Intergenic Sequences Research Articles

The \u03c354 system directly regulates bacterial natural product genes

Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutics and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery controlling their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explored this idea by analyzing a selected group of putative σ54 promoters identified in Myxococcus xanthus natural product gene clusters. We show that mutations in putative σ54-RNA polymerase binding regions and in putative Nla28 EBP binding sites dramatically reduce in vivo promoter activities in growing and developing cells. We also show in vivo promoter activities are reduced in a nla28 mutant, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the Nla28 binding sites are mutated. Together, our results indicate that M. xanthus uses σ54 promoters for transcription of at least some of its natural product genes. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product loci are located within genes and not in intergenic sequences.

Insights into the evolutionary history of the virulent factor HBHA of Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, heparin-binding hemagglutinin (HBHAMT) has a relevant role in infection. It is also present in non-virulent mycobacteria and ancient actinobacteria, such as Rhodococcus opacus. To have a better understanding of the underlying mechanisms that shaped the evolutionary divergence of these proteins, we performed a comprehensive phylogenetic analysis of the regulatory sequences that drive the expression of hbha in saprophytic and pathogenic mycobacterial species. The alignment of the hbha loci showed the appearance of intergenic sequences containing regulatory elements upstream the hbha gene; this sequence arrangement is present only in slow-growing pathogenic mycobacteria. The heterologous expression of HBHAMT in oleaginous R. opacus PD630 results in protein binding to lipid droplets, as it happens with HBHA proteins from saprophytic mycobacteria. We hypothesize that mycobacterial hbha gene cluster underwent functional divergence during the evolutionary differentiation of slow-growing pathogenic mycobacteria. We propose here an evolutionary scenario to explain the structural and functional divergence of HBHA in fast and slow-growing mycobacteria.

Comparative Mitogenomic Analysis Reveals Dynamics of Intron Within and Between Tricholoma Species and Phylogeny of Basidiomycota.

The genus of Tricholoma is a group of important ectomycorrhizal fungi. The overlapping of morphological characteristics often leads to the confusion of Tricholoma species classification. In this study, the mitogenomes of five Tricholoma species were sequenced based on the next-generation sequencing technology, including T. matsutake SCYJ1, T. bakamatsutake, T. terreum, T. flavovirens, and T. saponaceum. These five mitogenomes were all composed of circular DNA molecules, with sizes ranging from 49,480 to 103,090 bp. Intergenic sequences were considered to be the main factor contributing to size variations of Tricholoma mitogenomes. Comparative mitogenomic analysis showed that the introns of the Agaricales mitogenome experienced frequent loss/gain events. In addition, potential gene transfer was detected between the mitochondrial and nuclear genomes of the five species of Tricholoma. Evolutionary analysis showed that the rps3 gene of the Tricholoma species was under positive selection or relaxed selection in the evolutionary process. In addition, large-scale gene rearrangements were detected between some Tricholoma species. Phylogenetic analysis using the Bayesian inference and maximum likelihood methods based on a combined mitochondrial gene set yielded identical and well-supported tree topologies. This study promoted the understanding of the genetics, evolution, and phylogeny of the Tricholoma genus and related species.

Isolation and Characterization of Fungi strains associated with Mycotoxin Production from Bambara (Vigna subterranea (L) verdc) nuts Recovered in Nigeria

Fungal contamination of food commodities is a global food security challenge that impacts negatively on the health of consumers. Mycotoxins are produced as secondary metabolites by some pathogenic fungi and may contaminate agricultural products while on the field or during harvesting and storage. A field survey was conducted in order to isolate and identify the mycotoxin-producing fungal strains in Bambara nut collected from major markets in eight States in Nigeria including Oyo, Ogun, Lagos, Ekiti, Kwara, Kogi, Abuja, and Nasarawa. Samples per site were pooled, processed and screened for the presence of mycotoxin-producing fungi by culture technique and intergenic spacer sequencing (ITS). Mycotoxin production was determined using thin layer chromatography (TLC) with scanning densitometer. Twenty-four mycotoxin-producing fungal species were isolated from Bambara samples, which were identified as Talaromyces pinophilus, Macrophomina phaseolina (6), Fusarium oxysporum (2), Aspergillus foetidus, Aspergillus flavipes, Rhizopus oryzae (2), Phanerochaete chrysosporium, Aspergillus flavus (2), Aspergillus terreus, Aspergillus awamori, Aspergillus welwitschiae, Rhizopus microsporus, Ceriporia lacerate and Fusarium verticillioides (accession numbers of MN42329- MN4233323) from Bambara nuts. The genera Macrophomina were the most dominant. Some of the identified fungi are noted for the production of mycotoxins and mycotoxins exert toxic effects on animals and humans. The fungi associated with Bambara nut diseases were identified and can be taken as targets in varietal improvement of Bambara nut for resistance to fungal diseases in Nigeria.

