Glaesserella parasuis (G. parasuis) is a respiratory pathogen of swine and the etiological agent of Glässer’s disease. The structural organization of genetic information, antibiotic resistance genes, potential pathogenicity, and evolutionary relationships among global G. parasuis strains remain unclear. The aim of this study was to better understand patterns of genetic variation, antibiotic resistance factors, and virulence mechanisms of this pathogen.The whole-genome sequence of a ST328 isolate from diseased swine in China was determined using Pacbio RS II and Illumina MiSeq platforms and compared with 54 isolates from China sequenced in this study and 39 strains from China and eigtht other countries sequenced by previously. Patterns of genetic variation, antibiotic resistance, and virulence mechanisms were investigated in relation to the phylogeny of the isolates. Electrotransformation experiments were performed to confirm the ability of pYL1—a plasmid observed in ST328—to confer antibiotic resistance.6678 transposon harbouring a unique resistance determinant. It also contained a small broad-host-range plasmid pYL1 carrying aac(6’)-Ie-aph(2”)-Ia and blaROB-1; when transferred to Staphylococcus aureus RN4220 by electroporation, this plasmid was highly stable under kanamycin selection. Most (85.13–91.74%) of the genetic variation between G. parasuis isolates was observed in the accessory genomes. Phylogenetic analysis revealed two major subgroups distinguished by country of origin, serotype, and multilocus sequence type (MLST). Novel virulence factors  and drug resistance genes  in G. parasuis were identified. Resistance determinants -Ib, norA, bacA, ksgA, and bcr) were widespread across isolates, regardless of serovar, isolation source, or geographical location.The ST328 genome contained a novel TnG. parasuis isolates provides valuable insight into the emergence and transmission of G. parasuis in the swine industry. The result suggests the importance of transposon-related and/or plasmid-related gene variations in the evolution of G. parasuis.Our comparative genomic analysis of worldwide  Glaesserella parasuis, a gram-negative bacterium in the family Pasteurellaceae  and multiple target gene mutations. To date, two β-lactam resistance genes (blaROB−1 and blaTEM), an aminoglycoside-resistance gene (aac (6′)-Ib-cr) and a mutation in the six copies of the 23S rRNA gene, associated with macrolide resistance, have been reported in G. parasuis -Ib, aph(6)-Id, floR, sul1, and sul2 (hhdBA), iron acquisition genes , the restriction modification system hsdS, and genes involved in sialic acid utilization   . RecentlG. parasuis strains, the structural organization of genetic information, ARGs, potential pathogenicity determinants, and evolutionary relationships among global G. parasuis strains remain unclear. In this study, we sequenced a multidrug-resistant isolate from diseased swine in Dongguan, China, then compared this genome sequence with those of 54 isolates from China sequenced by us and 39 strains from China and eight other countries sequenced by other researchers in order to improve our understanding of genomic diversity in G. parasuis and provide information for gaining better control to treat these infections.Though significant effort has been focused on exploring ARGs, virulence factors and other genetic characteristics of various G. parasuis isolate HPS-1 examined in this study belongs to serotype 4 and was originally isolated from the lungs of a pig suffering from Glässer’s disease in a commercial pig farm in Dongguan city, Guangdong province, China, in 2017. Susceptibility to 19 antimicrobial agents was determined by the disc agar diffusion method and the broth microdilution method .Isolates were cultured on tryptic soy agar or in tryptic soy broth  supplemented with 10 mg/mL nicotinamide adenine dinucleotide and 5% bovine serum at 37 °C in 5% COCP040243. The plasmid pYL1 and transposon Tn6678 of HPS-1 were submitted to GenBank under accession number MK182379 and and MK994978, respectively. Genomic libraries of the other 53 genomes were generated and sequenced using the Illumina HiSeq 4000 system  as previously described  against 6 databases: Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), NCBI non-redundant protein database (NR), Swiss-Prot, Gene Ontology (GO), and TrEMBL.Among the 55 isolates, one multidrug-resistant isolate (HPS-1) and one sensitive isolate (HPS-2) from diseased swine in Guangdong were randomly selected for WGS using the PacBio RSII  and Illumina MiSeq  platforms as previously described . The genescribed . WGS datescribed . Gene prescribed , and a wescribed  searchesG. parasuis strains and 39 previously published genome sequences using single-copy core orthologs and single nucleotide polymorphisms (SNPs)  software to generate non-paralogous gene clusters .Two phylogenetic trees were constructed to assess the relatedness of the 55 s (SNPs) . Phyloges (SNPs) . The WAGE-value ≤ 1e−5, minimal alignment length percentage ≥ 80%) was performed against four databases for pathogenicity and drug resistance analysis: Pathogen Host Interactions (PHI), Virulence Factors of Pathogenic Bacteria (VFDB), Carbohydrate-Active enZYmes Database (CAZy), and Integrated Antibiotic Resistance Genes Database (IARDB).A whole-genome BLAST search -Ie-aph(2″)-Ia, which confer to β-lactams and aminoglycosides resistance. To determine the contributions of pYL1 to penicillin and aminoglycoside antibiotic resistance, electrotransformation experiments were performed using Staphylococcus aureus RN4220 as the recipient as previously described (aac(6′)-Ie-aph(2″)-Ia and blaROB−1 genes in transformants was confirmed by PCR amplification followed by DNA sequence analysis. The primers for blaROB−1 (494 bp) were 5′-CGCTTTGCTTATGCGTCCAC-3′ (forward) and 5′-ACTTTCCACGATGTTGGCGT-3′. The primers for aac(6′)-Ie-aph(2″)-Ia (412 bp) were 5′-AGAGCCTTGGGAAGATGAAGTT-3′ (forward) and 5′-TGCCTTAACATTTGTGGCATT-3′ (reverse). The primers were designed using NCBI Primer-BLAST. The PCR conditions were as follows: initial denaturation at 95 °C for 5 min, 30 cycles of amplification , followed by extension at 72 °C for 10 min. The PCR products were purified and sequenced by Majorbio Company . The MICs of S. aureus RN4220 and five transformants were determined by Etest (Liofilchems.r.l.) according to the manufacturer’s instructions.Plasmid pYL1 harboring two antimicrobial resistance genes, escribed . Transfoaac(6′)-Ie-aph(2″)-Ia and blaROB−1 was determined by serial passages for 15 consecutive days at 1:1000 dilutions into fresh BHI, with or without antibiotic (kanamycin) pressure. Serially diluted cultures were spread on BHI agar plates with or without kanamycin (8 µg/mL), and the resistance retention rate was determined by randomly picking at least 50 colonies from the BHI plates, spotting them onto new BHI plates with kanamycin (8 µg/mL), and calculating the ratio of resistant to total colonies. Both the resistant and susceptible colonies from the plates were randomly picked and subjected to PCR for detection of blaROB−1 and aac(6′)-Ie-aph(2″)-Ia.The stability of plasmids carrying G. parasuis gene content compared with the core genomes and the number of unique genes ranged from 0 to 103 indicating that 0–4.6% of the genome consists of strain-specific accessory genes  core biosynthesis, and galU and galE, resulting in impaired biofilm formation, were universally present in the G. parasuis isolates.Variation in virulence and stress resistance genes was observed among ubgroups . All 94 G. parasuis, including the β-lactam-resistant gene blaROB−1, tetracycline resistance genes tetB, aminoglycoside resistance genes aph(3′)-Ib and aac(6′)-Ie-aph(2″)-Ia, fluoroquinolone resistance gene norA, chloramphenicol resistance genes catIII and floR, sulfonamide resistance gene sul2 were discovered (sul2 and aph(3′)-Ib, and β-lactam-resistant genes pbp1a and pbp3a were universally present in the G. parasuis isolates , aminoglycosides (aph(3′)-Ib, aac(6′)-Ie-aph(2″)-Ia), and β-lactam (blaROB−1) (bcr) and multidrug resistance (acrB).Following sequencing and assembly, a 2,326,414-bp chromosome with an average G+C content of 40.03%, and a 7,777-bp small plasmid sequence (pYL1) with an average G+C content of 33.32% were identified in strain HPS-1 . HPS-1 elaROB−1) . Furtherhttps://transposon.lstmed.ac.uk/). This transposon harbours two 966-bp IS110 family transposases at both ends, two toxin genes pilT and phd, two genes associated with the two-component signal transduction system cpxA and cpxR, one efflux pump-associated gene bcr, and four genes encoding hypothetical proteins with unknown function (34)-methyltransferase MnmD/FAD-dependent 5-carboxymethylaminomethyl-2-thiouridine (34) oxidoreductase MnmC flanked the transposon to the right and left, respectively.We also identified a novel transposon in the ST328 isolate, designated Tn6678 in the Tn Number Registry , SH0165 , ZJ0906 , and str. Nagasaki ]. The only differences in these five chromosomes were in the transposases, but transposon Tn6678 had two complete inverted repeats of IS110 transposases flanked by 32-bp inverted repeats of ISNme5 at both ends  were identified in four oth ends , suggestbcr gene returned a large set of divergently related sequences using default parameters. These sequences were annotated as bicyclomycin/multidrug efflux system, Bcr/CflA family drug resistance efflux transporter, Bcr/CflA family multidrug efflux major facilitator superfamily (MFS) transporter or drug resistance transporter, and Bcr/CflA subfamily. Phylograms revealed that the bcr gene in HPS-1 was most closely related to homologs identified in other members of the Pasteurellaceae, particularly G. parasuis, Actinobacillus indolicus, Bibersteinia trehalosi, Actinobacillus , and Mannheimia (M. haemolytica and M. varigena), all of which are known causative agents of upper respiratory tract infections -Ie-aph(2″)-Ia. Four ORFs were identified to encode a 3′-truncated transposase protein ISApl1 (30 amino acids), a Rep-like protein (444 amino acids) involved in plasmid replication, and two Mob proteins, MobC (144 amino acids) and MobA (541 amino acids), associated with plasmid mobilization . This finding indicated that plasmid pYL1 carrying blaROB−1 and aac(6′)-Ie-aph(2″)-Ia contributed to the penicillin resistance and aminoglycoside antibiotic resistance in S. aureus RN4220 transformants. Furthermore, the plasmid showed low stability in S. aureus without antibiotic pressure, as only 52.5%, 30.48%, and 2.68% of transformants maintained the kanamycin resistance after five, six, and seven subcultures, respectively. However, the plasmid can be conserved in S. aureus cultured with kanamycin, as 100% of the colonies remained resistant to kanamycin after 10 subcultures, as confirmed by PCR mapping.Transformation of pYL1 into G. parasuis pan-genome is vast, and unique genes can be continuously be identified upon sequencing more G. parasuis genomes. However, the isolates in this study with ∼3.34% core genes, primarily isolated from China, displayed further diversity and higher variability than isolates with only ∼14% core genes, primarily obtained from the UK , in line with potentially virulent strains isolated from the nasal cavities of healthy pigs -Ib, tetB, tetD, aac(6′)-Ie-aph(2″)-Ia, catIII, and floR genes have previously been identified in G. parasuis  family, has been analysed to date. Efflux pump AcrB may play a role in multidrug resistance, and the acrAB gene cluster could affect the efflux of macrolides in G. parasuis  have been reported in G. parasuis -Ie-aph(2″)-Ia. The ROB-1 of plasmid pYL1 had a typical size of 305 bp, in line with functionally active members of the ROB-1 family from different plasmids in Pasteurellaceae species. AAC(6′)-Ie-APH(2′)-Ia, the most important aminoglycoside-resistance enzyme in gram-positive bacteria conferring resistance to almost all known aminoglycoside antibiotics in clinical use, also had a typical size of 479 amino acids in this family (aac(6′)-Ib-cr is considered the most prominent aminoglycoside-resistance gene in G. parasuis  and drug resistance genes  in G. parasuis. Resistance determinants -Ib, norA, bacA, ksgA, and bcr) were widespread across isolates, regardless of serovar, isolation source, or geographical location. Future research focused on a larger sample of G. parasuis isolates worldwide will further increase understanding of the rapid development of antibiotic resistance associated with mobile genetic elements in this important animal pathogen.In summary, our results shed new light on the importance of genomic variations, especially transposon-related and/or plasmid-related gene variations, in the evolution of 10.7717/peerj.9293/supp-1Table S1P. aeruginosa .MIC: minimum inhibitory concentration; R: resistant; S: susceptible. a Interpreted according to Clinical and Laboratory Standards Institution (CLSI) guidelines for Click here for additional data file.10.7717/peerj.9293/supp-2Table S2N.A. represents unknown information; ST: sequence type; UT: untypeable.Click here for additional data file.10.7717/peerj.9293/supp-3Table S3Click here for additional data file.10.7717/peerj.9293/supp-4Table S4Click here for additional data file.10.7717/peerj.9293/supp-5Table S5Click here for additional data file.10.7717/peerj.9293/supp-6Figure S1The tree was constructed with MEGA 7 using a maximum-likelihood optimality criterion as implemented in PhyML v3.0 with 1, 000 bootstrap replicates. The annotation rings surrounding the tree, from inside to outside, depict (1) serotype, (2) host, (3) geographic region and (4) year of sample collection. The branch colors denote two major lineages, lineage I (pink) and lineage II (green).Click here for additional data file.10.7717/peerj.9293/supp-7Figure S2Glaesserella sp. 15–184 was chosen as an outgroup. The branch colors denote two major lineages, lineage I (pink) and lineage II (green).Click here for additional data file.10.7717/peerj.9293/supp-8Figure S3From inside to outside, the first circle represents the genome size of HPS-1; the second circle represents the GC skew; the third circle represents the GC content; the fourth circle and the seventh circle represent the COG (cluster of orthologous groups) designation of each coding sequence (CDS); the fifth and sixth circles represent the position of CDS, tRNA, and rRNA on the genome.Click here for additional data file.10.7717/peerj.9293/supp-9Supplemental Information 1Click here for additional data file.10.7717/peerj.9293/supp-10Supplemental Information 2Click here for additional data file.10.7717/peerj.9293/supp-11Supplemental Information 3Click here for additional data file.10.7717/peerj.9293/supp-12Supplemental Information 4Click here for additional data file.10.7717/peerj.9293/supp-13Supplemental Information 5Click here for 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These fungi have worldwide distribution, but with the exception of Articulosporatetracladia, no aquatic hyphomycete taxon was previously recorded on Madeira Island. Aquatic hyphomycetes were sampled from 40 sites, distributed by 27 permanent streams in 2015, to provide the distribution of aquatic hyphomycetes in Madeira Island streams.Aquatic hyphomycetes were recorded belonging to three classes of Ascomycota. All taxa are new records for Madeira Archipelago, except Articulosporatetracladia and four are reported for the first time in Macaronesian biogeographic region.In this study, a total of 21 species of aquatic  Aquatice scarce . In factal zones . Despiteems e.g. .fungi started almost two centuries ago with the work of th century, many mycological studies increased the number of records for Madeira Archipelago (North Atlantic), including numerous descriptions of species new to science . Lying in the subtropical region, Madeira’s climate is influenced by winds from NE and the Canary Islands current. The Island has a mild oceanic climate, both in winter and summer with mild temperatures ranging from 15.9°C in February up to 22.3°C in August , relative humidity between 55 and 75% and annual rainfall between 500 and 1,000 mm  distributed by 27 permanent streams : 384 (1942)3690A495-A84F-50C3-A2E6-2BEDD6529505Cosmopolitan .Madeira distribution: Streams in agricultural and natural areas at low to moderate altitude: Ribeira de São Vicente (MAD04); Ribeira do Juncal (MAD15); Ribeira do Faial (MAD16); Ribeira Primeira (MAD18); Ribeira de São Jorge (MAD37).Habitat: Submerged leaf litter .(H.J. Huds.) L. Lombard & Crous, in Lombard, van der Merwe, Groenewald & Crous, Stud. Mycol. 80: 207 (2015)672BA860-8D1E-5A9B-9963-0C35130DCB8ECosmopolitan .Madeira distribution: Stream in agricultural and natural areas at low to moderate altitude: Ribeira de São Vicente (MAD04); Ribeira Brava (MAD07); Ribeira do Juncal ; Ribeira do Faial (MAD16); Ribeira da Janela (MAD21); Ribeira de São Roque do Faial (MAD33); Ribeira dos Arcos (MAD36); Ribeira de São Jorge (MAD17); Ribeira da Fonte do Bugio (MAD39).Habitat: Submerged leaf litter .(R.H. Petersen) Dyko, Trans. Br. mycol. Soc. 70 (3): 412 (1978)8A859C84-48D6-5382-BE6E-FC0C03AD9EDDCosmopolitan .Madeira distribution: Streams in natural areas at low to high altitude: Ribeira Brava (MAD07); Corgo da Ribeira de Anéis (MAD11); Ribeira do Córrego do Arrochete (MAD30); Ribeira de São Jorge (MAD37).Habitat: Submerged leaf litter [e.g. Ilexperado (xperado ].(Ingold) Sv. Nilsson ex Marvanová & Sv. Nilsson, Trans. Br. mycol. Soc. 57 (3): 532 (1971)15B4E8EC-42DF-52E6-B357-42CBE90D2558Cosmopolitan .Madeira distribution: Streams in natural areas at high altitude: Ribeira da Janela ; Ribeira do Córrego do Arrochete (MAD30).Habitat: Submerged leaf litter .(Sacc. & Therry) L. Lombard & Crous, in Lombard, van der Merwe, Groenewald & Crous, Phytopath. Mediterr. 53(3): 528 (2014)9B3C3176-C801-5961-B75C-48C31DFB8EEDCosmopolitan .Madeira distribution: Streams in urban, agricultural and natural areas at low to high altitude: Ribeira Brava (MAD02); Ribeira da Vargem (MAD03); Ribeira Brava ; Ribeira dos Socorridos (MAD09); Ribeira do Juncal ; Ribeira do Machico (MAD17); Ribeira Primeira (MAD18); Ribeira da Janela ; Ribeira do Alecrim (MAD28); Ribeira do Córrego do Arrochete (MAD30).Habitat: Submerged leaf litter .