ST Sequence Research Articles

Emergence and dissemination of three mild outbreaks of Neisseria gonorrhoeae with high-level resistance to azithromycin in Barcelona, 2016-18.

Neisseria gonorrhoeae (NG) isolates with high-level azithromycin resistance (HL-AziR) have emerged worldwide in recent decades, threatening the sustainability of current dual-antimicrobial therapy. This study aimed to characterize the first 16 NG isolates with HL-AziR in Barcelona between 2016 and 2018. WGS was used to identify the mechanisms of antimicrobial resistance, to establish the MLST ST, NG multiantigen sequence typing (NG-MAST) ST and NG sequence typing for antimicrobial resistance (NG-STAR) ST and to identify the clonal relatedness of the isolates with other closely related NG previously described in other countries based on a whole-genome SNP analysis approach. The sociodemographic characteristics of the patients included in the study were collected by comprehensive review of their medical records. Twelve out of 16 HL-AziR isolates belonged to the MLST ST7823/NG-MAST ST5309 genotype and 4 to MLST ST9363/NG-MAST ST3935. All presented the A2059G mutation in all four alleles of the 23S rRNA gene. MLST ST7823/NG-MAST ST5309 isolates were only identified in men who have sex with women and MLST ST9363/NG-MAST ST3935 were found in MSM. Phylogenomic analysis revealed the presence of three transmission clusters of three different NG strains independently associated with sexual behaviour. Our findings support the first appearance of three mild outbreaks of NG with HL-AziR in Spain. These results highlight the continuous capacity of NG to develop antimicrobial resistance and spread among sexual networks. The enhanced resolution of WGS provides valuable information for outbreak investigation, complementing the implementation of public health measures focused on the prevention and dissemination of MDR NG.

ST Sequence Miner: visualization and mining of spatio-temporal event sequences

As a promising field of research, event sequence analysis seems to assist in facilitating clear reasoning behind human decisions by mining reality behind the sequential actions. Mining frequent patterns from event sequences has proved to be promising in extracting actionable insights, which plays an important role in many application domains. Much of the related work challenges the problem solely from the temporal perspective omitting the information that could be gained from the spatial part. This could be in part due to the fact that analysis of event sequences with references to both time and space is attributed as a challenging task due to the additional variance in the data introduced by the spatial aspect. We propose a visual analytics approach that incorporates spatio-temporal pattern extraction leveraging an extended sequential pattern mining algorithm and a pattern discovery guidance mechanism operating on geographic query and selection capabilities. As an implementation of our approach, we introduce a visual analytics tool, namely ST Sequence Miner, enabling event pattern exploration in time-location space. We evaluate our approach over a credit card transaction dataset by adopting case study methodology. Our study unveils that patterns mined from event sequences can better explain possible relationships with proper visualization of time-location data.

Immunizations with Enterotoxigenic Escherichia coli Heat-Stable Toxin Conjugates Engender Toxin-Neutralizing Antibodies in Mice That Also Cross-React with Guanylin and Uroguanylin.

Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.

Different Contexts in the Oddball Paradigm Induce Distinct Brain Networks in Generating the P300.

Despite the P300 event-related potential (ERP) differences between distinct stimulus sequences, the effect of stimulus sequence on the brain network is still left unveiled. To uncover the corresponding effect of stimulus sequence, we thus investigated the differences of functional brain networks, when a target (T) or standard (S) stimulus was presented preceding another T as background context. Results of this study demonstrated that, when an S was first presented preceding a T (i.e., ST sequence), the P300 experiencing large amplitude was evoked by the T, along with strong network architecture. In contrast, if a T was presented in advance [i.e., target-to-target (TT) sequence], decreased P300 amplitude and attenuated network efficiency were demonstrated. Additionally, decreased activations in regions, such as inferior frontal gyrus and superior frontal gyrus were also revealed in TT sequence. Particularly, the effect of stimulus sequence on P300 network could be quantitatively measured by brain network properties, the increase in network efficiency corresponded to large P300 amplitude evoked in P300 task.

Characteristics and drug resistance of non-O157 Shiga toxin-producing E. coli in animal feces, from Shandong Province