Structural Insight Into the SARS-CoV-2 Nucleocapsid Protein C-Terminal Domain Reveals a Novel Recognition Mechanism for Viral Transcriptional Regulatory Sequences.

Coronavirus disease 2019 (COVID-19) has caused massive disruptions to society and the economy, and the transcriptional regulatory mechanisms behind the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are poorly understood. Herein, we determined the crystal structure of the SARS-CoV-2 nucleocapsid protein C-terminal domain (CTD) at a resolution of 2.0 Å, and demonstrated that the CTD has a comparable distinct electrostatic potential surface to equivalent domains of other reported CoVs, suggesting that the CTD has novel roles in viral RNA binding and transcriptional regulation. Further in vitro biochemical assays demonstrated that the viral genomic intergenic transcriptional regulatory sequences (TRSs) interact with the SARS-CoV-2 nucleocapsid protein CTD with a flanking region. The unpaired adeno dinucleotide in the TRS stem-loop structure is a major determining factor for their interactions. Taken together, these results suggested that the nucleocapsid protein CTD is responsible for the discontinuous viral transcription mechanism by recognizing the different patterns of viral TRS during transcription.

Distribution and therapeutic outcomes of intergenic sequence-ALK fusion and coexisting ALK fusions in lung adenocarcinoma patients

IntroductionPatients with ALK rearranged non-small-cell lung cancer (NSCLC) show survival benefits from tyrosine-kinase inhibitor (TKI). Widely application of DNA sequencing revealed various rearrangement pattern in addition to single EML4-ALK fusion. Here, we retrospectively analyzed the distribution and coexistence of ALK rearrangement and therapeutic outcome of patients with ALK rearranged NSCLC. MethodALK positive NSCLC patients were screened at West China Hospital. NGS was performed on pre-treatment samples. Clinical characteristics and therapeutic outcomes were collected to retrospectively analyzed. ResultsAmong the 89 patients with 22 ALK rearrangements, fusions of intergenic sequences with ALK were found in 15 (16.85 %). Non-EML4–ALK fusions were present in 18 patients (20.22 %). Coexistence of rearrangements were present in 16 patients (17.98 %). Intergenic sequence–ALK and non-EML4–ALK fusions occurred at higher rates in patients with at least two fusions (62.5 % versus 6.85 % for intergenic sequence–ALK, 62.5 % versus 10.96 % for non-EML4–ALK). There were 40 ALK-rearranged NSCLC patients receiving the first-line crizotinib. The median progression-free survival (PFS) was 9.7 months when excluding three lost patients. In the seven patients who had at least two fusions, the median PFS was 11.9 months, compared with 9.0 months among those with single (p = 0.336). No significant difference in median PFS was found between patients with and without intergenic-ALK fusion (12.0 months versus 9.6 months, p = 0.989). The median PFS was 9.0 months in patients harboring a single EML4-ALK fusion versus 13.0 months in those with other ALK alterations (P = 0.890). The PFS of patients with single intergenic sequence-ALK fusion reached to 2.9 months, 27 months, and 28.9 months respectively. ConclusionOur study reports the distribution of intergenic sequence–ALK and coexisting fusions in ALK-rearranged NSCLC. Intergenic sequence–ALK and non-EML4-ALK are prone to coexist with other fusions. Neither intergenic sequence–ALK nor coexistence of fusions had a significant effect on the therapeutic benefit of treatment with crizotinib.

First report of Seville root-knot nematode, Meloidogyne hispanica (Nematoda: Meloidogynidae) in the USA and North America.