(Grove) Ingold, Trans. Br. mycol. Soc. 25(4): 371(1942)29EA0E24-82EE-5239-85A5-1555AED4BAD6Cosmopolitan .Madeira distribution: Streams in agricultural areas at low to moderate altitude: Ribeira dos Socorridos (MAD01); Ribeira Brava (MAD02); Ribeira do Juncal (MAD15).Habitat: Submerged leaf litter [e.g. Clethraarborea, Ilexperado : 624 (1974)68D79614-DC15-5AF0-991B-F21A84FD1786Cosmopolitan .Madeira distribution: Streams in urban, agricultural and natural areas at low to high altitude: Ribeira dos Socorridos (MAD01); Ribeira de São Vicente (MAD04); Ribeira Grande (MAD05); Ribeira Brava ; Ribeira do Cidrão (MAD12); Ribeira do Juncal ; Ribeira do Faial (MAD16); Ribeira do Machico (MAD17); Ribeira Primeira ; Ribeira da Janela ; Ribeiro Frio (MAD29); Ribeira do Córrego do Arrochete (MAD30); Ribeira da Metade (MAD31); Ribeira das Lajes (MAD32); Ribeira de São Jorge (MAD35); Ribeira dos Arcos (MAD36).Habitat: Submerged leaf litter [e.g. Acaciamelanoxylon, Acerrubrum, Alnusglutinosa, Clethraarborea, Cryptomeriajaponica, Ilexperado, Pittosporumundulatum, Quercusrobur, Rhododendronmaximum : 346 (1975)F45FE042-099D-5651-8E96-FC87B0CB74C9Cosmopolitan .Madeira distribution: Streams in agricultural and natural areas at low to moderate altitude: Ribeira dos Socorridos (MAD01); Ribeira Brava (MAD08); Ribeira do Juncal (MAD15); Ribeira do Faial (MAD16); Ribeira do Machico (MAD17); Ribeira Primeira (MAD18); Ribeira da Janela (MAD21); Ribeiro Frio (MAD29); Ribeira do Córrego do Arrochete (MAD30); Ribeira das Lajes (MAD32); Ribeira Seca (MAD34); Ribeira de São Jorge (MAD35); Ribeira dos Arcos (MAD36); Ribeira de Santa Luzia (MAD38).Habitat: Submerged leaf litter [e.g. Acaciamelanoxylon, Alnusglutinosa, Clethraarborea, Ilexperado, Pittosporumundulatum, Quercusrobur : 152 (1943)E9A99FEC-221E-5C74-98E4-1A77CEA78B80Cosmopolitan .Madeira distribution: Streams in urban, agricultural and natural areas at low to high altitude: Ribeira dos Socorridos (MAD01); Ribeira Brava ; Ribeira de São Vicente (MAD04); Ribeira Grande ; Ribeira da Gomeira (MAD10); Ribeira do Cidrão (MAD12); Ribeira do Juncal ; Ribeira do Faial (MAD16); Ribeira do Machico (MAD17); Ribeira Primeira (MAD18); Ribeira da Janela (MAD21); Ribeira dos Cedros (MAD25); Ribeiro Frio (MAD29); Ribeira de São Roque do Faial (MAD33); Ribeira Seca (MAD34); Ribeira de São Jorge ; Ribeira dos Arcos (MAD36); Ribeira de Santa Luzia (MAD38).Habitat: Submerged leaf litter [e.g. Acaciamelanoxylon, Acerrubrum L., Alnusglutinosa, Clethraarborea, Eucalyptusglobulus, Ilexperado, Pittosporumundulatum, Quercusrobur, Rhododendronmaximum , Leotiomycetes encompassed 43% of the taxa recorded. At the order level, the 21 ascomycetes were distributed by Helotiales (12 spp.), Hypocreales (2 spp.), Leotiales (1 sp.), Microascales (1 sp.), Sordariales (1 sp.) and Incertae sedis (4 spp.), according to In the present study, we found a total of 21 aquatic Articulosporatetracladia, Clavariopsisaquatica, Lemonnieraaquatica, Lunulosporacurvula, Tetrachaetumelegans, Tetracladiummarchalianum, Tricladiumchaetocladium and Triscelophorusmonosporus, which were the most ubiquitous aquatic hyphomycetes in Madeira streams. Two taxa, Lemonniera sp. and Tetracladiumfurcatum, had a sporadic occurrence, being found at only one or two sampling sites. A maximum of 14 species was recorded in MAD15 and a minimum of one species in MAD13 and MAD20, with a mean richness of eight species per stream site. Higher altitude stream sites (> 800 m a.s.l.) in natural areas, such as MAD07, MAD08, MAD24 and MAD26, displayed higher taxa richness , compared with coastal (< 25 m a.s.l.) and urban stream sites, such as MAD13, MAD20, MAD32, MAD33, MAD37, MAD38, MAD39 and MAD40 (5.8 ± 1.2). All species, with the exception of Articulosporatetracladia reported by From the 21 species identified, none occurred in all 40 studied stream sites and only 8 taxa occurred in more than 50% of the stream sites: hyphomycetes in insular streams from Madeira Island. Twenty-one taxa are recorded for Madeira Island, which is lower than what is reported for the Azores archipelago  of the Archipelago. In this context, it is essential to increase the sampling effort for both archipelagos, as well as to survey multiple matrices  in order to find a greater diversity of aquatic hyphomycetes. To the best of our knowledge, no data of recorded species exist for the other Macaronesia archipelagos, such as Canary Islands and Cabo Verde Archipelago.Here we present the first study that explored the distribution of aquatic hyphomycetes species have a cosmopolitan distribution Aquatic Data typeData recordsBrief descriptionMetafileFile: oo_404009.xmlhttps://binary.pensoft.net/file/404009Raposeiro, P.M.; Faustino, H.; Ferreira, V.; Gonçalves, V.23207FAB-3574-536A-BFDF-036E130E693C10.3897/BDJ.8.e53690.suppl4Supplementary material 4Occurrence of the aquatic hyphomycete species found in this study in 15 geographic regions defined based on the geographic location and climatic influence in the western and eastern hemispheres Data typeTableBrief descriptionTableFile: oo_420217.csvhttps://binary.pensoft.net/file/420217Raposeiro, P.M.; Faustino, H.; Ferreira, V.; Gonçalves, V.


This is the first report of plasmid-mediated colistin resistance mcr-8 gene from a clinical Klebsiella pneumoniae K9 isolate recovered from Lebanon. The isolate was characterized phenotypically and genotypically through both short and long read whole-genome sequencing, plasmid typing and conjugation assays. k9 belonged to sequence type 15 and harbored 31 antimicrobial resistance genes. The mcr-8.1 variant was carried on a novel ~ 300 kb multireplicon plasmid having IncFIA, IncR and IncHI1B. The plasmid was conjugative and carried a plethora of antimicrobial resistance determinants. The introduction of novel mcr variants in Lebanon poses an alarming health concern. Surveillance and screening for colistin resistant Enterobacteriaceae and mcr in livestock, animal farms, imported meat and poultry is highly recommended along with monitoring antibiotic use.Colistin is considered as a last resort treatment for infections caused by multidrug-resistant Enterobacteriaceae. Plasmid-mediated mobile colistin resistance ( Klebsiella pneumoniae (CRKP) [mcr-1, was identified in 2015 in Enterobacteriaceae, mainly Escherichia coli and K. pneumoniae, collected from animals and humans in China [mcr variants have been described, designated as mcr-1 -9, isolated from humans, animals and the environment [Colistin is the last-line treatment option for infections caused by carbapenem resistant Enterobacteriaceae such as carbapenem-resistant e (CRKP) . The firironment .mcr-8 located on an IncFII-type conjugative plasmid was first described in 2018 in China in K. pneumoniae collected from both animals and humans [d humans . Shortlyd humans  and Alged humans .K. pneumoniae in Lebanon was in 2017 [mcr-1-positive E. coli from a human clinical isolate in Lebanon has been recently reported [mcr-1 was widely detected in Gram-negative bacilli isolated from poultry [mcr-8.1 gene variant in an ST-15 K. pneumoniae isolate recovered from Lebanon.The first report of a colistin resistant reported . On the  poultry , swine f poultry  and wate poultry . This isKlebsiella pneumoniae and designated as k9.The isolate was recovered from the urine sample of a 50-years old female patient on the 28th of August 2018 at the Clinical Microbiology Laboratory of the American University of Beirut Medical Centre (AUBMC), Lebanon. The identification of the isolate was performed using Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) system . The isolate was identified as E. coli transconjugant was further determined by using the broth microdilution method according to the recommendations from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [Antimicrobial susceptibility testing was performed using the disk diffusion assay on Mueller-Hinton agar and included a panel of 24 antibiotics belonging to 16 different classes (Table S(EUCAST) .DNA extraction was performed from fresh bacterial colonies using the Nucleospin® Tissue kit  according to the manufacturer’s instructions.mcr-8 was confirmed through PCR using MCR-8F 5′-AACCGCCAGAGCACAGAATT-3′ and MCR-8R 5′-TTCCCCCAGCGATTCTCCAT-3’primer pair as previously described [The presence of escribed .Plasmid characterization was performed using the DIATHEVA plasmid based replicon typing (PBRT) kit  through a polymerase chain reaction (PCR) based replicon typing method consisting of eight multiplex PCR assays for the amplification of 25 replicons: A/C, B/O, FIA, FIB, FIB-M, FIC, FII, FIIK, FIIS, HI1, HI2, HIB-M, I1, I2, K, L/M, N, P, R, T, U, W, X1, X2, and Y found in the family Enterobacteriaceae. Positive controls were included for all reactions. All PCR reactions were performed according to the manufacturer’s instructions.E. coli J53 strain. Selection of transconjuguants was done on Uriselect agar  supplemented with 100 mg/L sodium azide and 4 μg/ml colistin. E. coli transconjugants were typed by PBRT to detect plasmid replicons.Conjugation was performed with an azide-resistant Genomic libraries were constructed using the Nextera XT DNA library preparation kit with dual indexing (Illumina). The libraries were sequenced on an Illumina MiSeq with 250 bp × 2 read length. Genome assembly was performed de novo using Spades Genome Assembler Version 3.6.0 . Qualitymcr8-carrying plasmid was sequenced using PacBio long-read sequencing technology on the Sequel platform . Library preparation was performed according to the manufacturer’s instructions for microbial multiplexing. G-tubes  were used for DNA shearing, and no size selection was performed.The http://rast.nmpdr.org) [www.genomicepidemiology.org) were used to determine the ST, presence of resistance genes and plasmid content, respectively. The sequences encoding for plasmids were then extracted and aligned to references obtained from NCBI as previously described [https://www-is.biotoul.fr) [wzi gene was performed using Kaptive (www.kaptive.holtlab.net) [http://bigsdb.pasteur.fr. PlasmidsSpades was used to assemble the plasmid sequences [The assembled genomes were annotated using the RAST online server (pdr.org) . MLST 2.pdr.org) , ResFindpdr.org) , PointFipdr.org)  and Plaspdr.org)  availablescribed . The ISftoul.fr)  was usedlab.net) . The preequences . Plasmidequences .pmrA, pmrB, pmrC, pmrD, phoP, phoQ, mgrB, crrA and crrB genes were investigated in silico using both nucleic and amino acid alignments compared to the wild-type sequence of K. pneumoniae MGH 78578 (GenBank accession no. CP000647). SNAP [http://www.hiv.lanl.gov) was used to calculate synonymous and non-synonymous substitution rates based on the codon-aligned nucleotide sequences of pmrA and pmrB against the K. pneumoniae MGH 78578 references.The presence of known mutations conferring colistin resistance on the 7). SNAP   and fimbriae .K9 genome size was 5,656,813 bp with a GC content of 56.9%. The best match for the capsular locus was KL107 (Blastn identity: 89.60%) with idFinder  includinmcr-8.1 positive isolates recovered so far (Table R. ornithinolytica QDRO2 (Accession no. MK097469.1) [n = 31) than other mcr-8.1 positive