Objective: To understand the infection status, characteristics and drug resistance of non-O157 Shiga toxin-producing E. coli (STEC) in animal feces in Shandong Province. Methods: From 2015 to 2016, convient sampling method was used to collect 1 022 fresh feces of animals in Weishan county and Laizhou city, and 24 non-O157 STEC were isolated. The serotypes of non-O157 STEC strains were confirmed through serum agglutination test. The susceptibility was explored through the antimicrobial sensitivity experiments. ESBLs activity was confirmed by double-disc diffusion. PCR method was used to detect the resistance genes. PFGE typing was operated to assess the relatedness and variability of the strains. The multi-locus sequence typing (MLST) was adopted to get the allelic profile and ST sequence of strains. Analysis was made on the evolutionary relationship between different ST groups was made through CLC Sequence Viewer and Counting Express. Results: A total of 24 non-O157 STEC were isolated from animal feces. 23 strains were from pig feces, and 1 strain was from cow feces, and the serotypes were more dispersed. All of the 24 strains carried stx2 genes. The highest resistance rate was sulfamethoxazole(22 strains), the mount of cotrimoxazole and nalidixic acid was 18 strains, chloramphenicol was 13 strains, tetracycline was 19, and there was a phenomenon of multiple drug resistance. The drug resistance spectrum was sulfamethoxazole tetracycline-compound novammin-naphthidine-chloramphenicol. All strains were sensitive to cefepime and imipenem. The ESBLs confirmatory test showed that 4 strains of non O157 STEC produced beta lactamase. PCR detected 7 resistance genes, and 4 tetracycline resistance genes (Tet A, Tet B, tetC and tetD) were detected. The beta lactamase resistance genes (blaSHV-1, bla CTX-M, bla TEM) were all negative. 24 strains were divided into 15 PFGE types, and their clustering results were more dispersed and no dominant PFGE type. There were 11 kinds of MLST types, most of them are ST540 and ST5133 types, each of which was 4 strains, and clustered into 1 MLST genomes. Conclusion: The serotypes of non-O157 STEC in animal feces O157 STEC were dispersed, and the resistant rate to common antibiotic was high. MLST typing results presents obvious polymorphism. Surveillance and manage ment of these strains should be strengthened.

Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSA sT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult Ubc CreERT2; ROSA sT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult Ubc CreERT2; ROSA sT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1 CreERT2/+; ROSA sT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1 CreERT2/+; ROSA sT and Atoh1 CreERT2/+; ROSA sT; p53f lox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

A Novel B-Cell Epitope Identified within Mycobacterium tuberculosis CFP10/ESAT-6 Protein

BackgroundThe 10-kDa culture filtrate protein (CFP10) and 6-kDa early-secreted target antigen (ESAT-6) play important roles in mycobacterial virulence and pathogenesis through a 1∶1 complex formation (CFP10/ESAT-6 protein, CE protein), which have been used in discriminating TB patients from BCG-vaccinated individuals. The B-cell epitopes of CFP10 and ESAT-6 separately have been analyzed before, however, the epitopes of the CE protein are unclear and the precise epitope in the positions 40 to 62 of ESAT-6 is still unknown.MethodsIn the present study, we searched for the B-cell epitopes of CE protein by using phage-display library biopanning with the anti-CE polyclonal antibodies. The epitopes were identified by sequence alignment, binding affinity and specificity detection, generation of polyclonal mouse sera and detection of TB patient sera.ResultsOne linear B-cell epitope (KWDAT) consistent with the 162nd–166th sequence of CE and the 57th–61st sequence of ESAT-6 protein was selected and identified. Significantly higher titers of E5 peptide-binding antibodies were found in the sera of TB patients compared with those of healthy individuals.ConclusionThere was a B-cell epitope for CE and ESAT-6 protein in the position 40 to 62 of ESAT-6. E5 peptide may be useful in the serodiagnosis of tuberculosis, which need to be further confirmed by more sera samples.

Multiple origins of allopolyploid wheatgrass Elymus caninus revealed by RPB2, PepC and TrnD/T genes

We examined evolutionary mechanisms in the tetraploid Elymus caninus by comparing the phylogenetic relationships of 21 accessions suggested by sequence data from two single copy nuclear genes, the largest subunit of RNA polymerase II (RPB2) and phosphoenolpyruvate carboxylase (pepC), and one non-coding chloroplast region, TrnD/T. Elymus caninus is known combining two different genomes, an St genome and an H genome. Data from two single copy nuclear genes showed that there are two versions of the St genome in the species, St1 and St2. Most accessions combined one of these versions with an H genome version but two accessions had both versions of the St sequence for RPB2. This suggests that the RPB2gene may have been duplicated without chromosome doubling, possibly induced by transposable element. Our data also indicate that the H genome sequences in E. caninus have multiple origins, and a close phylogenetic relationship between Hordeum bogdanii and H sequences in some accessions of E. caninus. Thus, it is more likely that H. bogdanii is one of the major donors of the H copy in E. caninus. The maternal origin of E. caninus is the St genome species. There was no correlation between the geographic origin of the accessions and their sequence divergence.

PHOTODEGRADATION OF St-MMA COPOLYMERS: CORRELATION OF DEGRADATION, STYRENE CONTENT, AND PHOTOPHYSICS

A series of styrene(St)-methyl methacrylate(MMA) copolymers with compositions ranging from 0 to 100% St were synthesized and exposed to UV light for various periods of time. The changes in yellowing (yellowness index—YI) and absorbance of exposed films were evaluated as a function of photolysis time and St content. Both the changes in YI and absorbance at 400 nm were found to increase dramatically for polymer films with styrene (St) content greater than about 50%. A direct correlation between increases in the YI and absorbance at 400 nm with mean styrene sequence length was found with a maximum change being obtained for copolymers with mean sequence length of approximately 5 St units or greater. A small absorbance shoulder at 270–290 nm was observed for copolymers with St content less than 30% after relatively short irradiation times (e. g. 2 hours). Excimer formation of copolymers in dilute solution was found to increase with an increase in St content and St sequence length; in films, a plateau in the relative excimer emission was attained with a St content of 60%, corresponding to a mean St sequence length of 2.