A high number of second stage juveniles of the root-knot nematode were recovered from soil samples collected from a corn field, located in Pickens County, South Carolina, USA in 2019. Extracted nematodes were examined morphologically and molecularly for species identification which indicated that the specimens of root knot juveniles were Meloidogyne hispanica. The morphological examination and morphometric details from second-stage juveniles were consistent with the original description and redescriptions of this species. The ITS rRNA, D2-D3 expansion segments of 28S rRNA, intergenic COII-16S region, nad5 and COI gene sequences were obtained from the South Carolina population of M. hispanica. Phylogenetic analysis of the intergenic COII-16S region of mtDNA gene sequence alignment using statistical parsimony showed that the South Carolina population clustered with Meloidogyne hispanica from Portugal and Australia. To our best knowledge, this finding represents the first report of Meloidogyne hispanica in the USA and North America.

Fecal Carriage and Molecular Epidemiology of Carbapenem-Resistant Enterobacteriaceae from Inpatient Children in a Pediatric Hospital of Shanghai.

PurposeTo determine the epidemiology characteristics of intestinal colonization of carbapenem-resistant Enterobacteriaceae (CRE) among inpatients in a pediatric hospital in China.MethodsA retrospective study was conducted from April to December 2019. Medical records were reviewed to extract the clinical information. Antimicrobial susceptibility was performed by broth microdilution method. Drug resistance determinants and plasmid types were analyzed using polymerase chain reaction (PCR) assays. Multilocus sequence typing (MLST) and Enterobacterial repetitive intergenic consensus sequences PCR (ERIC-PCR) were employed to determine the genetic relationships between strains.ResultsA total of 90 CRE strains were isolated, with a fecal carriage rate of 8.6% (90/1052), and mainly distributed in E. aerogenes (n=30), K. pneumoniae (n=25) and E. coli (n=23). More than 50% of CRE colonizers had a history of invasive procedures and antibiotic exposures. As high as 91.1% (82/90) of CRE isolates carried carbapenemase genes, with blaNDM-5 (n=56) being the most common, and mainly found in E. aerogenes (51.8%, 29/56) and E. coli (32.1%, 18/56) isolates, which primarily belonged to ST4 (100%, 29/29) and ST692 (55.6%, 10/18), respectively. Followed by blaKPC-2 (n=12), and all found in K. pneumoniae ST11 isolates. Other carbapenemase genes including blaNDM-1, blaIMP-4 and blaIMP-26. Meanwhile, ESBL genes (blaCTX-M, blaTEM-1 and blaSHV) and AmpC genes (blaDHA-1 and blaEBC) were also detected. All CRE isolates showed high resistance to cephalosporins and carbapenemases (97.8%-100.0%) but remained susceptible to tigecycline (98.9%). IncX3 was a major plasmid type in NDM-containing strains (91.3%), and 91.7% of KPC-2-producing K. pneumoniae harboring IncFII and IncFIB plasmids. The ERIC-PCR revealed that several strains with identical STs were genetically similar.ConclusionThis study revealed a major intestinal colonization of ST4 NDM-5 E. aerogenes, ST11 KPC-2 K. pneumoniae and ST692 NDM-5 E. coli strains among inpatients in a pediatric hospital. Infection control measures should be implemented immediately to prevent the spread of these strains in clinical settings.

Histone modifications associated with gene expression and genome accessibility are dynamically enriched at Plasmodium falciparum regulatory sequences

BackgroundThe malaria parasite Plasmodium falciparum has an unusually euchromatic genome with poorly conserved positioning of nucleosomes in intergenic sequences and poorly understood mechanisms of gene regulation. Variant histones and histone modifications determine nucleosome stability and recruit trans factors, but their combinatorial contribution to gene regulation is unclear.ResultsHere, we show that the histone H3 acetylations H3K18ac and H3K27ac and the variant histone Pf H2A.Z are enriched together at regulatory sites upstream of genes. H3K18ac and H3K27ac together dynamically mark regulatory regions of genes expressed during the asexual life cycle. In contrast, H3K4me1 is depleted in intergenic sequence and dynamically depleted upstream of expressed genes. The temporal pattern of H3K27ac and H3K18ac enrichment indicates that they accumulate during S phase and mitosis and are retained at regulatory sequences until at least G1 phase and after cessation of expression of the cognate genes. We integrated our ChIPseq data with existing datasets to show that in schizont stages H3K18ac, H3K27ac and Pf H2A.Z colocalise with the transcription factor PfAP2-I and the bromodomain protein PfBDP1 and are enriched at stably positioned nucleosomes within regions of exposed DNA at active transcriptional start sites. Using transient transfections we showed that sequences enriched with colocalised H3K18ac, H3K27ac and Pf H2A.Z possess promoter activity in schizont stages, but no enhancer-like activity.ConclusionsThe dynamic H3 acetylations define P. falciparum regulatory sequences and contribute to gene activation. These findings expand the knowledge of the chromatin landscape that regulates gene expression in P. falciparum.

