A new insertion sequence for incremental Delaunay triangulation

Incremental algorithm is one of the most popular procedures for constructing Delaunay triangulations （DTs）. However, the point insertion sequence has a great impact on the amount of work needed for the construction of DTs. It affects the time for both point location and structure update, and hence the overall computational time of the triangulation algorithm. In this paper, a simple deterministic insertion sequence is proposed based on the breadth-first-search on a Kd-tree with some minor modifications for better performance. Using parent nodes as search-hints, the proposed insertion sequence proves to be faster and more stable than the Hilbert curve order and biased randomized insertion order （BRIO）, especially for non-uniform point distributions over a wide range of benchmark examples.

Extended role for insertion sequence elements in the antibiotic resistance of Bacteroides

The Bacteroides species are important micro-organisms, both in the normal physiology of the intestines and as frequent opportunistic anaerobic pathogens, with a deeply-rooted phylogenetic origin endowing them with some interesting biological features. Their prevalence in anaerobic clinical specimens is around 60%-80%, and they display the most numerous and highest rates of antibiotic resistance among all pathogenic anaerobes. In these antibiotic resistance mechanisms there is a noteworthy role for the insertion sequence(IS) elements, which are usually regarded as representatives of ‘selfish' genes; the IS elements of Bacteroides are usually capable of up-regulating the antibiotic resistance genes. These include the cep A(penicillin and cephalosporin), cfx A(cephamycin), cfi A(carbapenem), nim(metronidazole) and erm F(clindamycin) resistance genes. This is achieved by outwardoriented promoter sequences on the ISs. Although some representatives are well characterized, e.g., the resistance gene-IS element pairs in certain resistant strains, open questions remain in this field concerning a better understanding of the molecular biology of theantibiotic resistance mechanisms of Bacteroides, which will have clinical implications.

Insertion sequence transposition activates antimycobacteriophage immunity through an Isr2-silenced lipid metabolism gene island

Insertion sequences(ISs)exist widely in bacterial genomes,but their roles in the evolution of bacterial antiphage defense remain to be clarified.Here,we report that,under the pressure of phage infection,the IS1o96 transposition of Mycobacterium smegmatis into the Isr2 gene can occur at high frequencies,which endows the mutant mycobacterium with a broad-spectrum antiphage ability.Lsr2 functions as a negative regulator and directly silences expression of a gene island composed of 11 lipid metabolism-related genes.The complete or partial loss of the gene island leads to a significant decrease of bacteriophage adsorption to the mycobacterium,thus defending against phage infection.Strikingly,a phage that has evolved mutations in two tail-filament genes can re-escape from the Isr2 inactivation-triggered host defense.This study uncovered a new signaling pathway for activating antimycobacteriophage immunity by Is transposition and provided insight into the natural evolution of bacterial antiphage defense.

Increment of hFIX expression with endogenous intron 1 in vitro

This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i’IX, retroviral vector GINaCi’IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKG5i’IX, 151 "g/106 cells/24h; PA317/GINaCi’IX, 308ng/106 cells/24 h; C2C12/G1 NaCi’IX, 186 ng/106 cells/24 h; RSF/GINaCi’IX, 1929 ng/106 cells/24 h; HSF/GlNaCi’IX, 1646 ng/106 cells/ 24 h. These results indicated that hFIX minigene with nitron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from nitron splicing during viral production, a retroviral vector GlNaCi’IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/106 cells/24 h with 79% of bioactivity. PCR detection of HT/GlNaCi’IXR cells infected with PA317/ClNaCi’IXR supernatant confirmed the existence of nitron 1 sequence. These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene Human factor IX expression with nitron transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.

Understanding the rapid spread of antimicrobial resistance genes mediated by IS26

Insertion sequences(ISs)promote the transmission of antimicrobial resistance genes(ARGs)across bacterial populations.However,their contributions and dynamics during the transmission of resistance remain unclear.In this study,we selected is26 as a representative transposable element to decipher the relationship between ISs and ARGs and to investigate their transfer features and transmission trends.We retrieved 2656 translocatable IS26-bounded units with ARGs(tiS26-bUs-ARGs)in complete bacterial genomes from the NCBI RefSeq database.In total,124 ARGs spanning 12 classes of antibiotics were detected,and the average contribution rate of IS26 to these genes was 41.2%.We found that IS26-bounded units(IS26-bUs)mediated extensive ARG dissemination within the bacteria of the Gammaproteobacteria class,showing strong transfer po-tential between strains,species,and even phyla.The Is26-bUs expanded in bacterial populations over time,and their temporal expansion trend was significantly correlated with antibiotic usage.This wide dissemination could be due to the nonspecific target site preference of is26.Finally,we experimentally confirmed that the introduction of a single copy of is26 could lead to the formation of a composite transposon mediating the transmission of“passenger”genes.These observations extend our knowledge of the IS26 and provide new insights into the mediating role of ISs in the dissemination of antibiotic resistance.

