a set of 1,821 sporadic ALS cases and 2,258 controls from the U.S. and Europe. Survival analysis was performed using 1,014 deceased sporadic cases. Top results for susceptibility were further screened in an independent sample set of 538 ALS cases and 556 controls. SNP rs1541160 within the KIFAP3 gene (encoding a kinesin-associated protein) yielded a genome-wide significant result (P = 1.84 x 10(-8)) that withstood Bonferroni correction for association with survival. Homozygosity for the favorable allele (CC) conferred a 14.0 months survival advantage. Sequence, genotypic and functional analyses revealed that there is linkage disequilibrium between rs1541160 and SNP rs522444 within the KIFAP3 promoter and that the favorable alleles of rs1541160 and rs522444 correlate with reduced KIFAP3 expression. No SNPs were associated with risk of sporadic ALS, site of onset, or age of onset. We have identified a variant within the KIFAP3 gene that is associated with decreased KIFAP3 expression and increased survival in sporadic ALS. These findings support the view that genetic factors modify phenotypes in this disease and that cellular motor proteins are determinants of motor neuron viability.

High-throughput mutation screening using a single amplification condition.

Numerous innovative and high-throughput techniques have been established to identify human disease genes. However, DNA sequencing of candidate genes still remains as a major limitation in the identification of causative mutations. Much of this limitation is due to the time and labor needed for the polymerase chain reaction (PCR) optimization and reaction setup. Toward this end, we have established a simplified protocol that utilizes a single PCR amplification condition. PCR purification is accomplished via enzymatic digestion and all products can be sequenced using universal primers. This combination of a single amplification condition, single-step purification, and sequencing setup using universal primers all contribute to a simple and high-throughput mutation screen.

A common haplotype within the PON1 promoter region is associated with sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disorder of upper and lower motor neurons. Genetic variants in the paraoxonase gene cluster have been associated with susceptibility to sporadic ALS. Because these studies have yielded conflicting results, we have further investigated this association in a larger data set. Twenty SNPs spanning the paraoxonase gene cluster were genotyped on a panel of 597 case and 692 control samples and tested for association with risk of sporadic ALS and with ALS sub-phenotypes. Our study revealed two SNPs, rs987539 and rs2074351, within the paraoxonase gene cluster that are associated with susceptibility to sporadic ALS (uncorrected p=6.47E-04 and 7.87E-04, respectively). None of the 20 SNPs displayed significant associations with age of onset, site of onset or disease survival. Using a sliding window approach, we have also identified a 5-SNP haplotype that is significantly associated with risk of sporadic ALS (p=2.75E-05). We conclude that a common haplotype within the PON1 promoter region is associated with susceptibility to sporadic ALS.

Polymorphisms of human TAP2 detected by denaturing gradient gel electrophoresis.

The human transporter associated with antigen processing (TAP1 and TAP2) genes are located in the human leukocyte antigen (HLA) class II region of the genome and encode proteins that form a heterodimer essential for the transport of endogenous peptides into the endoplasmic reticulum for assembly with HLA class I molecules. Type 1 diabetes is an autoimmune disease that is associated with the HLA region of the genome, with HLA class II genes conferring the greatest statistical risk. The presentation of self-peptides by HLA class I molecules is defective in individuals with this disease, and both TAP1 and TAP2 are potential contributors to this defect. Denaturing gradient gel electrophoresis (DGGE) was applied to screen all 11 exons and the 3' flanking region of TAP2 for polymorphisms in individuals with type 1 diabetes patients and controls. Seventy polymorphisms, including 51 in introns, 4 in the 3' flanking region, and 15 in exons, were identified. Sequencing of polymorphic DNA fragments revealed several new polymorphisms, including a Gln --> Arg substitution at codon 611 and a GT --> GC polymorphism affecting the donor splice site of intron 4, that might be of functional significance. None of the polymorphisms examined differed in frequency between individuals with type 1 diabetes and controls.

Impaired processing and presentation by MHC class II proteins in human diabetic cells.

The biochemical processing of and Ag presentation by MHC class II molecules were examined in B cell lines derived from pairs of identical twins discordant for type 1 diabetes. MHC class II defects detected exclusively in cells derived from the twins with autoimmunity included increased rates of transport to and subsequent turnover at the cell surface, inadequate glycosylation, and a reduced display at the cell surface of antigenic peptides. These defects appeared to be secondary to a decreased abundance of the p35 isoform of the invariant chain (Ii), a human-specific chaperone protein for MHC class II normally generated by use of an alternative translation start site. Stable transfection of diabetic B cell lines with an Ii p35 expression vector corrected the defects in MHC class II processing and peptide presentation. A defect in the expression of Ii p35 may thus result in impairment of Ag presentation by MHC class II molecules and thereby contribute to the development of type 1 diabetes in at-risk genotypes.