Nanopore Sequencing of the Fungal Intergenic Spacer Sequence as a Potential Rapid Diagnostic Assay

Fungal infections are being caused by a broadening spectrum of fungi, yet in many cases, identification to the species level is required for proper antifungal selection. We investigated the fungal intergenic spacer (IGS) sequence in combination with nanopore sequencing for fungal identification. We sequenced isolates from two Cryptococcus species complexes, C. gattii and C. neoformans, which are the main pathogenic members of this genus, using the Oxford Nanopore Technologies MinION device and Sanger sequencing. There is enough variation within the two complexes to argue for further resolution into separate species, which we wanted to see if nanopore sequencing could detect. Using the R9.4.1 flow cell, IGS sequence identities averaged 99.57% compared to Sanger sequences of the same region. When the newer R10.3 flow cell was used, accuracy increased to 99.83% identity compared to the same Sanger sequences. Nanopore sequencing errors were predominantly in regions of homopolymers, with G homopolymers displaying the largest number of errors and C homopolymers displaying the least. Phylogenetic analysis of the nanopore- and Sanger-derived sequences resulted in indistinguishable trees. Comparison of average percent identities between the C. gattii and C. neoformans species complexes resulted in only a 74 to 77% identity between the two complexes. Sequencing using the nanopore platform could be completed in less than an hour, and samples could be multiplexed in groups as large as 24 sequences in a single run. These results suggest that sequencing the IGS region using nanopore sequencing could be a potential new molecular diagnostic strategy.

State-of-the-art genome inference in the human MHC.

The Major Histocompatibility Complex (MHC) on the short arm of chromosome 6 is associated with more diseases than any other region of the genome; it encodes the antigen-presenting Human Leukocyte Antigen (HLA) proteins and is one of the key immunogenetic regions of the genome. Accurate genome inference and interpretation of MHC association signals have traditionally been hampered by the region's uniquely complex features, such as high levels of polymorphism; inter-gene sequence homologies; structural variation; and long-range haplotype structures. Recent algorithmic and technological advances have, however, significantly increased the accessibility of genetic variation in the MHC; these developments include (i) accurate SNP-based HLA type imputation; (ii) genome graph approaches for variation-aware genome inference from next-generation sequencing data; (iii) long-read-based diploid de novo assembly of the MHC; (iv) cost-effective targeted MHC sequencing methods. Applied to hundreds of thousands of samples over the last years, these technologies have already enabled significant biological discoveries, for example in the field of autoimmune disease genetics. Remaining challenges concern the development of integrated methods that leverage haplotype-resolved de novo assembly of the MHC for the development of improved MHC genotyping methods for short reads and the construction of improved reference panels for SNP-based imputation. Improved genome inference in the MHC can crucially contribute to an improved genetic and functional understanding of many immune-related phenotypes and diseases.

First Identification and Characterization of Lactococcus garvieae Isolated from Rainbow Trout (Oncorhynchus mykiss) Cultured in Mexico.

Simple SummaryLactococcus garvieae is an emerging pathogen that causes lactococcosis, which is a septicemia disease that, as per reports, primarily affects rainbow trout and tilapia in various parts of the world. In rainbow trout, the clinical disease is very similar to other diseases caused by Gram-positive bacteria, including weissellosis, which has been previously reported in trout in Mexico. Following septicemia outbreaks that tested negative for weissellosis, a study was conducted to determine the etiology. This study confirms, for the first time, the presence of L. garvieae in processes of a septicemia disease affecting five rainbow trout farms in Mexico. Fourteen isolates were obtained from different geographical regions, but presented homogenous biochemical and protein profiles, as well as weak genetic heterogeneity. This study evidences the wide distribution and incidence of lactococcosis cases at rainbow trout farms in Mexico, demonstrating the need to develop vaccination programs against this disease.Lactococcosis is a hyperacute hemorrhagic septicemia disease caused by Lactococcus garvieae, which is an emerging pathogen in global fish farming. Between 2016 and 2018, rainbow trout (Oncorhynchus mykiss) from five farms that presented outbreaks were sampled as part of a Mexican surveillance program for the detection of fish diseases. Fourteen L. garvieae isolates were recovered from sampled fish, as confirmed by biochemical tests, 16S rRNA gene sequencing, and clinical and histological insights. The biochemical and protein profiles of the isolates obtained were homogeneous. Repetitive extragenic palindromic—(REP)—and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) analyses established weak genetic heterogeneity. Rainbow trout challenged with two of the isolates used at different bacterial concentrations (10−2 and 10−4 CFU/mL) showed melanosis, and hemorrhages were observed in the fins, liver, kidney, and spleen. Isolates were obtained from all of the organs sampled, including from surviving fish, as either pure or mixed cultures. The present study is the first to confirm the presence of L. garvieae as the agent of severe lactococcosis outbreaks in the two primary Mexican states for trout farming.

