Bactrocera dorsalis early embryos, we identified an autosomal-derived microRNA, miR-1-3p, that has predicted target sites in the transformer gene (Bdtra) required for female sex determination. We further demonstrate by both in vitro and in vivo tests that miR-1-3p suppresses Bdtra expression. Injection of a miR-1-3p mimic in early embryos results in 87–92% phenotypic males, whereas knockdown of miR-1-3p by an inhibitor results in 67–77% phenotypic females. Finally, CRISPR/Cas9-mediated knockout of miR-1-3p results in the expression of female-specific splice variants of Bdtra and doublesex (Bddsx), and induced sex reversal of XY individuals into phenotypic females. These results indicate that miR-1-3p is required for male sex determination in early embryogenesis in B. dorsalis as an intermediate male determiner.Regulation of male sexual differentiation by a Y chromosome-linked male determining factor (M-factor) is one of a diverse array of sex determination mechanisms found in insects. By deep sequencing of small RNAs from  Bdtra, which is required for female sex determination.In insects, Y chromosome-linked male determining factor can determine sex, but the sex determination factor of the Oriental Fruit fly is unknown. Here the authors show that an autosomal-derived miR-1-3p is enriched in males, suppressing the expression of  This is exemplified in dipteran insects, such as Drosophila melanogaster, in which the primary sex-determining signal consists of the dose of X chromosome-linked signal elements (XSE)4. A double XSE dose from XX diploid individuals leads to expression of Sex-lethal (Sxl), which directs female-specific splicing of transformer (tra) and doublesex (dsx) pre-mRNA whose gene products sequentially direct female differentiation. In the absence of Sxl expression from a single XSE dose in XY (or XO) individuals, male-specific tra pre-mRNA splicing results in nonfunctional TRA and the subsequent default mode of dsx pre-mRNA male-specific splicing, with a resulting DSX-male product that directs male differentiation7. Although the Drosophila Y chromosome is necessary for male fertility, it has no apparent role in determining somatic and germline sexual differentiation9. This is in contrast to other dipterans, including the mosquitoes Aedes aegypti, Anopheles gambiae and Anopheles stephensi, and the housefly, Musca domestica, in which a dominant male determining factor (M-factor) is the primary determinant of male differentiation13. However, M-factors identified in these species have no obvious sequence conservation, nor is their mode of function necessarily conserved. For example, in M. domestica the paternally inherited male determiner (Mdmd) CWC22 paralog in XY embryos prevents the zygotic activation of the positive Mdtra loop, resulting in the default male-specific splicing of Mdtra and Mddsx pre-mRNAs leading to male differentiation10. The M-factors Nix, Yob/gYG2 and Guy1 from A. aegypti, An. gambiae, and An. stephensi, respectively, exhibit early embryonic transcription and subsequent expression throughout development in their respective species, and are required to initiate male development but have no primary sequence homology13. Since a tra ortholog sequence appears not to be conserved, and is possibly lacking in mosquito genomes, the different M-factors could more directly effect male-specific splicing of dsx pre-mRNA.Primary signals that initiate sex determination during development appear to be highly divergent among many taxonomic groupsBactrocera dorsalis (Hendel), which is one of the most devastating and highly invasive agricultural pests of fruits and vegetables throughout the world14. In contrast to Drosophila, the tra gene in B. dorsalis as well as other dipteran, hymenopteran and coleopteran species20, is able to autoregulate its female-specific splicing, analogous to the Drosophila Sxl gene. As maternal tra transcripts initiate a positive autoregulatory feedback loop in the early zygote, continuous female-specific functional TRA protein is provided and, thus, acts as a cellular memory, maintaining the female-determining signal15. For male development, the presence of an M-factor, which is required to prevent activation of the tra autoregulatory loop, leads to a male-specific nonfunctional TRA protein.Y-linked M-factors have been inferred for several dipteran insects, but have not yet been identified in tephritid fruit flies, including the Oriental fruit fly, B. dorsalis, and other tephritid pests, the foremost biologically-based population control strategy is the sterile insect technique (SIT), which relies on the ability to generate sterile male-only populations for mass release into infested areas21. Understanding the genetic basis of sex determination and male fertility has therefore been central to the development of genetically modified strains for sex separation and sterility. Although Sxl is not involved in tephritid sex determination, orthologs of the Drosophila tra, transformer-2 (tra-2), and dsx genes are functionally homologous7. Therefore, the discovery of M-factors, or other upstream factors, remains fundamentally important for tephritid species. These factors may also include small RNAs as shown in the silkworm moth, Bombyx mori. In this lepidopteran species, a W chromosome-derived female-specific piRNA represses transcription of Masculinizer (Masc), leading to female-specific splicing of Bmdsx pre-mRNA in ZW females22. Thus, this piRNA acts as an essential primary feminizing factor in the ZW sex determination system. In view of this, we propose the hypothesis that small RNAs may also participate in sex determination by regulating the tra autoregulatory loop during early embryogenesis in B. dorsalis.For B. dorsalis, and show that a small autosomally derived RNA, miRNA-1-3p, induces male sex determination by suppressing Bdtra expression during this developmental stage. Embryonic injection of a synthetic miR-1-3p inhibitor, or disruption of miR-1-3p by CRISPR/Cas9, induces a sex reversal of XY chromosomal males into phenotypic females that exhibit female-specific splicing of both Bdtra and Bddsx pre-mRNAs. Conversely, embryonic injection of a synthetic miR-1-3p mimic results in masculinization of XX individuals. These data support the conclusion that