Antibody response against Escherichia coli heat-stable enterotoxin expressed as fusions to flagellin.

The heat-stable toxin (ST) produced by enterotoxigenic Escherichia coli strains causes diarrhoea by altering the fluid secretion in intestinal epithelial cells. Here, the effectiveness of a flagellin fusion protein of Salmonella containing a 19-amino-acid sequence derived from the ST sequence (FLA--ST) in generating antibodies capable of neutralizing the toxic activity of ST was evaluated. This fusion protein, and an alternative construction where two cysteine residues in the ST sequence were substituted by alanines (ST(mt)), were delivered to the immune system by three distinct strategies: (i) orally, using an attenuated Salmonella strain expressing FLA--ST; (ii) intraperitoneally, by injection of purified FLA--ST; (iii) orally, using attenuated Salmonella carrying a eukaryotic expression plasmid (pCDNA3) with the gene encoding FLA-ST. The results showed that the flagellin system can be used as a carrier to generate ST-neutralizing antibodies. However, it should be mentioned that humoral immune response against ST was only obtained when the mutated ST sequence was employed. FLA-ST was found to be non-immunogenic when delivered via the oral route with attenuated Salmonella strains. However, a flagellin antibody response was obtained by immunizing mice with Salmonella carrying pCDNA3/FLA-ST(mt). Oral immunization with Salmonella carrying the eukaryotic expression plasmid (pCDNA3/FLA--ST(mt)) seems to be a promising method to elicit an appropriate response against fusions to flagellin.

The thiol oxidoreductase ERp57 is a component of the MHC class I peptide-loading complex.

The proper folding and assembly of major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER) is an intricate process involving a number of components. Nascent heavy chains of MHC class I molecules, translocated into the ER membrane, are rapidly glycosylated and bind the transmembrane chaperone calnexin. In humans, after dissociation from calnexin, fully oxidized MHC class I heavy chains associate with beta 2-microglobulin (beta 2m) and the soluble chaperone calreticulin. This complex interacts with another transmembrane protein, tapasin, which is believed to assist in MHC class I folding as well as in mediating the interaction between assembling MHC class I molecules and the transporter associated with antigen processing (TAP). The TAP heterodimer (TAP1-TAP2) introduces the final component of the MHC class I molecule by translocating peptides, predominately generated by the proteasome, from the cytosol into the ER where they can bind dimers of beta 2M and the MHC class I heavy chain. Recently, the thiol oxidoreductase ERp57--also known as GRP58, ERp61, ER60, Q2, HIP-70, and CPT and first misidentified as phospholipase C-alpha--has been shown to bind in conjunction with calnexin or calreticulin to a number of newly synthesized ER glycoproteins when their N-linked glycans are trimmed by glucosidases I and II. It was speculated that ERp57 is a generic component of the glycan-dependent ER quality control system. Here, we show that ERp57 is a component of the MHC class I peptide-loading complex. ERp57 might influence the folding of MHC class I molecules at a critical step in peptide loading.

Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis

A competitive nested PCR-temperature gradient gel electrophoresis protocol (nPCR/TGGE) has been established for the quantification of human cytomegalovirus (HCMV) target sequences. The measurement was achieved by co-amplification of a defined copy number of an internal standard (st) and separation of st and wild-type (wt) amplimers by temperature gradient gel electrophoresis (TGGE). The number of HCMV target sequences could be precisely determined within wt/st ratios of 0.1 to 10. With 50 copies of the st sequence the detection limit of nPCR/TGGE was found to be five to 10 copies of the target sequence. Effects of sample preparation on quantitative HCMV PCR were minimized by the additional quantification of beta-globin target sequences and calculation of the ratio of HCMV copies/beta-globin copies. Serial peripheral blood leukocyte specimens of 17 renal allograft recipients positive in a qualitative nested HCMV PCR were tested using nPCR/TGGE. Thirty healthy blood donors served as negative controls. Positive results were obtained by nPCR/TGGE in nine renal allograft recipients but in none of the healthy blood donors. Five of five patients with an HCMV pp65 antigenaemia and positive for HCMV IgM were positive in nPCR/TGGE. The highest HCMV/beta-globin ratios (10,000 to 8000 copies HCMV/10(6) copies beta-globin) were found in transplant recipients experiencing acute clinically symptomatic HCMV infection. HCMV DNA levels in asymptomatic patients ranged from 900 to 200 copies HCMV/10(6) beta-globin.