The 5S rRNA genes in Alexandrium: their use as a FISH chromosomal marker in studies of the diversity, cell cycle and sexuality of dinoflagellates

Chromosomal markers of the diversity and evolution of dinoflagellates are scarce because the genomes of these organisms are unique among eukaryotes in terms of their base composition and chromosomal structure. Similarly, a lack of appropriate tools has hindered studies of the chromosomal localization of 5S ribosomal DNA (rDNA) in the nucleosome-less chromosomes of dinoflagellates. In this study, we isolated and cloned 5S rDNA sequences from various toxin-producing species of the genus Alexandrium and developed a fluorescence in situ hybridization (FISH) probe that allows their chromosomal localization. Our results can be summarized as follows: 1) The 5S rDNA unit is composed of a highly conserved 122-bp coding region and an intergenic spacer (IGS), the length and sequence of which are variable even within strains. 2) Three different IGS types, one containing the U6 small nuclear RNA (snRNA) gene, were found among four of the studied species (A. minutum, A. tamarense, A. catenella and A. pacificum). 3) In all strains investigated by FISH (A. minutum, A. tamarense, A. pacificum, A. catenella, A. andersonii and A. ostenfeldii), 5S rDNA gene arrays were separate from the nucleolar organizer region, which contains the genes for the large 45S pre-ribosomal RNA. 4) One to three 5S rDNA sites per haploid genome were detected, depending on the strains/species. Intraspecific variability in the number of 5S rDNA sites was determined among strains of A. minutum and A. pacificum. 5) 5S rDNA is a useful chromosomal marker of mitosis progression and can be employed to differentiate vegetative (haploid) vs. planozygotes (diploid) cells. Thus, the FISH probe (oligo-Dino5Smix5) developed in this study facilitates analyses of the diversity, cell cycle and life stages of the genus Alexandrium.

Detection of Klebsiella pneumoniae complex in carrots

Abstract Background Klebsiella pneumoniae (Kp) is an opportunistic pathogen now acknowledged as an urgent threat to human health because it is multi-drug resistant bacteria. The role of food as reservoir or carrier of Kp is under investigation, at the same time the optimization of the method to detect Kp in food is ongoing. The study was aimed to detect Kp in carrots using conventional method and to screen Real-time PCR performances, targeting intergenic sequence between zur and khe, based on INRA and Pasteur Institute method Methods A total of 60 samples of carrots were tested. The samples (25 g) were added to 225 ml of Buffered peptone water and incubated at 37 °C and at 44 °C for 24h. After incubation broths were subjected to Real-Time PCR, and one loop streaked on SCAI + inositol agar. Plates were then incubated at 37 °C and at 44 °C for 48 h. After incubation a maximum of 5 typical colonies were selected, subcultered on Nutritive agar and identified by MALDI-TOF MS. Results The RT PCR gave 29 positive samples, according to the presence of Kp detected at least in one of the two temperature conditions of broths incubation and 31 negative samples. Conventional culture gave, instead, 13 positive and 47 negative samples. Discordance was highlighted in 16 samples, all positives in RT-PCR and negatives with culture method. Conclusions Real-Time PCR gave not false negative results, then PCR decrease the time needed to perform the detection in negative samples. The discordance of results could be linked to lack of selectivity of the SCAI agar + inositol, not able to distinguish colonies of Klebsiella spp. Vs similar colonies not Klebsiella spp., the presence of DNA and not viable cells. The process of validation of the detection method is still ongoing, trying to improve selectivity using two selective media for plating. It's necessary to do new trials and to select more typical colonies to increase the probability to detect Kp colonies. Key messages Real-Time PCR is able to detect all positive samples, and to shorten the length of the analytical method. To improve sensibility of the analytical method more selective media need to be used.

