Dual effects of single-walled carbon nanotubes coupled with near-infrared radiation on <Emphasis Type="Italic">Bacillus anthracis</Emphasis> spores: inactivates spores and stimulates the germination of surviving spores

Background

Bacillus anthracis is a pathogen that causes life-threatening disease--anthrax. B. anthracis spores are highly resistant to extreme temperatures and harsh chemicals. Inactivation of B. anthracis spores is important to ensure the environmental safety and public health. The 2001 bioterrorism attack involving anthrax spores has brought acute public attention and triggered extensive research on inactivation of B. anthracis spores. Single-walled carbon nanotubes (SWCNTs) as a class of emerging nanomaterial have been reported as a strong antimicrobial agent. In addition, continuous near infrared (NIR) radiation on SWCNTs induces excessive local heating which can enhance SWCNTs’ antimicrobial effect. In this study, we investigated the effects of SWCNTs coupled with NIR treatment on Bacillus anthracis spores.

Results and discussion

The results showed that the treatment of 10 μg/mL SWCNTs coupled with 20 min NIR significantly improved the antimicrobial effect by doubling the percentage of viable spore number reduction compared with SWCNTs alone treatment (88% vs. 42%). At the same time, SWCNTs-NIR treatment activated the germination of surviving spores and their dipicolinic acid (DPA) release during germination. The results suggested the dual effect of SWCNTs-NIR treatment on B. anthracis spores: enhanced the sporicidal effect and stimulated the germination of surviving spores. Molecular level examination showed that SWCNTs-NIR increased the expression levels (>2-fold) in 3 out of 6 germination related genes tested in this study, which was correlated to the activated germination and DPA release. SWCNTs-NIR treatment either induced or inhibited the expression of 3 regulatory genes detected in this study. When the NIR treatment time was 5 or 25 min, there were 3 out of 7 virulence related genes that showed significant decrease on expression levels (>2 fold decrease).

Conclusions

The results of this study demonstrated the dual effect of SWCNTs-NIR treatment on B. anthracis spores, which enhanced the sporicidal effect and stimulated the germination of surviving spores. SWCNTs-NIR treatment also altered the expression of germination, regulatory, and virulence-related genes in B. anthracis.

     

<Emphasis Type="Italic">Sfi</Emphasis>NX: a method for assembly of protein coding sequences with high success rates

Objective

Concatenation of two NdeI–XhoI gene fragments via an oligonucleotide linker on a plasmid vector with an SfiI site was performed to evaluate success rates in construction of polycistronic genes expressible in Escherichia coli.

Results

A series of plasmids with an SfiI site between the selection marker and the replication origin were constructed. The three wheat eEF1B subunit genes inserted between the NdeI and XhoI sites of pET-22b were transferred to the SfiI-containing plasmid with a spectinomycin-resistance gene. Then, the marker gene in the resultant plasmids was substituted with the ampicillin-resistance gene. These plasmids were used for concatenation of two different genes via a linker oligonucleotide containing a ribosome-binding site. During these operations, 42 clones were picked up out of which 41 had the intended product plasmid.

Conclusion

This method, named as the SfiNX method, is useful for trial-and-error based testing of different combinations of fusion and co-expression partners for optimization of recombinant protein production.

     

Silencing of <Emphasis Type="Italic">Mythimna separata chitinase</Emphasis> genes via oral delivery of <Emphasis Type="Italic">in planta</Emphasis>-expressed RNAi effectors from a recombinant plant virus

Objectives

To evaluate transient expression of RNA interference (RNAi) effectors in Nicotiana benthamiana plants by using recombinant virus vectors and also oral delivery of the effectors for silencing of Mythimna separata endogenous gene expression.

Results

Mythimna separata is a serious pest of corn production in China. To evaluate RNAi approaches to target specific RNAs in M. separate, we cloned fragments of the M. separata chitinase sequences into a virus vector in order to produce RNAi effectors during virus infection and replication in plants. When the infected plants were fed to M. separata, expression levels of target MseChi1 and MseChi2 genes were down-regulated by 76 and 45 %, respectively, and sequence-specific siRNAs were detected in recipient insects. RNAi-based silencing of chitinase genes also led to body weight decreases by 43 %.

Conclusion

Our research demonstrates target mRNA knockdown and suggests a promising application for controlling of M. separata by in planta expression of RNAi effectors using a recombinant plant virus.

     

Effect of 1-aminocyclopropane-1-carboxylic acid (ACC)-induced ethylene on <Emphasis Type="Italic">cellulose synthase A</Emphasis> (<Emphasis Type="Italic">CesA</Emphasis>) genes in flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L. ‘Nike’) seedlings

Introduction

Cellulose microfibril is a major cell wall polymer that plays an important role in the growth and development of plants. The gene cellulose synthase A (CesA), encoding cellulose synthases, is involved in the synthesis of cellulose microfibrils. However, the regulatory mechanism of CesA gene expression is not well understood, especially during the early developmental stages.

Objective

To identify factor(s) that regulate the expression of CesA genes and ultimately control seedling growth and development.