the miR-1-3p microRNA has a necessary intermediary function required for the induction of male sex determination in B. dorsalis.In this study, we identify the key embryonic stages in which male sex determination is initiated in B. dorsalis sex determination, we first determined when Bdtra-f and Bdtra-m isoforms appear during embryogenesis. This was achieved by analyzing Bdtra sex-specific splicing isoforms in individual embryos at distinct hours post-oviposition (hpo) time points, thereby distinguishing the chromosomal sex of the embryos and their sexual fate. At 5 hpo, only female-specific Bdtra transcript was apparent in individual embryos, which was most likely due to maternal deposition of Bdtra-f in both XX and XY embryos. Male-specific Bdtra-m transcript was first detectable at 6 hpo while Bdtra-f transcript was not detectable at 7 hpo, presumably in XY male embryos. Embryos that expressed only Bdtra-f transcripts at 6 and 7 hpo were presumably XX . The three libraries produced a total of 6,178,809, 5,695,996, and 9,255,550 clean reads, respectively  . Sixty-five miRNAs were differentially expressed at 5, 6, and 7 hpo, according to a Fisher’s test and a chi-squared test  for co-transfection with the dual-luciferase reporter and miRNA mimics. Whereas co-transfection with the miR-8-3p, miR-10-5p, miR-308-3p, miR-10-3p and miR-305-5p mimics did not significantly alter the luciferase signal, co-transfection with the miR-276a-3p, miR-1-3p and miR-12-5p mimics resulted in approximately 33%, 46% and 32% reductions in luminescence compared to controls, respectively  with corresponding miRNA agomirs (mimics), antagomirs (inhibitors)  had a similar effect on Bdtra expression compared to the dsBdDicer-1 treatment alone   Fig. . A PCR-bes) Fig.  and a T7es) Fig. . This iswhite/brown pupae strain developed for B. dorsalis sexing in the sterile insect technique28. In this strain, XX individuals homozygous for the recessive white pupae (wp−) mutation develop as non-pigmented white pupae, whereas XY individuals, having a wild type wp allele (wp+) translocated to the Y chromosome, develop as normally pigmented brown pupae  mated normally, both the number of eggs per female and their hatching rate were similar to control injected (egfp sgRNA/Cas9) females  brown phenotypic males, 5 phenotypic XX (wp−) white females, and 8 XX (wp−) white phenotypic males  are small noncoding RNAs that regulate spatio-temporal gene expression in various biological processes, including the degradation of maternal mRNAs in the developing embryowhite/brown pupae sexing strain having a Y-linked marker in B. dorsalis. The latter wp strain, in particular, showed that the miR-1-3p specific mutation in XY individuals results in female expression of Bdtra and complete adult feminization, whereas XX females developed normally. This proposed role for miR-1-3p in B. dorsalis male sex determination would be the converse of the proposed role for the Fem piRNA in B. mori, as the primary female-determining factor in the ZW/ZZ sex determination system. Here, the Fem piRNA represses Masc mRNA expression required for male Bmdsx pre-mRNA splicing and male sexual development22. Nevertheless, the roles of miR-1-3p miRNA and Fem piRNA in the regulation of tra and Masc, respectively, indicate that small RNAs, in different orders of insects, have essential roles as post-transcriptional factors during the initiation of sex determination in early embryogenesis. However, miR-1-3p is a non-Y chromosome-linked miRNA with higher transcript levels in early XY embryos, which differs from Fem piRNA which is expressed from the female-specific W chromosome in B. mori22. The dominant component of the silkworm W chromosome is female-enriched piRNAs gene clusters40, whereas the major component of tephritid Y chromosomes is repeat-rich sequences, as reported for C. capitata42. Indeed, the diversity of heterogametic sexual determinants between B. mori and B. dorsalis may explain, in part, the differing roles for small RNAs and mRNAs in regulating sex determination in the two species. Our study extends this pathway of interactions, at least for a tephritid species, by showing that miR-1-3p is a transducer of the Y-linked primary signal for male sex determination and may play a more direct role as a male-determiner by promoting male-specific tra pre-mRNA splicing and, hence, its functional inactivation in XY embryos.In order to verify this hypothesis for the requirement of miR-1-3p for male sex determination, miR-1-3p-specific mutations were generated in a wild type strain and B. dorsalis , whereas miR-1-3p has a more direct role in inhibiting maternal Bdtra expression. The inhibition of maternal Bdtra expression would result in male-specific splicing of zygotic Bdtra pre-mRNA, producing a truncated BdTRA unable to maintain the threshold level needed to sustain the feedback loop of Bdtra expression. Thus, a lack of BdTRA protein in XY would lead to Bddsx-m splicing and the production of the BdDSXM protein that programs male sexual differentiation. However, due to low expression levels of miR-1-3p in female embryos, insufficient inhibition of maternal Bdtra transcript would allow it to translate into functional BdTRA protein to initiate the female-specific autoregulatory loop in the zygotes. BdTRA, in concert with BdTRA-2, would then result in female-specific splicing of the downstream Bddsx gene pre-mRNA, thus promoting female sexual differentiation.These results lead us to propose an updated model for the sex determination pathway in lis Fig. , that miB. dorsalis through a combination of miR-1-3p knockdown, knockout and over expression experiments. This is the first report of a direct role for a miRNA-mediated sex determination mechanism in insects, and the second finding of a role for a small RNA, although the B. mori piRNA and miR-1-3p act conversely. This suggests that small RNAs may have an expanded and varying repertoire of regulatory functions for sex specification in insect species, and perhaps other organisms. For B. dorsalis in particular, manipulation of miR-1-3p may be used for sexing in SIT, for example, by producing male-only populations by its conditional ectopic overexpression in XX females during embryogenesis.In summary, we have shown that miR-1-3p is essential in determining the male sex in B. dorsalis wild type and white/brown pupae sexing  strains were reared at the Institute of Horticultural and Urban Entomology at Huazhong Agricultural University . Adult flies were fed an artificial diet consisting of yeast extract and sugar and cultured at 28 °C under a 12 h light: 12 h dark photoperiod in cages, and hatched larvae were maintained on bananas18. The sex ratio of normally bred adult females to males is approximately 1:1 for wild type and 1:3 for the white pupae strain. We have complied with all relevant ethical regulations for B. dorsalis testing and research.