Cell division factor ZapE regulates Pseudomonas aeruginosa biofilm formation by impacting the pqs quorum sensing system

Pseudomonas aeruginosa is one of the leading nosocomial pathogens that causes both severe acute and chronic infections.The strong capacity of P.aeruginosa to form biofilms can dramatically increase its antibiotic resistance and lead to treatment failure.The biofilm resident bacterial cells display distinct gene expression profiles and phenotypes compared to their free-living counterparts.Elucidating the genetic determinants of biofilm formation is crucial for the development of antibiofilm drugs.In this study,a highthroughput transposon-insertion site sequencing(Tn-seq)approach was employed to identify novel P.aeruginosa biofilm genetic determinants.When analyzing the novel biofilm regulatory genes,we found that the cell division factor ZapE(PA4438)controls the P.aeruginosa pqs quorum sensing system.The ΔzapE mutant lost fitness against the wild-type PAO1 strain in biofilms and its production of 2-heptyl-3-hydroxy-4(1H)-quinolone(PQS)had been reduced.Further biochemical analysis showed that ZapE interacts with PqsH,which encodes the synthase that converts 2-heptyl-4-quinolone(HHQ)to PQS.In addition,site-directed mutagenesis of the ATPase active site of ZapE(K72A)abolished the positive regulation of ZapE on PQS signaling.As ZapE is highly conserved among the Pseudomonas group,our study suggests that it is a potential drug target for the control of Pseudomonas infections.

Genetic environment of β-lactamase genes of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates from patients with lower respiratory tract infection in China

Background Extended-spectrum β-lactamase （ESBL）-producing Klebsiella pneumoniae （K.pneumoniae） is one of the most popular pathogens that cause refractory respiratory tract infection.The genetic environment,including insertion sequences and the types of promoter,plays a key role in exploration of the mechanism of prevalence and dismission of the ESBL-producing K.pneumoniae isolates.The aim of the investigation was to target analysis the genetic environment and promoter sequences of blaCTX-M,blaSHV and blaTEM,the most popular β-lactamase genes harbored by ESBL-producing K.pneumoniae isolates.Methods From February 2010 to July 2011,158 of 416 K.pneumoniae isolates producing ESBL from patients with lower respiratory tract infection were collected from seven tertiary hospitals from Beijing,Anhui,Fujian,Liaoning,Hebei and Inner Mongolia Autonomous Region in China.The genetic environment including promoters of 10 types of blaCTX-M,18 types of blaSHVand 2 types of blaTEM were analyzed by amplification and direct sequencing with various sets of PCR primers.Results ISEcp1 was located upstream of the 5＇ end of the blaCTX-M gene in 130 （97.0％） out of 134 K.pneumoniae isolates harboring blaCTX-M and provided a conserved promoter to blaCTX-M.A non-coding sequence preceded by kdpC and recF was identified in all of the blaSHV genes except blaSHV-12 and blaSHV-2a.IS26 was also found upstream of 1 blaCTX-M-15,10 blaSHV-1 strains,4 blaTEM-1 and all of the blaSHV-2,blaSHV-2a,blaSHV-5 and blaSHV-12.Eighty-seven of 91 strains harboring blaTEM-1 carried a copy of Tn2 and IS26-blaTEM-1 fragments were also detected in 4 strains.With respect to K.pneumoniae,the genetic environment of blaCTX-M-38,blaSHV-142 and blaTEM-135 were firstly elaborated,and four kinds of novel genetic environment of blaCTX-M-3,blaCTX-M-15 and blaTEM-1 have been detected as well.Conclusions Perfective implementation of the genetic environment information of β-lactamase gene needs to be further explored and supplemented.ISEcp1 and IS26 elements are widespread upstream of the blaCTX-M,blaSHV and blaTEM genes and contribute to horizontal transmission and genetic expression.

Pooled Plasmid Sequencing Reveals the Relationship Between Mobile Genetic Elements and Antimicrobial Resistance Genes in Clinically Isolated Klebsiella pneumoniae

Plasmids remain important microbial components mediating the horizontal gene transfer(HGT)and dissemination of antimicrobial resistance.To systematically explore the relationship between mobile genetic elements(MGEs)and antimicrobial resistance genes(ARGs),a novel strategy using single-molecule real-time(SMRT)sequencing was developed.This approach was applied to pooled conjugative plasmids from clinically isolated multidrug-resistant(MDR)Klebsiella pneumoniae from a tertiary referral hospital over a 9-month period.The conjugative plasmid pool was obtained from transconjugants that acquired antimicrobial resistance after plasmid conjugation with 53 clinical isolates.The plasmid pool was then subjected to SMRT sequencing,and 82 assembled plasmid fragments were obtained.In total,124 ARGs(responsible for resistance to b-lactam,fluoroquinolone,and aminoglycoside,among others)and 317 MGEs[including transposons(Tns),insertion sequences(ISs),and integrons]were derived from these fragments.Most of these ARGs were linked to MGEs,allowing for the establishment of a relationship network between MGEs and/or ARGs that can be used to describe the dissemination of resistance by mobile elements.Key elements involved in resistance transposition were identified,including IS26,Tn3,IS903 B,ISEcp1,and ISKpn19.As the most predominant IS in the network,a typical IS26-mediated multi-copy composite transposition event was illustrated by tracing its flanking 8-bp target site duplications(TSDs).The landscape of the pooled plasmid sequences highlights the diversity and complexity of the relationship between MGEs and ARGs,underpinning the clinical value of dominant HGT profiles.