isolates with 16, 18 and 23 antimicrobial resistance genes (AMRs) detected in isolates KP95, KP91 and QDRO2, respectively. The latter isolates represented different STs and were recovered from both humans and animal sources [k9 was compared to all other 97469.1)  KP91, KP97469.1) , respect sources .Table 1k9 had a colistin MIC of 10 μg/mL , aminoglycosides (aac(6′)-Ib-cr, aadA1, aadA16, aadA2b and aph(3′)-Ia), quinolones (aac(6′)-Ib-cr, oqxA, oqxB, qnrB2, qnrB4, and qnrB52), sulphonamides (sul1 and sul3), fosfomycin (fosA), macrolides (mph(A),mph(E),msr(E)), tetracycline (tetD), trimethoprim (dfrA27), phenicol , rifampicin (arr-3) and colistin (mcr-8).Studying antibiotic resistance mechanisms revealed the presence of 31 different resistance genes on the plasmid including resistance to: β-lactams  revealed a dN/dS ratio of 1.131 for pmrA and 1.181 for pmrB indicating a positive selection for non-synonymous mutations , ompK36 sequence (Accession no. Z33506.1) and ompK37 (Accession no. AJ011502.1). We detected a premature stop codon in ompK35 (Y76*), and 22 different amino acids in ompK36 and two in ompK37. A premature stop codon was also detected in ramR (K194*); a positive regulator of the AcrAB efflux system, compared to an intact ramR reference genes (Accession no. KY465996.1).Mutations in mcr variant was mcr-8.1 showing 100% identity to mcr-8.1 carried by K. pneumoniae plasmid pKP91 (Accession no. MG736312), a ~ 90-kb IncFII-type plasmid obtained from swine fecal material in China [Raoutella ornithinolytica QDRO2 (Accession no. MK097469.1) [Blast analysis revealed that the in China  and in R97469.1) mcr-8.1 recovered from k9 was further compared with other mcr-8 variants. The amino acid sequences and the genetic environment of mcr-8.1 was most similar to mcr-8.3 and mcr-8.4 then to mcr-8.2, the latter being recovered from K. quasipneumoniae in China [mcr-8.2 also harboured more mutations compared to mcr-8.1 than the other variants  and pKPN-065  isolated from China and USA [rep genes also found in pKP91 and IncHI1B rep gene also found in pKPN-065. IncFIA was bracketed by two IS1 insertion sequences while IS6 was present downstream of IncR. Parts of pk9 plasmid carrying various AMR determinants also aligned to pR50–74 (Accession no. CP040362.1) collected from a rabbit fecal sample in China in 2017, pYDC676 collected from patients in the USA in 2014 (Accession no. KT225462.1) [ and USA , respectsul1, aadA16, dfrA27, arr-3 and aac(6′)-Ib-cr5. It closest identity  to Enterobacter hormaechei plasmid pM206-NDM1 collected in Japan (Accession no. AP018830.1) [sul1, qnr, sul1, aadA16 and aac(6′)-Ib-cr5 showing 100% identity and 98% coverage to K. pneumoniae Kp_29407 (Accession no. LR736030.1). Other resistance determinants present on the pk9 included tetA, qnrB4, blaDHA-1, apha1b, sul3, aadA1–3, cmlA, floR and ampC. Various sulfonamide resistance genes were identified including four copies of sul1 in addition to sul3 and bcr (bicyclomycin resistance protein).A plethora of resistance genes were detected on pk9. An AMR cassette was located between 14,200 bp and 18,300 bp encoding 18830.1) . Anothermcr gene variants represents a major health concern worldwide. In this study, we identified a multireplicon plasmid carrying the mcr-8.1 gene variant in a K. pneumoniae clinical isolate recovered from a urine sample in Lebanon. This represents the first report of mcr-8 from Lebanon. mcr-8.1 showed 100% gene sequence similarity to mcr-8.1 encoded by pKP91 plasmid originating from a K. pneumoniae KP91 collected in 2018 from swine fecal material in China [mcr variants have been described (mcr-1 to mcr-9) from human, animal and environmental sources [mcr gene in the wildlife and particularly in surface water samples [The plasmid-mediated spread of in China . The detin China , 32. Sin sources , showing samples .mcr-8.1 was located on a 324,283 bp multireplicon plasmid showing highest nucleotide similarity (99–100%) to plasmids pF10AN_1 (Accession no. CP026154.1) and pKPN-065 (Accession no. CP015026.1) isolated from China and USA [rep genes also found in pKP91 [rep gene also found in pKPN-065. Recently, mcr-8.2 was detected on a large, hybrid plasmid containing IncQ, IncR, and IncFII replicons [mcr gene spread [ and USA , respecteplicons  confirmie spread .mcr gene family could be transmitted via the food chain, and so its prudent use in both human and veterinary medicine is of paramount importance [mcr-8 in livestock and animal farms in Lebanon as well as in imported meat and poultry is recommended to track the environmental influences contributing to the development and dissemination of resistance determinants propagating on large mobile genetic vehicles.Colistin resistant bacteria and the portance . Polymyxportance . FurtherAdditional file 1: Table S1. Antimicrobial susceptibility and MIC results of K. pneumoniae k9. Antimicrobial susceptibility was performed using the disk diffusion technique; MICs of ertapenem, imipenem and meropenem were determined using the E-test methodology; MIC of colistin was determined using the broth microdilution method. Antibiotics were divided by their drug categories. GM: gentamycin 10μg; IP: imipenem; MEM: meropenem 10μg; IPM: imipenem 10μg; PM: cefepime; CTX: cefotaxime 30μg; CXM: cefuroxime 30μg; FEP: cefepime 30μg; CZD: ceftazidime 10μg; CZN: cefazolin 30μg; SCF: cefoperazone/sulbactam 75/30μg; FOX: cefoxitin 30μg; NOR: norfloxacin 5μg; CIP: ciprofloxacin 5μg; FAD: fusidic acid 10μg; TGC: tigecycline 15μg; CMN: clindamycin 2μg; ERY: erythromycin 15μg; ATM: aztreonam 30μg; LZD: linezolid 30μg; AMX: amoxicilin 25μg; PIR: pipercacillin 100μg; AMP: amipicillin 10μg; TZP: pipercacillin/tazobactam 100/10μg; FOS: fosfomycin 200μg; TE: tetracycline 30μg; TS: trimethoprim/sulfamethoxazole; CO: colistin; Dark blue: resistant, light blue: intermediate resistance, white: susceptible; MICs are in μg/mL; NA: not available.Additional file 2: Figure S1. SNAP plots with potential synonymous and non-synonymous substitutions in pmrA (A) and pmrB (B). Alignment was performed against the query sequence of K. pneumoniae MGH 78578 wild-type chromosomal genes.


Since electronic cigarettes (e-cigarettes) entered the U.S. marketplace in 2007, the landscape has evolved to include different product types  and flavored e-liquids , which have contributed to increases in youth use , from 7.7 million to 17.1 million units per 4-week interval. ; however, within the context of this general increase, sales fluctuated . During Among total e-cigarette unit sales during September 2014–August 2019, the proportion that were prefilled cartridges increased from 47.5% to 89.4% (AIPC = 1.0) (p<0.05) . The proAmong total e-cigarette unit sales during September 2014–August 2019, the proportion accounted for by mint products increased from 0.01% to 43.4% (AIPC = 10.5) (p<0.05) . During Among prefilled cartridge sales during September 2014–August 2019, the percentage that were mint increased from <0.1% to 47.6% (AIPC = 14.1) (p<0.05) . During Among disposable e-cigarette sales during September 2014–May 2020, the percentage of sales of tobacco-flavored and menthol-flavored products decreased; sales of tobacco-flavored e-cigarettes accounted for 17.2% and menthol-flavored accounted for 10.2% of all disposable e-cigarette sales in May 2020, (p<0.05). . During 1,2).During November 2016–August 2019, total e-cigarette unit sales in the U.S. increased nearly 300%. Although prefilled cartridges remained the leading product type sold, disposable sales increased beginning in August 2019, reaching 19.8% of total sales by May 2020. Among prefilled cartridge sales, the proportion of mint-flavored products declined beginning in August 2019; by May 2020, menthol (61.8%) and tobacco (37.1%) flavors dominated the market. Among disposable e-cigarette sales, tobacco-flavored and menthol-flavored sales decreased during September 2014–May 2020; during the same period, the proportion of sales that were mint and all other flavors increased, with mint reaching 10.5% and all other flavors reaching 62.1% of total sales by May 2020. Continued monitoring of e-cigarette sales could inform strategies to reduce use among U.S. youths, including strategies that address youth-appealing product innovations and flavors  products. Flavored e-cigarette sales patterns by product type are likely influenced by multiple factors. For example, JUUL voluntarily removed mango, creme, fruit, and cucumber flavored cartridges from retail stores (November 2018) and online (October 2019)The findings in this report are subject to at least three limitations. First, sales data did not include purchases from the Internet or “vape shops,” which accounted for approximately one half of U.S. e-cigarette sales in 2019;1,2). In the U.S., e-cigarette use is markedly higher among youths than adults; in 2018, current use of e-cigarettes was 20.8% (past 30-day use) among high school students, 7.6% (everyday/someday use) among adults aged 18–24 years, and 3.2% (everyday/someday use) among adults aged ≥18 years  entered the U.S. marketplace in 2007, the landscape has evolved to include disposable e-cigarettes and rechargeable e-cigarettes with prefilled cartridges and flavored e-liquids .During September 2014–May 2020, e-cigarette sales increased by 122.2%. Sales of prefilled cartridges increased during September 2014–August 2019; since then, sales of disposable products have increased. Prefilled mint cartridge e-cigarette sales increased from September 2014 to August 2019, then decreased, as menthol sales increased during August 2019–May 2020.Continued monitoring of e-cigarette sales and use is critical to inform strategies to minimize risks. As part of a comprehensive approach, such strategies could include those that address youth-appealing product innovations and flavors.