® Reverse Transcriptase  with the oligo (dT) adapter primer. Female and male transcripts from Bdtra were amplified by RT-PCR from 5, 6, 7, 8, and 9 h post-oviposition (hpo) embryo cDNA with Bdtra sex-specific exon primers . To identify the small RNAs involved in B. dorsalis primary sex determination, three small RNA libraries were constructed from the 5, 6 and 7 hpo samples. Total RNAs were extracted using RNAiso Plus reagent  according to the manufacturer’s protocol, with the quantity and purity examined using a Bioanalyzer 2100 .Embryo total RNAs were extracted using RNAiso Plus reagent  according to the manufacturer’s protocol. First strand cDNA was synthesized from 1ug total RNA using PrimeScriptSmall RNA libraries were generated from the three RNA samples using the TruSeq Small RNA Sample Prep Kits, following manufacturer’s guidelines . The 18-30 nt length RNA fractions were separated by gel electrophoresis and ligated sequentially to 3′ and 5′ adapters. Ligation products were then reverse transcribed using a primer to the 3′ adapter with 15 PCRs amplified for the first strand synthesis production with both adapter sequence primers. Ultimately, PCR products were purified, validated and sequenced using an Illumina HiSeq 2500 System .B. dorsalis mRNA database44, RFAM and Repbase to identify possible mRNA, rRNA, tRNA, snRNA, snoRNA and other ncRNAs. The remaining clean reads were aligned to the miRNA precursors/mature miRNA sequences in miRBase and previously identified miRNAs of B. dorsalis45, with the matched or single mismatch sequences, were identified as known miRNAs. Unmapped sequences were aligned to the whole genome sequence (WGS) of B. dorsalis (NCBI Assembly# ASM78921v2) and hairpin RNA structures containing sequences were predicted from flanking 80 nt sequences using RNAfold software (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) complying with criteria from pre-miRNA statistics in miRBase to identify potentially novel miRNAs.After removing low quality reads and adapter sequences from the raw data, large amounts of clean small RNAs were analyzed by BLAST against the Bdtra gene. The abundance of each miRNA in the libraries was transformed through a modified global normalization. Normalization calculation was performed to adjust for the three sequencing libraries. The normalized data were used to calculate fold-change values [=log2(X/Y)] and the log2-ratio plot. The statistically significant difference between each library was tested with a Fisher’s test and a chi-squared test.As male sex determination occurs within 5, 6, and 7 hpo, the expression patterns of miRNAs during these three key embryonic stages were compared to identify differentially expressed miRNAs which target the tra gene in B. dorsalis, three different target prediction programs were used: miRanda46, RNAhybrid47, and TargetScan23. To characterize the interaction of the Bdtra gene (GenBank ID: KP769559.1) with differentially expressed miRNA candidates, the full length sequence of the 3′ UTR of Bdtra and miRNA sequences were submitted on-line to RNAhybrid, an algorithm taking into account the free energy level of RNA-RNA duplexes and degree of miRNA target sequence complementarity using default parameters47.For the prediction of miRNAs targeting the 2.HEK293T cells provided by Prof. Shengbo Cao  were cultured and maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum , and 1% penicillin/streptomycin  at 37 °C in 5% COBdtra 3′ UTR (332 bp) was amplified by PCR from B. dorsalis cDNA and inserted into the psiCHECK-2 vector  which contains the firefly and renilla luciferase gene using Not I and Xho I sites, respectively . After growth in 96-well plates with serum-containing medium for 12 h (104 seeded cells per well), cells were transfected with 50 nM miRNA agomir or the negative control (NC) and 100 ng luciferase reporter vector psiCHECK-2, containing the Bdtra 3′ UTR, in each well using 0.3 μl FuGENE® HD . 48 h after transfection, cells were lysed and the activity of firefly and renilla luciferase was determined with the Dual-Luciferase® Assay System  according to the manufacturer’s protocol. Firefly luciferase values were normalized to renilla, and the ratio of firefly/renilla was presented.The Bdtra gene and a 643 bp fragment of the BdDicer-1 gene (RNase III coding region) were produced by PCR, and a control egfp fragment was amplified with primers from the egfp-containing vector pB[PUbnlsEGFP]48 , purified in elution buffer by MEGAclear  and stored at −20 °C until use. miRNA unique RNA-based mimics (agomirs), inhibitors (antagomirs) and negative control (NC) non-target oligonucleotides were synthesized by GenePharma . miRNA agomir and antagomir sequences are listed in Supplementary Table A 598 bp fragment of the Dicer-1/dstra, or a mixture of 100 μM agomir/antagomir and 2 μg/ul dsRNA according to the transformation procedure described by Sharma et al.10. Equivalent volumes of NCs and dsegfp were injected for controls. Ten hours post-injection, ten embryos from each treatment group were assayed for expression of miR-1-3p and Bdtra that was replicated at least three times for each treatment group. In miR-1-3p agomir/antagomir injection experiments embryos were incubated in an oxygen-saturated container at 28 °C. Surviving larvae were collected and placed on banana media until eclosion. Adults were collected and their emergence rate, sex ratio, and morphological changes were recorded.Newly oviposited embryos (0–1 hpo) were collected and microinjected with 100 uM miRNA agomir/antagomir and mutuant agomir/antagomir or 2 ug/ul ds25. and Zhang et al.26. To design the miR-1-3p specific