Methods

The presence of cis-elements in the promoter region of the eight CesA genes identified in flax (Linum usitatissimum L. ‘Nike’) seedlings was verified, and three kinds of ethylene-responsive cis-elements were identified in the promoters. Therefore, the effect of ethylene on the expression of four selected CesA genes classified into Clades 1 and 6 after treatment with 10^?4 and 10^?3 M 1-aminocyclopropane-1-carboxylic acid (ACC) was examined in the hypocotyl of 4–6-day-old flax seedlings.

Results

ACC-induced ethylene either up- or down-regulated the expression of the CesA genes depending on the clade to which these genes belonged, age of seedlings, part of the hypocotyl, and concentration of ACC.

Conclusion

Ethylene might be one of the factors regulating the expression of CesA genes in flax seedlings.

     

A study of candidate genes for day blindness in the standard wire haired dachshund

Background

A genetic study was performed to identify candidate genes associated with day blindness in the standard wire haired dachshund. Based on a literature review of diseases in dogs and human with phenotypes similar to day blindness, ten genes were selected and evaluated as potential candidate genes associated with day blindness in the breed.

Results

Three of the genes, CNGB3, CNGA3 and GNAT2, involved in cone degeneration and seven genes and loci, ABCA4, RDH5, CORD8, CORD9, RPGRIP1, GUCY2D and CRX, reported to be involved in cone-rod dystrophies were studied. Polymorphic markers at each of the candidate loci were studied in a family with 36 informative offspring. The study revealed a high frequency of recombinations between the candidate marker alleles and the disease.

Conclusion

Since all of the markers were at the exact position of the candidate loci, and several recombinations were detected for each of the loci, all ten genes were excluded as causal for this canine, early onset cone-rod dystrophy. The described markers may, however, be useful to screen other canine resource families segregating eye diseases for association to the ten genes.

     

Adherent/invasive <Emphasis Type="Italic">Escherichia coli</Emphasis> (AIEC) isolates from asymptomatic people: new <Emphasis Type="Italic">E. coli</Emphasis> ST131 O25:H4/H30-Rx virotypes

Background

The widespread Escherichia coli clone ST131 implicated in multidrug-resistant infections has been recently reported, the majority belonging to O25:H4 serotype and classified into five main virotypes in accordance with the virulence genes carried.

Methods

Pathogenicity Islands I and II (PAI-I and PAI-II) were determined using conventional PCR protocols from a set of four E. coli CTX^R ST131 O25:H4/H30-Rx strains collected from healthy donors’ stool. The virulence genes patterns were also analyzed and compared them with the virotypes reported previously; then adherence, invasion, macrophage survival and biofilm formation assays were evaluated and AIEC pathotype genetic determinants were investigated.

Findings

Non-reported virulence patterns were found in our isolates, two of them carried satA, papA, papGII genes and the two-remaining isolates carried cnfI, iroN, satA, papA, papGII genes, and none of them belonged to classical ST131 virotypes, suggesting an endemic distribution of virulence genes and two new virotypes. The presence of PAI-I and PAI-II of Uropathogenic E. coli was determined in three of the four strains, furthermore adherence and invasion assays demonstrated higher degrees of attachment/invasion compared with the control strains. We also amplified intI1, insA and insB genes in all four samples.

Interpretation

The results indicate that these strains own non-reported virotypes suggesting endemic distribution of virulence genes, our four strains also belong to an AIEC pathotype, being this the first report of AIEC in México and the association of AIEC with healthy donors.

     

Heterologous expression of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> serotype 1 O-antigen analog in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 W3110 by transferring a minimum number of genes involved in O-polysaccharide biosynthesis

Objective

To heterologously produce the Shigella dysenteriae serotype 1 O-polysaccharide (O-PS, O-antigen) in Escherichia coli by transferring the minimum number of genes instead of the entire O-PS gene cluster.

Results

The three glycosyltransferase genes (rfbR, rfbQ and rfp) responsible for the formation of the O-repeat unit were introduced into E. coli K-12 W3110 to synthesize S. dysenteriae 1 O-PS. The specific O-antigen ladder type with different chain lengths of O-repeat units was observed in the recombinant E. coli strain by SDS-PAGE silver staining and western blotting using S. dysenteriae 1 lipopolysaccharide antiserum. Analysis by mass spectrometry and ion chromatography suggested generation of the specific S. dysenteriae 1 O-repeat unit structure with an extra glucose residue attached.

Conclusions

Recombinant E. coli expressing specific glycosyltransferase genes can generate the O-PS of S. dysenteriae 1 and might be able to synthesize heterologous O-antigens of various pathogenic bacteria for vaccine preparation.

     

A versatile <Emphasis Type="Italic">Trichoderma reesei</Emphasis> expression system for the production of heterologous proteins

Objectives

To develop a versatile Trichoderma reesei (teleomorph Hypocrea jecorina) expression system for the high-purity production of heterologous proteins.