The First Mitochondrial Genome for Geastrales (Sphaerobolus stellatus) Reveals Intron Dynamics and Large-Scale Gene Rearrangements of Basidiomycota.

In this study, the mitogenome of artillery fungus, Sphaerobolus stellatus, was assembled and compared with other Basidiomycota mitogenomes. The Sphaerobolus stellatus mitogenome was composed of circular DNA molecules, with a total size of 152,722 bp. Accumulation of intergenic and intronic sequences contributed to the Sphaerobolus stellatus mitogenome becoming the fourth largest mitogenome among Basidiomycota. We detected large-scale gene rearrangements in Basidiomycota mitogenomes, and the Sphaerobolus stellatus mitogenome contains a unique gene order. The quantity and position classes of intron varied between 75 Basidiomycota species we tested, indicating frequent intron loss/gain events occurred in the evolution of Basidiomycota. A novel intron position classes (P1281) was detected in the Sphaerobolus stellatus mitogenome, without any homologous introns from other Basidiomycota species. A pair of fragments with a total length of 9.12 kb in both the nuclear and mitochondrial genomes of Sphaerobolus stellatus was detected, indicating possible gene transferring events. Phylogenetic analysis based on the combined mitochondrial gene set obtained well-supported tree topologies (Bayesian posterior probabilities ≥ 0.99; bootstrap values ≥98). This study served as the first report on the mitogenome from the order Geastrales, which will promote the understanding of the phylogeny, population genetics, and evolution of the artillery fungus, Sphaerobolus stellatus.

Genetic Diversity and Phylogenetic Analyses of Turkish Cotton (Gossypium hirsutum L.) Lines using ISSR Markers and Chloroplast trnL-F Regions

ABSTRACT In this study, genetic diversity and phylogenetic relationships of 22 cotton (Gossypium hirsutum L.) lines widely cultivated in Turkey were investigated. For this purpose, 20 ISSR primers were used, and five of which generated amplification products. The polymorphic locus ratio was calculated as 87.21% and a moderate level of genetic diversity was found between the genotypes. Percentage of the highest polymorphic locus was calculated as 12.79% for the Beren line, whereas ADN 98, ADN 710, and Özbek 105 lines showed the lowest value with 0.00%. trnL-F intergenic spacer sequences were used in order to determine phylogenetic relationships, and a comparison was carried out between both sequences of our lines and the sequences obtained from NCBI-GenBank database. The sequences of G. hirsutum, G. hirsutum subsp. latifolium, and G. hirsutum cv. “Hainansijimian” were described as the most similar sequences according to specifications of coverage, similarity, and maximum score ranges. Then, a phylogenetic tree was established by using the sample sequences from the prominent families cultivated, especially from Malvaceae. While the members of Malvaceae family were clustered as one group, the other families were comprised of different groups located in different positions according to their genetic similarities.

A Forward Genetic Approach to Mapping a P-Element Second Site Mutation Identifies DCP2 as a Novel Tumor Suppressor in Drosophila melanogaster.

The use of transposons to create mutations has been the cornerstone of Drosophila genetics in the past few decades. Second-site mutations caused by transpositions are often devoid of transposons and thereby affect subsequent analyses. In a P-element mutagenesis screen, a second site mutation was identified on chromosome 3, wherein the homozygous mutants exhibit classic hallmarks of tumor suppressor mutants, including brain tumor and lethality; hence the mutant line was initially named as lethal (3) tumorous brain [l(3)tb]. Classical genetic approaches relying on meiotic recombination and subsequent complementation with chromosomal deletions and gene mutations mapped the mutation to CG6169, the mRNA decapping protein 2 (DCP2), on the left arm of the third chromosome (3L). Thus the mutation was renamed as DCP2l(3)tb. Fine mapping of the mutation further identified the presence of a Gypsy-LTR like sequence in the 5′UTR coding region of DCP2, along with the expansion of the adjacent upstream intergenic AT-rich sequence. The mutant phenotypes are rescued by the introduction of a functional copy of DCP2 in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. With the increasing repertoire of genes being associated with tumor biology, this is the first instance of mRNA decapping protein being implicated in Drosophila tumorigenesis. Our findings, therefore, imply a plausible role for the mRNA degradation pathway in tumorigenesis and identify DCP2 as a potential candidate for future explorations of cell cycle regulatory mechanisms.