This study was aimed at exploring the effect of long noncoding RNA LINC00324 (LINC00324) on gastric cancer (GC) and the potential molecular mechanisms.  The expression of LINC00324 and miR-3200-5p in GC tissues and cells was detected by qRT-PCR. LINC00324 was silenced in GC cells by transfection of si-LINC00324. Then, the proliferation, migration, and invasion of GC cells were analyzed by MTT, wound healing, and transwell assays, respectively. The interactions between LINC00324 and miR-3200-5p and between miR-3200-5p and BCAT1 were determined by a dual-luciferase reporter and/or RNA pull-down assay.  The expression of LINC00324 was upregulated in GC cells and tissues, but miR-3200-5p was downregulated. Silencing of LINC00324 inhibited the proliferation, migration, and invasion of GC cells. LINC00324 directly targeted miR-3200-5p, and miR-3200-5p directly targeted BCAT1. si-LINC00324 negatively regulated BCAT1 expression via binding to miR-3200-5p. Furthermore, silencing of LINC00324 reversed the promoting effects of BCAT1 on the proliferation, migration, and invasion of GC cells.  Silencing of LINC00324 inhibited the proliferation, migration, and invasion of GC cells through regulating the miR-3200-5p/BCAT1 axis. Gastric cancer (GC), also known as stomach cancer, is the third most frequent malignancy worldwide . Less thLong noncoding RNAs (lncRNAs) are noncoding RNA transcripts, taking part in the occurrence and progression of diverse digestive organ cancers including esophageal cancer , pancreaMicroRNAs (miRNAs) are small noncoding RNAs which mediate target genes by interacting with the 3′ untranslated regions (3′ UTRs) . Some miBranched-chain amino acid transaminase 1 (BCAT1) is a cytosolic BCAT isozyme expressed in specific tissues . BCAT1 tHere, the effects of LINC00324 on the proliferation, migration, and invasion of GC cells were evaluated. Then, we investigated whether miR-3200-5p is a downstream target of LINC00324 and the association between miR-3200-5p and BCAT1. This study may reveal a hopeful therapeutic target for GC and the underlying mechanisms for the treatment of GC.In total, 60 GC patients  were collected from our hospital between April 2016 and May 2018. Fresh GC tissues and adjacent normal tissues were obtained from GC patients who underwent surgery at our hospital. The tissues were frozen in liquid nitrogen immediately after resection and stored at -80°C for future use. This study was permitted by the ethics committee of Taian City Central Hospital, and written informed consent was obtained from all the patients.2.Three human GC cell lines, AGS, MGC803, and MKN-45, and the human normal gastric epithelial cell line GES-1 were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco's Modified Eagle's Medium  containing 10% fetal bovine serum  at 37°C with 5% COThe siRNA-negative control (si-NC), si-LINC00324-1, si-LINC00324-2, mimics NC, miR-3200-5p mimics, miR-3200-5p inhibitor, inhibitor NC, pcDNA3.1-NC (pcDNA-NC), and pcDNA3.1 BCAT1 (pcDNA-BCAT1) were purchased from Shanghai GenePharma . MGC803 and MKN-45 cells grown to 80% confluence were transfected with these above agents using the Lipofectamine 3000 reagent . MGC803 and MKN-45 cells were divided into si-NC, si-LINC00324-1, si-LINC00324-2, mimics NC, miR-3200-5p mimics, inhibitor NC, miR-3200-5p inhibitor, si-NC+pcDNA-NC, si-NC+pcDNA-BCAT1, and si-LINC00324+pcDNA-BCAT1 groups. Cells without transfection were considered the control or blank groups.β-actin were used for the normalization of LINC00324, miR-3200-5p, and BCAT1, respectively. The primer sequences are shown in ΔΔCt- method.Total RNA was extracted from tissues and cells using the TRIzol reagent . RNA was quantified with a NanoDrop ND-1000 spectrophotometer . A total of 300 ng RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit . PCR reaction was performed on the ABI 7500HT Fast Real-Time PCR System  with the following conditions: 95°C for 3 min and 40 cycles of 95°C for 15 s and 60°C for 30 s. GAPDH, U6, and 3 cells/well). After 24, 48, 72, and 96 h of culturing, 20 μL MTT  was added into each well, and the cells were incubated for 4 h at 37°C with 5% CO2. After the supernatant was discarded, 150 μL dimethyl sulfoxide  was added to dissolve the formazan. The absorbance at 450 nm was measured using a microplate reader .MGC803 and MKN-45 cells were seeded into 96-well plates . The wound healing rate was calculated by the fraction of cell coverage across the line.When MGC803 and MKN-45 cells were cultured at 80% confluence, an artificial scratch was created using a 10 μm pore size) . MGC803 and MKN-45 cells (2 × 105) in serum-free medium were added to the upper chamber precoated with Matrigel . Roswell Park Memorial Institute- (RPMI-) 1640  medium with 10% FBS (Invitrogen) was added to the lower chamber. After 48 h of incubation at 37°C, cells were removed from the upper chamber with a cotton swab, and cells in the lower chamber were fixed in methanol and stained with 0.5% crystal violet for 2 min. Five random fields were photographed.The transwell assay was used to determine the cell invasion using a transwell chamber . The vectors were then cotransfected with mimics NC, miR-3200-5p mimics, and miR-3200-5p mimics+LINC00324 into MGC803 and MKN-45 cells using Lipofectamine 3000 (Invitrogen) for 48 h. The luciferase activity was detected by a dual-luciferase reporter gene assay system (Promega).Bio-LINC00324-Wt, Bio-LINC00324-Mut, and Bio-NC (GenePharma) were transfected into GC cells (MGC803 and MKN-45) using Lipofectamine 3000 (Invitrogen). After culturing for 48 h, cells were incubated with Dynabeads M-280 streptavidin beads  for 1 h. The qRT-PCR assay was employed to assess the enrichment of BCAT1.t-test. The correlation was determined by the Pearson correlation analysis. A P value < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 23.0 . Data were presented as mean ± standard deviation (SD). The differences among multiple groups were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. The differences between two groups were assessed using Student's P < 0.001) (P < 0.01) (P < 0.05), lymphoma metastasis (P < 0.01), and TNM stage in GC patients (P < 0.01). However, LINC00324 expression had no connection with age, gender, and histological grade in GC patients (P > 0.05) (P < 0.01)  . Additio < 0.01) . The cor > 0.05) . qRT-PCR < 0.01) . MGC803 P < 0.01)  (P < 0.01) (P < 0.01)  . si-LINC < 0.05) . The wou < 0.01) . The tra < 0.01) .P < 0.01) (P < 0.01) (P < 0.01) (P < 0.001)  (P < 0.01)  . The dua < 0.01) . In addi < 0.01) . The exp< 0.001) . There w < 0.01) . qRT-PCR < 0.01) .P < 0.01), and overexpression of LINC00324 reversed the reducing effect of miR-3200-5p mimics on the relative luciferase activity (P < 0.05) (P < 0.01) (P < 0.01) (P < 0.001)   (The downstream target of miR-3200-5p was predicted by TargetScan. A binding site of miR-3200-5p on the 3′ UTR of BCAT1 was predicted . Subsequ < 0.05) . The RNA < 0.01) . In addi < 0.01) . These r< 0.001) . There w < 0.01)  and a po < 0.05) .P < 0.01). The expression of BCAT1 in the si-LINC00324+pcDNA-BCAT1 group was markedly decreased compared with that in the si-NC+pcDNA-BCAT1 group (P < 0.01)  and 72 h (P < 0.01) postculturing was markedly increased compared with that in the si-NC+pcDNA-NC group. Silencing of LINC00324 markedly reversed the promoting effect of BCAT1 on the proliferation of MGC803 cells at 72 h postculturing (P < 0.01) (P < 0.01). Silencing of LINC00324 markedly reversed the promoting effects of BCAT1 on the migration and invasion of MGC803 cells (P < 0.01)  . Rescue  < 0.01) . Wound h Figures .Abnormal expression of lncRNAs is related to the tumorigenesis of GC , 21. SomPrevious researches have confirmed that LINC00324 takes part in cell proliferation, invasion, and migration in cancer. Wu et al. have proven that LINC00324 increases the proliferation and migration of osteosarcoma cells via targeting WDR66 . Pan et lncRNAs can serve as molecular sponges to competitively regulate miRNAs in GC. For instance, lncRNA-RMRP promotes cell proliferation by acting as a sponge of miR-206 in GC . lncRNA The expression of BCAT1 is usually upregulated in diverse cancers, such as ovarian cancer , breast In conclusion, the expression of LINC00324 was upregulated in GC tissues and cells. Silencing of LINC00324 inhibited the proliferation, migration, and invasion of GC cells through regulating the miR-3200-5p/BCAT1 axis. Our research indicated that LINC00324 might act as a potential therapeutic target for GC. However, the detailed action mechanisms of LINC00324 on GC remain limited, and further researches are still needed.