Results

The versatile T. reesei expression system is based on xyn1 and xyn2 promoters, A824V transition in XYRI, and a bicomponent carbon source strategy. Red fluorescent protein gene rfp and alkaline endoglucanase EGV gene egv3 from Humicola insolens were used as reporter genes to test our versatile expression system

Conclusions

The versatile T. reesei expression system can be applied to produce heterologous proteins with high purity and high yield.

     

<Emphasis Type="Italic">PiggyBac</Emphasis> transposon-mediated mutagenesis and application in yeast <Emphasis Type="Italic">Komagataella phaffii</Emphasis>

Objective

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.

Results

In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.

Conclusions

Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

     

Functional analysis of alcohol dehydrogenase (<Emphasis Type="Italic">ADH</Emphasis>) genes in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To characterize the genes responsible for ethanol utilization in Pichia pastoris.

Results

ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism.

Conclusion

The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.

     

Production of caffeoylmalic acid from glucose in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli.

Results

We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli–E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L.

Conclusions

Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.

     

Analysis of the conservation of synteny between Fugu and human chromosome 12

Background

The pufferfish Fugu rubripes (Fugu) with its compact genome is increasingly recognized as an important vertebrate model for comparative genomic studies. In particular, large regions of conserved synteny between human and Fugu genomes indicate its utility to identify disease-causing genes. The human chromosome 12p12 is frequently deleted in various hematological malignancies and solid tumors, but the actual tumor suppressor gene remains unidentified.

Results

We investigated approximately 200 kb of the genomic region surrounding the ETV6 locus in Fugu (fETV6) in order to find conserved functional features, such as genes or regulatory regions, that could give insight into the nature of the genes targeted by deletions in human cancer cells. Seven genes were identified near the fETV6 locus. We found that the synteny with human chromosome 12 was conserved, but extensive genomic rearrangements occurred between the Fugu and human ETV6 loci.

Conclusion

This comparative analysis led to the identification of previously uncharacterized genes in the human genome and some potentially important regulatory sequences as well. This is a good indication that the analysis of the compact Fugu genome will be valuable to identify functional features that have been conserved throughout the evolution of vertebrates.

     

Time-course microarray analysis for identifying candidate genes involved in obesity-associated pathological changes in the mouse colon

Background

Obesity is known to increase the risk of colorectal cancer. However, mechanisms underlying the pathogenesis of obesity-induced colorectal cancer are not completely understood. The purposes of this study were to identify differentially expressed genes in the colon of mice with diet-induced obesity and to select candidate genes as early markers of obesity-associated abnormal cell growth in the colon.

Methods

C57BL/6N mice were fed normal diet (11% fat energy) or high-fat diet (40% fat energy) and were euthanized at different time points. Genome-wide expression profiles of the colon were determined at 2, 4, 8, and 12 weeks. Cluster analysis was performed using expression data of genes showing log₂ fold change of ≥1 or ≤?1 (twofold change), based on time-dependent expression patterns, followed by virtual network analysis.

Results

High-fat diet-fed mice showed significant increase in body weight and total visceral fat weight over 12 weeks. Time-course microarray analysis showed that 50, 47, 36, and 411 genes were differentially expressed at 2, 4, 8, and 12 weeks, respectively. Ten cluster profiles representing distinguishable patterns of genes differentially expressed over time were determined. Cluster 4, which consisted of genes showing the most significant alterations in expression in response to high-fat diet over 12 weeks, included Apoa4 (apolipoprotein A-IV), Ppap2b (phosphatidic acid phosphatase type 2B), Cel (carboxyl ester lipase), and Clps (colipase, pancreatic), which interacted strongly with surrounding genes associated with colorectal cancer or obesity.

Conclusions

Our data indicate that Apoa4, Ppap2b, Cel, and Clps are candidate early marker genes associated with obesity-related pathological changes in the colon. Genome-wide analyses performed in the present study provide new insights on selecting novel genes that may be associated with the development of diseases of the colon.

     

New eight genes identified at the clinical multidrug-resistant <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> DMS06669 strain in a Vietnam hospital

Background

Acinetobacter baumannii is an important nosocomial pathogen that can develop multidrug resistance. In this study, we characterized the genome of the A. baumannii strain DMS06669 (isolated from the sputum of a male patient with hospital-acquired pneumonia) and focused on identification of genes relevant to antibiotic resistance.

Methods

Whole genome analysis of A. baumannii DMS06669 from hospital-acquired pneumonia patients included de novo assembly; gene prediction; functional annotation to public databases; phylogenetics tree construction and antibiotics genes identification.

Results

After sequencing the A. baumannii DMS06669 genome and performing quality control, de novo genome assembly was carried out, producing 24 scaffolds. Public databases were used for gene prediction and functional annotation to construct a phylogenetic tree of the DMS06669 strain with 21 other A. baumannii strains. A total of 18 possible antibiotic resistance genes, conferring resistance to eight distinct classes of antibiotics, were identified. Eight of these genes have not previously been reported to occur in A. baumannii.

Conclusions

Our results provide important information regarding mechanisms that may contribute to antibiotic resistance in the DMS06669 strain, and have implications for treatment of patients infected with A. baumannii.