sgRNA, miR-1-3p mature regions were searched with the protospacer-adjacent motifs (PAMs) of the recognition site for Streptococcus pyogenes Cas9 (NGG) , which revealed no off-target sites suggesting a high specificity of the miR-1-3p sgRNA. The dsDNA template for sgRNA synthesis was generated from template-free PCR, where one specific forward primer and one universal reverse primer overlapped . In vitro transcription of sgRNA and Cas9-mRNA were carried out following the instructions of the MEGAscript® Kit . The sgRNA and Cas9-mRNA were purified with the MEGAclear Kit  and stored at -80 °C until use. Embryos were microinjected with a mixture of purified Cas9-coding mRNA and miR-1-3p sgRNA according to the microinjection protocol described above. Final concentrations of Cas9-coding mRNA and miR-1-3p sgRNA were 300 ng/ul and 150 ng/ul respectively and Cas9-coding mRNA and egfp sgRNA were used as controls. Surviving larvae were placed on banana media until adult eclosion at which time G0 adults were collected with their emergence rate, sex ratio and morphology changes recorded.A miRNA-targeted genomic mutation was created by CRISPR/Cas9 using a miR-1-3p specific sgRNA according to the methods of Ling et al.wp+) brown pupae and phenotypic males that developed from chromosomal female (wp−) white pupae were reared and tested for fertility under standard laboratory conditions. Each female was backcrossed to three virgin normal XY males and each male was backcrossed to three virgin normal XX females in separate cages. Matings were observed at 30 min intervals during the dark photoperiod, and eggs from each cage were collected and counted continuously for 5 days. The newly hatched larvae were counted and then transferred onto banana until adult emergence. To molecularly characterize CRISPR/Cas9 induced mutations, genomic DNA was extracted from G0 individuals following the E.Z.N.A.® Insect DNA Kit protocols . The miR-1-3p target loci were amplified by PCR using the following conditions: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, followed by a final extension at 72 °C for 10 min. PCR products were cloned into pEASY®-T1 vector  and sequenced using GenScript . The PCR products were also used for the T7 endonuclease I (T7EI) assay according to the manufacturer’s instructions . For expression analysis of the Bdtra and Bddsx genes, total RNA was extracted and cDNA prepared from a G0 individual. Primers that were designed to detect mutagenesis in targeted sites and sex-specific expression of Bdtra and Bddsx are listed in Supplementary Table Phenotypic females that developed from chromosomal male , with 200 ng for each sample subjected to reverse transcription of mRNA using the PrimeScriptTM RT Master Mix . Reverse transcription reactions for mature miRNAs were conducted with 1 ug total RNA using miRNA specific stem-loop primers  using the cycling conditions: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. Three biological and three technical replicates were performed with expression data analyzed by the 2−ΔΔCt method49. Dissociation curves were determined for each mRNA and miRNA to confirm unique amplification. The expression of ribosomal protein 49 (rp49) was used as internal controls to normalize the expression of mRNA and the small noncoding RNA U6 was used as internal controls to normalize the expression of miRNA.Quantitative real-time PCR (qPCR) was used to assay the developmental transcript expression profiles of Western blots were performed on normal and miR-1-3p mutant female and male using TRA-specific rabbit polyclonal antibody peptide. The TRA antibody epitope sequences are as follows: SKRWRKERHISTDSSSPERYRKYQNNQKKESEIEPSDKTIRRTKTTKPISDDKYAARRNASPSPNYRRKTPEKIPYFVDQVRERDRIRRKYGSTRNKSPPASSKFRRRRSISRSRSRSHSRDSLKTKQRSPARRTNYRRRSISVDREWGGNSKREREREKSRADKDLHGSPRHYQHRSDDRTKNVRRSRSSRTHSRSRTRSRERSSRIGTQNSERHKYRYNENEEQNGN. Rabbit anti-β-Actin polyclonal antibody was utilized as an endogenous control. Total sample proteins were extracted by RIPA lysis Buffer , with the quantity determined using the BCA Kit . Proteins were subjected to polyacrylamide gel (10%) electrophoresis and transferred onto polyvinylidene difluoride membranes . Blocking was performed in 5% (weight per volume [w/v]) skimmed milk at room temperature for 1 h. The membranes were incubated with primary antibodies  in 5% (w/v) skimmed milk at 4 °C overnight. Secondary antibody   was incubated at room temperature for 1 h. Immunological blot detection was performed using an ECL Luminol Substrate Reagent .t tests using Bonferroni post-tests for multiple comparisons, or with the Student’s t test. For all tests, differences were considered significant when P < 0.05.All experiments were repeated at least three times, except for the small RNA sequencing, and analyzed using GraphPad Prism 5.0  or Microsoft Excel . Results are expressed as the mean ± SEM. Data was compared with either a two-way ANOVA, with subsequent Further information on research design is available in the Supplementary InformationPeer review FileReporting SummarySupplementary Data 1Supplementary Data 2Supplementary Data 3


Aspergillus fumigatus in Taiwan. Resistance emerged mainly from the environment  but occasionally during azole treatment. A high mortality rate observed for azole-resistant aspergillosis necessitates diagnostic stewardship in healthcare and antifungal stewardship in the environment.In a multicenter study, we determined a prevalence rate of 4% for azole-resistant  Aspergillus fumigatus since the late 2000s threatens human health  were defined as azole-resistant A. fumigatus and examined for resistance mechanisms, microsatellite-based phylogenetic relatedness, and growth rates following previously described methods , TR34/L98H/S297T/F495I , and TR46/Y121F/T289A (1 isolate) mutations. This observation is consistent with the estimated global prevalence of azole resistance in Aspergillus (3%–6%) and the predominance of environmental resistance mechanisms in azole-resistant A. fumigatus   recovered in 2018, which colonized a patient without overseas travel, was genetically identical to a clone prevalent in the Netherlands and Tanzania   belongedd Europe  Table 4.34/L98H/S297T/F495I, TR34/L98H, and TR46/Y121F/T289A isolates exhibited cross-resistance to