Orthodontic management of a non-syndromic patient with concomitant bimaxillary hypohyperdontia: a case report. Dental Press J. Orthod. [Internet]. 2020 Jan [cited 2020 Apr 15] ; 25( 1 ): 36-46.How to cite this article”: Now the article should have the following “Ei Hsu Hlaing Ei, Ishihara Yoshihito, Fujisawa Atsuro, Yamashiro Takashi, Kamioka Hiroshi. Orthodontic management of a non-syndromic patient with concomitant bimaxillary hypohyperdontia: a case report. Dental Press J. Orthod. [Internet]. 2020 Jan [cited 2020 Apr 15] ; 25( 1 ): 36-46.


Azadirachta indica, Azadirachta excelsa, Azadirachta siamens, Melia azedarach, Melia toosendan, and Melia volkensii, have been subject to botanical pesticide evaluation. This review focuses on Melia volkensii, which has not been intensively studied. M. volkensii, a dryland tree species native to East Africa, has shown activity towards a broad range of insect orders, including dipterans, lepidopterans and coleopterans. Its extracts have been reported to have growth inhibiting and antifeedant properties against Schistocerca gregaria, Trichoplusia ni, Pseudaletia unipuncta, Epilachna varivestis, Nezara viridula, several Spodoptera species and other insect pests. Mortality in mosquitoes has also been reported. Several limonoids with a wide range of biological activities have been isolated from the plant, including volkensin, salannin, toosendanin, trichilin-class limonoids, volkendousin, kulactone among others. This paper presents a concise review of published information on the phytochemical composition and potential of M. volkensii for application in insect pest management.Due to potential health and environmental risks of synthetic pesticides, coupled with their non-selectivity and pest resistance, there has been increasing demand for safer and biodegradable alternatives for insect pest management. Botanical pesticides have emerged as a promising alternative due to their non-persistence, high selectivity, and low mammalian toxicity. Six Meliaceae plant species,  The continuous and indiscriminate use of synthetic pesticides in crop protection has led to an increase in pest resistance, health and environmental concerns . This haMeliaceae plant family has been reported to produce a wide range of compounds, including flavonoids, chromones, coumarins, benzofurans, mono-, sesqui-, di-, and triterpenoids, but tetranortriterpenoids with a β-substituted furanyl ring at C17α are the best known for the production of limonoids [Azadirachta indica A. Juss (Indian neem tree) is reported as a potential plant for the control of vector mosquitoes [Melia azedarach Linnaeus (chinaberry) has been reported outlining its use in folk medicine having antifertility, antiviral, cytotoxic, antibacterial, immunomodulatory, repellent, antifeedant, antilithic and anthelmintic activity from various parts of the plant [A. indica has reported its use in agriculture for application as manure, fertilizer, soil conditioner, fumigant, and as botanical pesticide [Melia volkensii (Gurke) has also been identified as one of the pesticidal plants in Africa [The imonoids . Limonoiimonoids . Alkaloiimonoids . Reviewssquitoes . Reviewssquitoes ,7. A phyhe plant ,9. A revesticide . Melia vn Africa . Anothern Africa . Detailen Africa .A. indica (Indian neem tree), Azadirachta excelsa Jack (Philippine neem tree), Azadirachta siamens Valeton (Siamese neem tree), M. azedarach (chinaberry), Melia toosendan Siebold and Zucc., and M. volkensii [A. indica (neem tree) and M. azedarach (chinaberry) [A. indica, have been effective growth regulators and feeding deterrents for a wide range of insect species [M. azedarach have also shown antifeedant activity against the juvenile and adult Xanthogaleruca luteola Muller (elm leaf beetles) and mortality against its larvae [M. azedarach are also effective against Napomyza lateralis Fallen (agromyzid leafminers) and Trialeurodes vaporariorum Westwood (whiteflies) [M. azedarach which has been commercialized in China, is an effective growth inhibitor against Ostrinia nubilalis Hübner (European corn borer), effective repellent against Pieris brassicae Linnaeus (cabbage moth) and an oviposition deterrent against Trichoplusia ni Hübner (cabbage looper) [Several plant species of the Meliaceae have shown promising bioactivity against a variety of insects . Their iolkensii . Howevernaberry) . Azadira species . Azadira species . Extracts larvae . Fruit eteflies) . Toosend looper) . Toosend looper) .M. volkensii, a dryland tree species native to East Africa has, however, not been intensively studied [M. volkensii. These include: 1-O-cinnamoyltrichilin, meliavolkinin, 1,3-diacetylvilasinin, meliavolkin, volkensin, volkensinin, 12β- and 6β-hydroxykulactone, meliavolkenin, meliavolin, meliavolen, melianinone, meliavolkensin A and B, melianin C, (E)- and (Z)-volkendousin, meliavosin, 2-9-epoxymeliavosin [M. volkensii seed kernel extracts have more insect growth inhibitory and acute lethal toxicity than azadirachtin-containing fractions from neem seed kernel extracts [M. volkensii dried fruit powder and residual fruit cake obtained after extraction with ethanol are used as goat feed, their growth and performance are not negatively affected, indicating that both fruit powder and its cake could be used as safe ruminant feed supplement [M. volkensii have also traditionally been used to control ticks and fleas in goats [M. volkensii offers a key indigenous tree species that can be used to mitigate against desertification in arid and semi-arid lands [M. volkensii in insect pest management. studied . It is a studied . The tre studied . It rema studied . Severalextracts . It has pplement . Its usepplement , and trapplement . Aqueousin goats . M. volkid lands , while aid lands . This paM. volkensii have been reported to pose activity towards a broad range of insect orders including Diptera, Lepidoptera, Coleoptera among others [Schistocerca gregaria Forsk. (desert locusts) [S. gregaria when seed extract was applied to their preferred host plants mainly Schouwia thebaica Webb, Fagonia olivieri DC (fagonbush plant) and Hyoscyamus muticus Linnaeus (Egyptian henbane) in a field trial experiment [M. volkensii extracts could affect hormone levels in S. gregaria larvae [S. gregaria, 80% mortality was recorded 48 hours after injection with crude ethanolic and methanolic extracts at a concentration of 30 μg/g of the insect [S. gregaria, M. volkensii and neem oil have been reported to cause mortality of up to 91% and 92%, respectively, after 14 days in a comparative study [Crude fruit extracts from g others  as shownlocusts) . Repelleperiment . Althouga larvae . In fifte insect . When spve study . In contve study . Trichoplusia ni Hübner (cabbage looper) and Pseudaletia unipuncta Haworth (true armyworm) [T. ni (dietary EC50 = 7.6 ppm) and feeding deterrent (DC50 = 0.9 μg/cm2) [M. volkensii extracts has been observed to lead to a decrease in antifeedant response when tested against T. ni implying that the insect could develop tolerance to the extracts [Plutella xylostella Linnaeus (diamondback moth) and P. unipuncta, there was no significant decrease in feeding deterrent response to the extracts following continuous exposure [M. volkensii are responsible for the insecticidal activity in T. ni [M. volkensii extracts, other Meliaceae plant extracts  and commercial botanical insecticides  when tested against T. ni and P. unipuncta [Antifeedant and larval growth inhibitory effects of fruit extracts have been observed in rmyworm) ,28. Crud μg/cm2) . Prolongextracts . Howeverexposure . It has in T. ni . Comparanipuncta . M. volkensii fruit extracts when tested at concentrations ranging from 1 to 50 μg/μL showed feeding deterrence, growth disruption and mortality against Nezara viridula Linnaeus (stink bug), a polyphagous pest which attacks a variety of crops, including nuts, corn, cotton, grains and tomatoes [N. viridula [N. viridula when exposed to fruit extracts, with 10 μg/μL causing malformation in up to 85.70% of surviving adults [Coranus arenaceus Walker even though there were no deformities in resultant adults after topical application of the M. volkensii extracts at 1, 5, and 10 μg/μL [tomatoes . The disviridula . Furtherg adults . A delay10 μg/μL . M. volkensii fruit extract showed potent antifeedant properties against Epilachna varivestis Mulsant (Mexican bean beetle) [P. unipuncta (dietary EC50 = 12.5 ppm) with refined seed extracts to the leaf discs in a choice bioassay [P. unipuncta and P. xylostella, and adults of E. varivestis  [M. volkensii seed extracts have been recorded to have stronger antifeedant activity compared to pure allelochemicals: digitoxin, cymarin, xanthotoxin, toosendanin, thymol and trans-anethole against P. unipuncta, P. xylostella and E. varivestis [Spodoptera litura Fabricius, neem, rotenone, M. volkensii extract, toosendanin, Annona squamosal L. extract and pyrethrum at 1% concentration recorded larval growth (% relative to control) of 4.1, 97.5, 26.2, 48.3, 61.4, and 56.6%, respectively after 96 h in a comparative study [When applied to cabbage leaf disks in a choice bioassay,  beetle) . Growth bioassay . The seectively) . In factrivestis . When apM. volkensii fruit extracts have shown growth-inhibiting activity against Aedes aegypti Linnaeus (yellow fever mosquito) larvae at 2 μg/mL in water, whilst recording high mortality during the molting and melanization process with LC50 of 50 μg/mL in 48 h [A. aegypti could be a result of synergistic effects of a plethora of limonoid compounds or a single active compound exerting these effects [Dried  in 48 h . At a hi in 48 h . Growth  effects . M. volkensii fruit kernel extract showed growth-inhibiting activity against Anopheles arabiensis Giles with an LC50 of 5.4 μg/mL in 48 h [50 of 34.72 μg/mL