Prevalence, antibiotic susceptibility and characterization of Vibrio parahaemolyticus isolates in China.

Vibrio parahaemolyticus is a marine and estuarine bacterium that poses a major threat to human health worldwide. In this study, from 2017 to 2019, we evaluated 900 food samples collected from China in 2017, with the aim of determining the incidence and features of V. parahaemolyticus in ready-to-eat (RTE) foods, shrimp and fish in China. The contamination rates in these were 3.67, 19.33 and 10.67%, respectively, and the prevalence of V. parahaemolyticus was higher in summer than in winter. In addition, 101V. parahaemolyticus strains were isolated. Our results suggested that most of the isolates were resistant to aminoglycosides based on the antimicrobial resistance patterns of these aquatic product isolates against 14 antimicrobial agents. Furthermore, most of the isolates were multidrug-resistant. Serotyping showed that the isolates of the O2 serotype comprised the maximum proportion. Enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR results indicated that the isolates (n=101) could be classified into 12 clusters. There were 82 STs suggesting genetic variation and relatedness among these isolates. Our findings demonstrated the presence of V. parahaemolyticus in foods from Chinese retail markets and show that this methodology can be used for microbiological risk assessment in China.

Prevalence and antimicrobial resistance of retail-meat-borne Salmonella in southern China during the years 2009–2016: The diversity of contamination and the resistance evolution of multidrug-resistant isolates

Salmonella, one of the most important foodborne pathogens, can be the cause of bacterial food-borne illness and is commonly associated with the consumption of retail meat. Multidrug-resistant Salmonella isolates with high adaptability, have been responsible for many foodborne disease outbreaks. Here we present an investigation on the contamination and the antimicrobial resistance of Salmonella in retail meat obtained from supermarkets and from open markets in Guangxi, China. From the years 2009 to 2016, a total of 604 Salmonella isolates were recovered from a total of 3340 meat samples including 797 beef, 911 pork, 942 chicken and 690 duck, representing 18.08% of the samples tested. Pork was the most contaminated meat. Salmonella was detected in 322 samples from supermarkets and the positive rate of 21.03% was higher than that of 15.70% in 284 samples from open markets (P<0.05). The prevalence of Salmonella in retail meat in the summer and fall months: June (2015, 40.63%), October (2012, 34.6%; 2016, 43.75%) was higher than in other seasons of the year. One hundred and twenty-seven serotypes were identified among the 604 Salmonella enterica isolates, and S. Derby (28.48%), S. Agona (9.77%), S. London (4.97%) and S. Enteritidis (4.47%) were the most common serotypes. Tests of susceptibility to 21 antimicrobial agents showed that 87.58% of the isolates were resistant to at least one antimicrobial, and 57.79% exhibited multidrug resistance (MDR), as they were resistant to at least three antimicrobials. The presence of most of the antimicrobial-resistant genes tested was consistent with the resistant phenotypes found. Among all the antimicrobial resistant genes (ARGs) examined in this study, blaTEM-1, aadA1, cmlA, tetA, sul1 and sul2 were the most prevalent resistant genes in the multidrug resistant isolates. Our findings show that there was a trend that the Salmonella contamination in retail meat had increased and isolates showed an MDR phenotype and that the MDR had become more and more serious. Twenty-one isolates of S. Agona were randomly analyzed by using the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and six different types were found, indicating the existence of cross-contamination in the food market. The results indicate that the hazard analysis of the critical control points (HACCP) system for the whole food chain of retail meat should be further analyzed and improved.

Detecting and molecular typing of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a prevalent food-borne pathogen in many Asian countries and has been spreading rapidly following the appearance of pandemic strains. This article reviews conventional and molecular methods for culturing, identifying, enumerating and rapidly detecting V. parahaemolyticus and its virulence factors, such as thermostable direct hemolysin (TDH), TDH-related hemolysin and thermolabile hemolysin. It also discusses various molecular typing methods, namely, pulsed-field gel electrophoresis, ribotyping, random amplified polymorphic DNA and three other polymerase chain reaction methods based on specific conserved nucleotide sequences, such as ribosomal gene spacer, repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequences, for use in epidemiological investigations of this pathogen.