difenoconazole and tebuconazole (both triazole fungicides) without fitness cost, demonstrated by normal growth rates  (Appendix Table 2). The prevalence of TR34/L98H/S297T/F495I isolates in Taiwan might be attributed to widespread use of prochloraz over the past 3 decades. Studies have suggested an association between use of imidazole fungicides and emergence of azole-resistant A. fumigatus with TR34/L98H/S297T/F495I mutations  with A. fumigatus renal abscesses who was receiving voriconazole for >3 months in whom an initial urine isolate was susceptible to azole; all 5 isolates were genetically identical.Six azole-resistant cdr1B (a drug efflux transporter) and an S269P mutation in hmg1 (a hydroxymethylglutaryl-CoA reductase) were identified in 4 resistant isolates but not in the initial susceptible isolate (>5) from a single culture (Cyp51A overexpression and an F262 deletion in hmg1(hmg1F262_del) were identified in these 2 resistant isolates. Although hmg1F261_del was recently reported in azole-resistant A. fumigatus from a voriconazole-exposed patient  and for those from a previous report (Overall, 4 patients harboring azole-resistant s report  Table 6.In conclusion, we report a health threat that arose from clinical and environmental use of azoles; environmental use contributed at a larger and global scale. These data necessitate diagnostic stewardship in the clinic and antifungal stewardship in the environment.Aspergillus fumigatus clinical isolates, Taiwan.Additional information on multicenter study of azole-resistant


Diabetes mellitus (DM) might increase the incidence and mortality of cardiac failure after acute myocardial infarction (AMI) in patients. We attempted to investigate whether Caveolin-3 showed beneficial effects in DM patient post-MI injury through the cAMP/PKA and BDNF/TrkB signaling pathways. The activity of ADRB2 and cAMP/PKA signaling were impaired in nondiabetic ischemia-reperfusion (I/R) group compared with the sham and DM groups and were more impaired in diabetic I/R group than in the I/R group. In H9C2 cells, high-glucose (HG) stimulation further enhanced H/R injury by promoting cell apoptosis, inhibiting cell viability, and suppressing TrkB and Akt signaling; in contrast, the ADRB2 agonist isoprenaline (ISO) significantly attenuated the above-described effects of HG stimulation. Caveolin-3 overexpression promoted the localization of ADRB2 on the membrane of the HG-stimulated H9C2 cells, subsequently inhibiting apoptosis and promoting cell viability. Under HG stimulation, Caveolin-3 overexpression enhanced the activity of the cAMP/PKA and BDNF/TrkB signaling pathways, whereas ADRB2 silencing reversed the effects of Caveolin-3 overexpression. In conclusion, ADRB2 agonist promoted the activity of the BDNF/TrkB and cAMP/PKA signaling pathways, mitigating the HG-aggravated H/R injuries in H9C2 cells. Caveolin-3 exerts a protective effect on diabetic hearts against I/R damage through the β2AR, cAMP/PKA, and BDNF/TrkB signaling pathways. Diabetes mellitus (DM) is regarded as a metabolic disease. Deficiencies in insulin secretion, hyperglycemia, and hyperlipidemia are the major characteristic features of DM. Recently, the incidence of diabetes has gradually increased, and the major cause of the death in diabetic patients is cardiovascular complications . EpidemiIncreasing evidence has shown that neurotrophic factors, including nerve growth factor, can promote myocardial neovascularization and improve myocardial blood flow and heart function to exert beneficial effects on the heart after myocardial infarction . Brain-d2|FC| > 0.56, p < 0.05), including γ-aminobutyric acid receptor, cholinergic receptor, urotensin receptor, endothelin receptor, adrenergic receptor β2 (ADRB2), melanocortin 5 receptor and oxytocin receptor were significantly different. ADRB2 was significantly downregulated  [However, this essential upstream signaling is impaired in the diabetic heart post-MI, mainly due to inhibition of the β-adrenergic pathway that produces cAMP . Under hp <0.05) . Moreovein vitro and in vivo, respectively, through the β2AR, cAMP/PKA, and BDNF/TrkB signaling pathways. In summary, we provide a solid experimental basis for a novel mechanism of Caveolin-3 protecting against myocardial I/R injury in DM hearts.Caveolin-3 is a protein known to protect against myocardial I/R injury. Caveolin-3 regulates cardiomyocyte β2AR (ADRB2) localization on the membrane and mediates downstream cAMP signaling pathways –14. In vRats were subjected or not subjected to induction of DM and evaluated for blood sugar and body weight once every 10 days for 60 days. As shown in As we have mentioned, the cAMP/PKA pathway is impaired in DM, mainly because the upstream β-adrenergic pathway that produces cAMP is inhibited . AccordiSince the β-adrenergic pathway and ADRB2 expression are impaired in diabetic I/R rats, we next, constructed cell models to further investigate the underlying molecular mechanism. H9C2 cells were subjected to H/R injury in the absence or presence of HG stimulation and the ADRB2 agonist ISO and examined for related indexes. Cell apoptosis was significantly promoted by both single H/R injury and the H/R + HG combination and showed a greater increase with the H/R + HG combination –3B; the Regarding the cAMP/PKA signaling pathway, H/R injury induced sharp stress-responsive increases in the cAMP concentrations, p-PKA/PKA ratios, TrkB protein levels, and p-Akt/Akt ratios while the H/R + HG combination significantly attenuated these increases –3G; the Since Caveolin-3 has been reported to promote the localization of β2AR on the cytomembrane and mediate the downstream cAMP signaling –14, we nNext, we investigated whether Caveolin-3 exerts its effects by modulating BDNF/TrkB signaling via ADRB2 and cAMP. We transfected si-ADRB2 to conduct ADRB2 silencing in H9C2 cells and performed Immunoblotting to verify the transfection efficiency –5B. NextHere, we established an I/R model and diabetic I/R model in rats and found that I/R injury in the I/R + DM group was more severe than that in the I/R group. Online microarray profile and PCR-based analysis revealed that the activity of ADRB2 (β2AR) and cAMP/PKA signaling were impaired in the single I/R group compared with the sham group and were more