in 48 h) and oviposition deterrence was observed in second-instar larvae of Culex quinquefasciatus Say (Southern house mosquito) when treated with refined methanolic fruit extracts [M. volkensii fruit acetone extract showed S- and U-shaped postures and frequent stretching in C. quinquefasciatus; such postures and stretching are a characteristic of mosquito larvae reared in M. volkensii fruit extract [C. quinquefasciatus within 36 h [M. volkensii seeds have recorded growth inhibitory effects and equal toxicity (LD50 of 30 μg/mL) for larvae and pupae of C. pipiens f. molestus Forskål (London underground mosquito) [M. azedarach seed extracts recorded lower toxicity (LD50 of 40 μg/mL) while pure azadirachtin A recorded higher toxicity (LD50 of 1–5 μg/mL) against C. pipiens when compared with M. volkensii extracts [M. volkensii may indicate the presence of saponins as toxic principles thus making it an interesting candidate for application against aquatic insects such as mosquitoes and other vectors of diseases [A column chromatography-purified fraction of  in 48 h . Mortaliextracts . The graosquito) . M. azedextracts . The watdiseases .M. volkensii, as shown in M. volkensii. This indicates that insect control bioactivity is, therefore, based on other compounds than azadirachtin [M. volkensii fruit is more lipophilic than azadirachtin [Insect antifeedants have been found in major classes of secondary metabolites—alkaloids, phenolics, and terpenoids . Howeverirachtin . It is pirachtin . Botanicirachtin .1) and salannin (2) have been isolated from seed extracts of M. volkensii [1) and salannin (2) were isolated from the whole fruits of M. volkensii [1) has shown antifeedant activity against Spodoptera frugiperda Smith  larvae with an ED50 of 3.5 μg/cm2 [2) has also shown antifeedant activity against insect pests such as Acalymma vittata Fabricius (striped cucumber beetle), Musca domestica Linnaeus (housefly), Epilachna varivestis Mulsant (Mexican bean beetle), Heliothis virescens Fabricius (tobacco budworm), S. frugiperda and Spodoptera littoralis Boisduval (cotton leafworm) [2) has also been reported to cause feeding suppression against larvae of Earias insulana Boisduval (Egyptian stemborer), weight reduction (59%–89%) in Cnaphalocrocis medinalis Guenee (rice leafroller) and reduction in activities of acid phosphatases (ACP), alkaline phosphatases (ALP) and adenosine triphosphatases (ATPase), implying that gut enzyme activities were affected. 2’,3’-Dihydrosalannin (3), 1-detigloyl-1-isobutylsalannin (4) and 1α,3α-diacetylvilasinin (5) have also been isolated from the plant [The insect antifeedants volkensin  . Salannihe plant .M. volkensii seed extracts, extracted in cold water, have been reported to contain unsaturated fatty acids (oleic acid (6), linoleic acid (7) and gadoleic acid (8)) and saturated fatty acids , stearic acid (10) and arachidic acid (11)) as shown in 6), linoleic acid (7), linolenic acid, and ricinoleic acid have enhanced insecticidal activity of organophosphates and carbamates when applied against sucking insects and defoliating insects [ insects . M. volkensii are shown in 12), which has been isolated from the root bark of M. volkensii [O. nubilalis, an effective repellent against P. brassicae and an oviposition deterrent against T. ni [13) has shown antifeedant activity towards S. littoralis having minimum antifeedant concentration (MAC) value of 1000 mg/L and a significant antibacterial activity against Porphyromonas gingivalis ATCC 33277 with minimum inhibitory concentration (MIC) value of 15.6 μg/mL [14) has been reported to have antifeedant activity against several Spodoptera species  [13, 1-acetyltrichilinin 15, 1-tigloyltrichilinin 16) were tested against Spodoptera eridania Stoll (Southern armyworm) where the 12α-OH function was the most potent, followed by 12β-OH, 12-desoxy, and 12α-acetoxy groups in order of decreasing potency [13, 15, 16) [17, which has weak activity with marginally significant selectivity for breast cancer cell line (MCF-7), has also been isolated from the root bark of M. volkensii [18), isolated from methanolic extract of M. volkensii fruits [19), both insect antifeedants have also been reported [Other chemical compounds that have been isolated from various parts of olkensii , has beest T. ni . 1-Cinnaolkensii . Ohchinii fruits , and melreported . M. volkensii with different biological activities. These include volkensinin, as isolated from ethanolic extracts of M. volkensii root bark [50 = 57 μg/mL) and weak cytotoxicity against six human tumor cell lines with ED50 values of 27.90, 28.35, 33.56, 29.55, 8.43, and 28.51 μg/mL in A-498 (human kidney carcinoma), PC-3 (prostate adenocarcinoma), PACA-2 (pancreatic carcinoma), A-549 (human lung carcinoma), MCF-7 (human breast carcinoma), and HT-29 (human colon adenocarcinoma), respectively [Escherichia coli Migula and Aspergillus niger Tiegh. with respective minimum inhibitory concentration (MIC) values of 12.5 and 6.25 μg/mL [M. volkensii, showed cytotoxicity against six human solid tumor cell lines: A-549, MCF-7, HT-29, A-498, PACA-2, and PC-3 [M. volkensii root bark led to the isolation of meliavolkenin which showed moderate cytotoxicity against three human tumor cell lines with a respective ED50 value of 10.33 μg/mL, 4.30 μg/mL, and 0.67 μg/mL in A-549, MCF-7, and HT-29 cells [M. volkensii [50 values of 0.49 μg/mL and 0.25 μg/mL, respectively [E)-volkendousin, isolated from M. volkensii root bark, also showed activity against six human tumor cell lines  [50 of 11.25 μg/mL, 0.57 μg/mL and 6.65 μg/mL in A-549, MCF-7 and HT-29 cells, respectively [M. volkensii root bark following activity-directed fractionation with brine shrimp test [M. volkensii and exhibited significant activity against E. coli and A. niger with a respective minimum inhibitory concentration (MIC) value of 12.5 and 6.25 μg/mL [Mycobacterium tuberculosis Zopf resulted in the isolation of 12β-hydroxykulactone, 6β-hydroxykulactone and kulonate from M. volkensii seeds with MIC values of 16 μg/mL, 4 μg/mL, and 16 μg/mL, respectively [50 = 0.57 μg/mL), MCF-7 (ED50 = 0.26 μg/mL), and HT-29 (ED50 = 0.12 μg/mL) [M. volkensii include 3-episapelin, meliavolen, melianinone [M. volkensii include scopoletin [Other compounds have also been isolated from oot bark , which sectively . Toosend25 μg/mL . Melianiand PC-3 . Bioacti29 cells . The bioolkensii , have shectively . (E)-volnd PC-3) . Meliavo25 μg/mL . Bioactiopoletin , melianiM. volkensii have proved to be effective insect antifeedants and growth inhibitors. Extensive research has been done on mosquito control using M. volkensii; however, more research needs to be done on insect pests of agricultural importance. M. volkensii has no reported adverse effect on the environment or mammals, making it a potential botanical pesticide for the biosafe application in integrated pest management. The availability of renewable resources from the tree, such as fruits, stem bark, and leaves makes this plant a potential candidate for insect control with minimal interference on the plant. In this regard, M. volkensii could be further exploited as a source of natural insecticide. Extracts and pure compounds isolated from


Escherichia coli isolate INF13/18/A, which was isolated from Universiti Sains Malaysia (USM) Hospital. This isolate was identified as an extended-spectrum β-lactamase-producing Escherichia coli strain harboring the antimicrobial resistance genes TEM, CTX-M-1, and CTX-M-9.We describe here the draft genome sequence and basic characteristics of  Escherichia coli isolate INF13/18/A, which was isolated from Universiti Sains Malaysia (USM) Hospital. This isolate was identified as an extended-spectrum β-lactamase-producing Escherichia coli strain harboring the antimicrobial resistance genes TEM, CTX-M-1, and CTX-M-9.We describe here the draft genome sequence and basic characteristics of  Escherichia coli is one of the most versatile and widely studied species in the world  Hospital.Gram-negative he world . The emehe world . The precrobials . In thisThe clinical sample was collected aseptically and incubated into the Bactec 9240 blood culture system . Gram staining and biochemical testing . The isoE. coli , with read-trimming and filtered read length threshold values of 30 and 50 bp, respectively. The filtered and trimmed reads  were assembled de novo using SPAdes v3.9.0  (E. coli isolates. The complete genome sequences of E. coli O157 strain AR-0427 (GenBank accession no. CP044148.1) and E. coli strain AR216.2b (GenBank accession no. CP043942), which were the top two hits, were selected to scaffold the draft genome using a graph-based approach with Medusa v1.6  v4.10  (usa v1.6 . The assP) v4.10 , which pEscherichia coli INF/13/18/A has been deposited in GenBank under the accession no. WRPL01000000 (BioProject accession no. PRJNA593306 and BioSample accession no. SAMN13474918). The raw reads are available under the SRA accession no. SRR10587526.The whole-genome project for the ESBL-producing strain