impaired in I/R + DM group than in I/R group. In H9C2 cells, HG stimulation further enhanced H/R injury by promoting cell apoptosis, inhibiting cell viability, and suppressing TrkB and Akt signaling; in contrast, ADRB2 agonist ISO significantly attenuated the effects of HG stimulation on the H/R injured H9C2 cells. Caveolin-3 overexpression promoted the localization of ADRB2 to the membrane in HG-stimulated H9C2 cells, subsequently inhibiting the apoptosis and promoting the cell viability of H9C2 cells. Under H/R+HG stimulation, Caveolin-3 overexpression enhanced the activity of the cAMP/PKA and BDNF/TrkB signaling pathways, whereas ADRB2 silencing reversed the effects of Caveolin-3 overexpression.Increasing epidemiological and clinical evidence has revealed that the hearts of diabetic patients show increased susceptibility to I/R damage –17. Repoin vitro experiments revealed that HG stimulation further enhanced H/R injuries in H9C2 cells, whereas the ADRB2 agonist ISO improved the H/R injuries in H9C2 cells under HG stimulation, as shown by the inhibited cell apoptosis and enhanced cell viability. In addition, the activity of the BDNF/TrkB and cAMP/PKA signaling pathways was partially reversed by ISO administration in the H/R-injured H9C2 cells under HG stimulation, suggesting that the ADRB2 agonist could activate the BDNF/TrkB and cAMP/PKA signaling pathways, therefore mitigating the HG-aggravated H/R injuries in H9C2 cells.The protective effects of the BDNF/TrkB and cAMP/PKA signaling pathways against I/R injury have been reported. Hang et al.  demonstrCaveolin-3 is considered a master type of caveolin family protein in cardiomyocytes that forms caveolae  and actsAn ADRB2 agonist promotes the activity of the BDNF/TrkB and cAMP/PKA signaling pathways, therefore mitigating the HG-aggravated H/R injuries in H9C2 cells. Caveolin-3 exerts a protective effect on diabetic hearts against I/R damage through the β2AR, cAMP/PKA, and BDNF/TrkB signaling pathways.Male Sprague-Dawley rats (SD rats) aged 2-3 months (with an average weight of 300 g) were obtained from the SLAC experimental animal center . DM was induced in SD rats via a single peritoneal injection of STZ at a dose of 40 mg/kg –33 folloBlood samples were collected from the tail vein for the determination of blood glucose levels prior to induction of MI and at the end of study using Accutrend Plus test strips and meters  as described previously –33 to fuTransthoracic echocardiographic images of the hearts from the sham, DM, I/R and I/R + DM groups of rats were obtained at 4 weeks post-MI or sham using an ultrahigh-resolution ultrasound scanner  under isoflurane anesthesia. LVIDs, LVIDd, LVEF and LVFS were recorded as described previously –33. The ® RT Reagent kit with gDNA Eraser (Invitrogen) in accordance with the recommended protocol. RT-PCR was performed using SYBR®Premix Ex Taq™ II (Invitrogen). ABI7500 real-time PCR detection system  was used for detection. β-actin expression levels were used as endogenous control. Finally, the data were processed using the 2-ΔΔCt methods.Total RNA from cardiac tissues was extracted using TRIzol reagent , following the manufacturer’s procedures. Complementary DNA was synthesized from extracted RNA using the Prime ScriptTissues or cells were lysed in RIPA lysis buffer . For membrane protein isolation, cells were lysed by membrane and Cytosol Protein Extraction Kit, according to the manufacture’s instruction (Beyotime). Proteins extracted from tissues or cells were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane , and then incubated with the primary antibody followed by anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase . The primary antibodies used were as follows: anti-TrkB (ab18987), anti-Akt (ab32505), anti-p-Akt (ab81283), anti-Bax, anti-Bcl-2anti-ADRB2 (ab182136), anti-PKA , anti-p-PKA (ab75991), and anti-Caveolin-3 (ab2912). β-actin  was used as an endogenous control for total protein. Na/K ATPase  was used as an endogenous control for membrane protein. All antibodies were obtained from Abcam unless otherwise stated.5 cells/well. After treatment and washing with ice-cold PBS, cells were lysed with RIPA buffer (Beyotime). The supernatant was obtained by a centrifugation and examined for the concentration of cAMP using cAMP ELISA kit.The intracellular cAMP concentration was determined by a rat cAMP ELISA kit . Target cells were plated in a 24-well plate at a cell density of 5 × 102.H9C2 cells, rat embryonic myoblasts, were obtained from ATCC  and cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37°C in 5% COThe Caveolin-3 overexpression vector, empty pcDNA3.1 vector, si-NC and si-AADRB2 were purchased from Genetop Medical Tech.co. ltd . 1 μg/ml vector or 20 nM siNRA were transfected into H9C2 cells using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were harvested for further experiments. The primers for plasmid construction and sequence for interference were listed in 2, 5% CO2 and 1% O2 for 5 min, subjected to hypoxia for 12 h at 37°C and reoxygenated for 12 h by exposing the cells to a cell incubator. The cells were stimulated with NG (5 mmol/L glucose) or HG (30 mmol/L glucose) in the presence or absence of the ADRB2 agonist isoproterenol  during reoxygenation.H9C2 cells were placed in a hypoxic vessel filled with the mixture of 94% NA modified MTT assay was used to evaluate cell viability following the previously described methods . DMSO waCell apoptosis was determined using an Annexin V-FITC apoptosis detection kit  following methods described previously . PropidiADRB2 protein content and distribution were analyzed by performing IF staining using anti-ADRB2. The secondary antibody FITC-conjugated donkey anti-rabbit IgG (1:500) was obtained from Abcam. The nucleus was stained with DAPI. The green fluorescence represents the ADRB2 protein and blue fluorescence represents the nucleus.P < 0.05 were considered statistically significant.All data from at least three independent experiments were analyzed using SPSS 13.0 software  and are presented as the mean ± SD. The differences between groups were analyzed by one-way ANOVA , and multiple comparisons were performed by the Bonferroni correction. Differences with The protocol in the study was approved by the Institutional Animal Care and Use Committee at The Second Xiangya Hospital. The animal experiments were approved by our institutional review board.All data generated or analyzed during this study are included in this published article. Further details are available on request.Consent for publication was obtained from the participants.Supplementary Figure 1Supplementary Table 1