Human immunodeficiency virus (HIV) is associated with co-morbid affective and stress-sensitive neuropsychiatric disorders that may be related to dysfunction of the hypothalamic-pituitary-adrenal (HPA) stress axis. The HPA axis is perturbed in up to 46% of HIV patients, but the mechanisms are not known. The neurotoxic HIV-1 regulatory protein, trans-activator of transcription (Tat), may contribute. We hypothesized that HPA dysregulation may contribute to Tat-mediated interactions with oxycodone, a clinically-used opioid often prescribed to HIV patients. In transgenic male mice, Tat expression produced significantly higher basal corticosterone levels with adrenal insufficiency in response to a natural stressor or pharmacological blockade of HPA feedback, recapitulating the clinical phenotype. On acute exposure, HIV-1 Tat interacted with oxycodone to potentiate psychomotor and anxiety like-behavior in an open field and light-dark transition tasks, whereas repeated exposure sensitized stress-related psychomotor behavior and the HPA stress response. Pharmacological blockade of glucocorticoid receptors (GR) partially-restored the stress response and decreased oxycodone-mediated psychomotor behavior in Tat-expressing mice, implicating GR in these effects. Blocking corticotrophin-releasing factor (CRF) receptors reduced anxiety-like behavior in mice that were exposed to oxycodone. Together, these effects support the notion that Tat exposure can dysregulate the HPA axis, potentially raising vulnerability to stress-related substance use and affective disorders. Human immunodeficiency virus type 1 (HIV-1) continues to be a serious health concern with over 1 million infected individuals in the U.S. . HoweverA common, but under-investigated neurological complication of HIV involves disruption of the hypothalamic-pituitary-adrenal (HPA) stress axis. Despite treatment with cART, 14–46% of HIV+ patients demonstrate a dysregulated HPA axis characterized by elevated basal glucocorticoids with seemingly paradoxical adrenal insufficiency in response to a stressor ,8,9. ComThe mechanisms of HIV-mediated HPA dysregulation are not known but may involve neurotoxic HIV-1 proteins. One important therapeutic target that may contribute to these effects is the regulatory protein, trans-activator of transcription (Tat). HIV-1 Tat exerts direct effects on neurons to promote excitotoxic injury and activates monocyte-derived cells (and astrocytes to a lesser extent) to promote neuroinflammation via cytokine production . We haveIn the present study, we hypothesized that HIV-1 Tat and/or oxycodone would interact to promote HPA axis dysregulation, potentiating oxycodone-mediated effects including psychomotor stimulation and affective dysfunction in a conditionally-inducible Tat transgenic mouse model. We further expected pharmacological blockade of GR and/or CRF receptors to restore HPA function and to ameliorate behavioral deficits.In Experiment 1, Tat-tg male mice had HIV-1 Tat expression induced (or not) via doxycycline administration for five days. After two days of doxycycline washout (to limit non-specific anti-inflammatory effects), mice were (or were not) exposed to a 15-min swim stress. Following stress, mice were acutely administered an injection of saline  or oxycodone  and psychomotor and anxiety-like behavior were assessed 15 min later A.F = 5.00, p < 0.05]  = 5.00, p < 0.05]  = 5.04, p < 0.05]  = 27.16, p < 0.05]  = 27.34, p < 0.05] of travel and decreased the frequency  and time spent rearing , compared to saline administration  = 8.69, p < 0.05] and made fewer transitions between compartments  compared to Tat(−) controls  = 5.15, p < 0.05]  controls, Tat(+) mice traveled a significantly greater distance [ < 0.05] B; see *  < 0.05]  in an op < 0.05] . Irrespe < 0.05] A; see † stration . In a licontrols . No signcontrols . When ci < 0.05] C. Tat(+)F = 13.83, p < 0.05] and speed  of travel; however, no differences were observed between Tat(−) and Tat(+) mice  = 13.88, p < 0.05]  mice D, nor we(+) mice . Similar(+) mice . As expe < 0.05] E; see *.While the initial opioid response is indicative of later abuse liability, many HIV+ patients are prescribed oxycodone and are exposed repeatedly. How repeated oxycodone exposure modifies the HPA stress response in Tat-exposed mice was of interest. In Experiment 2, Tat-tg male mice had HIV-1 Tat expression induced (or not) via doxycycline administration for five days (with two days of washout). During this time, mice received daily injections of saline  or oxycodone . Mice were (or were not) exposed to a 15-min swim stress prior to testing A.F = 46.98, p < 0.05]  = 46.68, p < 0.05]  = 6.08, p < 0.05]  = 11.18, p < 0.05]  = 4.05, p = 0.05]; no such effect was observed on Tat(−) controls  = 3.99, p = 0.05]  = 4.93, p = 0.05]  = 14.79, p < 0.05]  mice spent significantly less time in the brightly-lit compartment, compared to Tat(−) mice [ = 0.05] . Irrespe = 0.05] . Tat(+)  < 0.05] C; see *.F = 7.01, p < 0.05]  = 7.10, p < 0.05]  = 3.86, p = 0.05]  = 9.60, p < 0.05]  mice. As seen in Experiment 1, overall motor behavior was reduced following swim stress compared to that observed in non-stressed mice. Repeated oxycodone and genotype interacted such that oxycodone–administered Tat(+) mice traveled a significantly greater distance [ < 0.05] D; see ‡  < 0.05]  than did = 0.05] . No diff = 0.05] . As obse < 0.05] E; see §.In order to begin identifying the important aspects of the HPA axis that are involved in the psychomotor, anxiety-like, and glucocorticoid response to oxycodone and Tat, mice were administered glucocorticoid receptor (GR) and/or corticotrophin-releasing factor-receptor (CRF-R) inhibitors concurrent with doxycycline administration A. To bloF = 2.86, p < 0.05] and speed  traveled. As before, oxycodone significantly increased the distance . All Tat(+) mice administered a GR and/or CRF-R inhibitor demonstrated a modest, but significant, reduction in the distance  mice  = 11.54, p < 0.05]. Pretreatment with RU-486  produced a significant and large increase in circulating corticosterone among Tat(−) mice  = 6.11, p < 0.05]. Irrespective of genotype, at t5 oxycodone-administered mice exhibited significantly lower circulating corticosterone compared to saline-administered mice  and promoting adrenal insufficiency in response to HPA activation to a natural or pharmacological challenge. HPA dysregulation occurred concurrent with anxiety-like behavior and combined Tat and oxycodone-mediated psychostimulation. Repeated oxycodone exposure was able to sensitize the Tat-suppressed HPA response, suggesting potential for pharmacological therapeutic intervention. Blocking GR attenuated combined Tat- and oxycodone-mediated psychostimulation and produced a proportional increase in corticosterone, indicating its importance in these effects. Thus, therapeutics with the capacity to restore the HPA axis may be important mediators of HIV-1 Tat/opioid interactions.Procedures were preapproved by the Institutional Animal Care and Use Committee (IACUC) at the University of Mississippi . All experiments were carried in accordance with ethical guidelines defined by the National Institutes of Health (NIH Publication No. 85-23).n = 324) were bred in the vivarium at the University of Mississippi . Mice were housed 2–5 per cage and be kept in a temperature-and humidity-controlled environment on a 12:12 h light:dark cycle (lights off at 09:00 h) with ad libitum access to food and water. HIV-1 Tat1–86 is conditionally expressed by administration of doxycycline as described previously  via the administration of doxycycline QD for 5 days i.p. Given that doxycycline can attenuate neuroinflammation and could therefore mask potential effects of Tat, 2 days of doxycycline washout was carried out . All behn = 8–9), Tat(+)/saline (n = 7–8), Tat(−)/oxycodone (n = 12), Tat(+)/oxycodone (n = 8–9), and the following swim-stressed groups were yielded: Tat(−)/saline (n = 8), Tat(+)/saline (n = 9), Tat(−)/oxycodone (n = 8), Tat(+) oxycodone (n = 9).To begin to determine the HPA-axis interactions involved in exposure to HIV-1 Tat and acute oxycodone, mice were randomly-assigned to undergo a 15-min swim stress (or not) followed by administration of vehicle  or oxycodone  only once prior to behavioral testing. Fifteen minutes after drug administration, mice were assessed in an open field to determine their psychomotor response followed immediately by assessment in a light-dark transition test to determine anxiety-like behavior A. Acrossn = 7–8), Tat(+)/saline (n = 10), Tat(−)/oxycodone (n = 8), Tat(+)/oxycodone (n = 10), and the following swim-stressed groups were yielded: Tat(−)/saline (n = 8), Tat(+)/saline (n = 8), Tat(−)/oxycodone (n = 8), Tat(+) oxycodone (n = 9).Given that most patients are exposed to opioids on a repeated dosing schedule, some mice were administered sterile saline (0.9%) or oxycodone (3 mg/kg) daily throughout the 7-day doxycycline-induction/washout schedule A. As befn = 8), Tat(+)/saline/vehicle (n = 7–8), Tat(−)/oxycodone/vehicle (n = 8–9), Tat(+)/oxycodone/vehicle (n = 9), Tat(−)/saline/antalarmin (n = 8), Tat(+)/saline/antalarmin (n = 8–9), Tat(−)/oxycodone/antalarmin (n = 8–9), Tat(+)/oxycodone/antalarmin (n = 10), Tat(−)/saline/RU-486 (n = 8–9), Tat(+)/saline/RU-486 (n = 7–8), Tat(−)/oxycodone/RU-486 (n = 8–9), Tat(+)/oxycodone/RU-486 (n = 7–8), Tat(−)/saline/antalarmin+RU-486 (n = 9), Tat(+)/saline/antalarmin+RU-486 (n = 8), Tat(−)/oxycodone/antalarmin+RU-486 (n = 9–10), Tat(+)/oxycodone/antalarmin+RU-486 (n = 9–10).To begin to identify the important receptor sites involved in HIV-1 Tat- or oxycodone-mediated disruption of the HPA axis, some mice were pretreated with the GR antagonist, RU-486, and/or the CRF-R antagonist, antalarmin, prior to testing. RU-486 was administered daily throughout the 7-day doxycycline-induction/washout schedule and 30 min prior to behavioral testing A. Antalan = 5/group) in order to assess the time-course for HPA activation. Prior work revealed peak HPA circulating corticosterone levels in HIV-1 Tat females within 30 min of testing  as the between-subjects factors. For Experiment 3, behavioral and steroid analyses were assessed via separate three-way ANOVAs with pretreatment , drug condition , and genotype  as the between-subjects. For Experiment 4, corticosterone steroid analyses were assessed via repeated measures ANOVA with drug condition  and genotype  as the between-subjects factors and time  as the within-subjects factor. Main effects were followed by Fisher’s Protected Least Significant Difference