Pseudomonas aeruginosa. However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL  showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14.Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of  Pseudomonas aeruginosa. However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL  showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14. Spontaneous mutants selected on Pmx-B, harboring loss of function mutations in the PhoQ sensor kinase gene, showed increased Pmx-B MICs and arnB operon expression (4-amino-l-arabinose addition to lipid A), but remained susceptible to dendrimers. Two mutants carrying a missense mutation in the periplasmic loop of the PmrB sensor kinase showed increased MICs for Pmx-B (8-fold) and G3KL (4-fold) but not for the dendrimer T7 . The pmrB mutants showed increased expression of the arnB operon as well as of the speD2-speE2-PA4775 operon, located upstream of pmrAB, and involved in polyamine biosynthesis. Exogenous supplementation with the polyamines spermine and norspermine increased G3KL and T7 MICs in a phoQ mutant background but not in the PA14 wild type. This suggests that both addition of 4-amino-l-arabinose and secretion of polyamines are required to reduce susceptibility to dendrimers, probably neutralizing the negative charges present on the lipid A and the 2-keto-3-deoxyoctulosonic acid (KDO) sugars of the lipopolysaccharide (LPS), respectively. We further show by transcriptome analysis that the dendrimers G3KL and T7 induce adaptive responses through the CprRS two-component system in PA14.Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of  Paenibacillus polymyxa (2+ ions that stabilize the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria  represent the most diverse class of antibiotics. They are synthesized by animals, plants, and microorganisms and show a broad spectrum of activity , 2. Theipolymyxa . The posbacteria –7. Subseacteria –, 9. The acteria ––12.Pseudomonas aeruginosa clinical isolates as well as Acinetobacter baumannii and Enterobacteriaceae harboring extended-spectrum β-lactamases (ESBLs)  , 22. G3K (ESBLs)  and IC50HO cells . G3KL anHO cells . FurtherHO cells . G3KL-liSince the physicochemical properties of the synthetic dendrimer antimicrobial peptides differ substantially from natural AMPs like polymyxins, we wondered whether dendrimers and polymyxins were affected by similar resistance mechanisms. Surprisingly, attempts to select spontaneous mutants with G3KL were unsuccessful; however, mutants selected on Pmx-B, harboring specific mutations in the PmrB sensor kinase, showed decreased susceptibility to both G3KL and Pmx-B. We characterized these mutants and found that two different mechanisms affecting the surface charge of the LPS are required to achieve decreased susceptibility to G3KL.P. aeruginosa reference strains and MDR clinical isolates (MIC = 8 mg/liter) (P. aeruginosa strain PA14 on agar plates using polymyxin B (Pmx-B) as a comparator . We thus performed a selection in liquid medium in a microtiter plate format, exposing PA14 to increasing G3KL and T7 concentrations. After six serial passages, we were still unable to obtain a stable mutant showing increased MICs to G3KL, although such mutants emerged readily with Pmx-B. However, one mutant colony (2P4) was obtained after six passages with the dendrimer T7.G3KL and T7 are representatives of third-generation dendrimer molecules . They dig/liter) . To evalmparator . While PTo investigate a potential cross-resistance between the different AMPs, we tested colonies obtained during the initial Pmx-B selection on agar plates for their susceptibilities to G3KL and T7. Among seventeen randomly selected colonies from the Pmx-B-supplemented plates (4× the MIC of Pmx-B), fifteen were resistant to Pmx-B alone (4.1 to 4.17) and only two colonies (4.18 and 4.19) showed increased MICs for both Pmx-B and G3KL . The single colony selected on T7 showed decreased susceptibility to Pmx-B and to the other dendrimer G3KL . Hence, pmrB gene, encoding another TCS sensor kinase . The remaining colony displaying cross-resistance to Pmx-B and G3KL (4.19) carried a single nt change in pmrB, resulting in a Phe-to-Cys substitution at position 124, located again in the periplasmic loop of PmrB  was submitted to whole-genome sequencing. Mutant 4.13, which is resistant only to Pmx-B (MIC = 8 mg/liter), showed a 4-nucleotide (nt) insertion at position 1,105, resulting in a frameshift mutation at codon Val369 of PhoQ, a two-component system (TCS) sensor kinase frequently mutated in colistin-resistant clinical isolates  25). Th. Th25). r kinase . This mur kinase . The mut of PmrB . Based o of PmrB . We concphoQ mutant 4.13, and the two pmrB mutants 4.18 and 2P4 at 6× the MIC of either Pmx-B or G3KL. Pmx-B caused a 2-log reduction in CFU after 24 h for the wild-type, while CFU of the phoQ and the two pmrB mutants remained unaffected  and its addition to the lipid A sugar portion of the LPS  arnB levels were increased 4- to 12-fold. The expression of arnB was induced in the presence of 0.25× MIC of G3KL in the wild-type strain as well as in the two cross-resistant mutants 4.18 and 4.19, but not in mutant 2P4. This suggests that modification by the arn operon could affect susceptibility to G3KL and T7. We therefore constructed a deletion in the arnT gene, encoding the l-Ara4N transferase. In the PA14 wild type this resulted in a weak but reproducible 2-fold decrease in G3KL MICs, without affecting Pmx-B MICs , which have been reported to be regulated by the PmrAB TCS , but not in the wild-type PA14  and norspermidine (C6H17N3), both carrying three amine groups, had no effect on AMP MICs. Instead, we observed that spermidine increased susceptibility to Pmx-B and G3KL by 2- to 4-fold, but only for the pmrB mutants  and norspermine (C9H24N4), both harboring four amine groups, increased MIC values of Pmx-B and G3KL in the phoQ mutant 4.13 but not in the wild type  of Pmx-B, G3KL, or T7. Overall, Pmx-B exposure resulted in a broader response compared to dendrimers. Indeed, PA14 responded to Pmx-B by upregulating 73 and downregulating 108 genes. In comparison, G3KL and T7 induced, respectively, upregulation of 29 and 21 genes and downregulation of 18 and 27 genes . Not unexpectedly, genes that were upregulated by all three AMPs included the complete arn operon genes as well as those of the speD2 operon (arn operon (8- to 12-fold change), while Pmx-B and G3KL elicited a lower response (3- to 4-fold change). Expression of the speD2 operon was induced 4- to 15-fold by Pmx-B and T7 and only 3-fold by G3KL. As expected from our quantitative real-time PCR (qPCR) data , a gene located next to the cprRS operon, and PA14_41280 (PA1797), located next to the parRS operon. While the former was induced only by the dendrimers, the latter responded strongly (54-fold upregulation) to Pmx-B but not to the dendrimers (<2-fold). We also observed an 8- to 9-fold induction by Pmx-B of the mexXY genes, encoding the MexXY efflux pump involved in aminoglycoside export  and shown to be involved in Pmx-B resistance in P. aeruginosa strain PAK  data , expresse export . Two othrain PAK  and (ii)eostasis . In summprRS TCS .P. aeruginosa by G3KL compared to Pmx-B, or to the requirement of specific mutations located in the periplasmic domain of the PmrB sensor kinase gene. The fact that 20% of spontaneous mutants selected on Pmx-B also showed decreased susceptibility to G3KL, but not to T7, indicates that only partial cross-resistance exists between Pmx-B and certain dendrimers. Indeed, analysis of additional dendrimer peptides revealed heterogeneity in their MICs for the selected mutants (data not shown). All of the mutants resistant only to Pmx-B carried exclusively loss-of-function mutations in the sensor kinase PhoQ of the PhoPQ two-component system, as observed in polymyxin-resistant clinical isolates of P. aeruginosa   is cotranscribed with speE2  core sugars available for potential interactions with polyamines  . These m2 operon . The speth speE2  and is rermidine , 37, 38.lyamines . The secycosides , polymyxycosides . Upon coarn and speD2 operons) as those overexpressed in the pmrB mutants. However, the response to G3KL was different between the three pmrB mutants. While the mutant strains 4.18 (PmrB Thr132Pro) and 4.19 (PmrB Phe124Cys) still responded to G3KL, as indicated by induction of the arnB gene, the PmrB D47E mutation present in mutant 2P4 resulted in constitutive high-level expression of arnB independent of G3KL  at 37°C with agitation (250 rpm). E. coli strain DH10B was used as a host for cloning experiments.Bacterial strains and plasmids used in this study are shown in Dendrimers were synthesized and purified as described , 42. PolP. aeruginosa mutants resistant to polymyxin B (Pmx-B) were selected on Mueller-Hinton broth (MHB) agar plates supplemented with 4 mg/liter of Pmx-B. An 150-μL aliquot of P. aeruginosa culture grown in LB medium was spread on these plates and incubated overnight at 37°C and then at room temperature until the appearance of P. aeruginosa colonies.Spontaneous 600) of 1.0). DNA libraries were sequenced on an Illumina HiSeq instrument (100-bp paired-end reads) at the iGE3 Genomics platform of the University of Geneva.Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen) from bacterial strains grown in LB medium to midexponential phase  and added at the indicated concentrations.MICs for all strains were determined in Mueller-Hinton broth (MHB) by broth microdilution in accordance to the Clinical and Laboratory Standards Institute (CLSI) guidelines . AMP con600 = 2.0. Fifteen microliters from these bacterial suspensions were added to the first row of a microtiter plate containing 135 μl of M9-salts medium  supplemented with 2 mM MgSO4 and either 6 mg/liter of Pmx-B or 48 mg/liter of G3KL. The microtiter plates were incubated at 37°C. At each time point , serial dilutions were spotted in triplicate onto LB agar plates and CFU counts were determined after incubation at 37°C.Bacteria were grown overnight in LB medium. For the Pmx-B and G3KL preincubation, bacteria were grown with 0.25× MIC of the corresponding compound. Next, 500 μl of culture was centrifuged at 5,000 rpm for 3 min and pellets were resuspended in M9-salts medium and adjusted to ODFor RNA extraction, the strains were grown under MIC assay conditions in microtiter plates in MH broth, supplemented or not with 0.25× the MIC of Pmx-B, G3KL, or T7. Three wells were pooled to form one sample. RNA was extracted using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions. Genomic DNA was removed by treatment with RNase-free DNase (Promega). Five hundred nanograms of RNA was reverse transcribed using ImProm-II reverse transcriptase (Promega).rpsL housekeeping gene (Gene expression was measured using a Rotor-Gene SYBR Green PCR kit (Qiagen), with primers specific to the genes of interest and the ing gene . qPCRs wNC_008463.1Pseudomonas aeruginosa UCBP-PA14 reference genome using BWA aligner v.0.7.17 software. The counts were normalized according to the relative number of reads for each library, and genes having a count above 1 count per million reads (cpm) in at least 3 samples were kept for the analysis. The starting raw gene number of the set was 6,100. The final filtered data set consisted of 6,043 genes after excluding poorly or nonexpressed genes.Approximately 1.3 μg of total RNA for each replicate sample was ribodepleted using the Ribo-Zero rRNA removal kit for bacteria (Epicentre) according to the manufacturer’s protocol. The TruSeq total RNA stranded kit (Illumina) was used for the preparation of libraries. A Quibit spectrophotometer measured their quantity and a Tapestation on a DNA High-sensitivity chip (Agilent Technologies) determined their quality. The 12 generated libraries were pooled at equimolarity and loaded at 7 pM for clustering. An Illumina HiSeq 4000 instrument was used to sequence 100-bp single reads per sample (iGE3 Genomics platform of the University of Geneva). FastQC v.0.11.5 was used for sequencing quality control and reads were mapped to the NCBI E. coli mobilizing strain S17-λpir. After conjugation into P. aeruginosa, mero-diploids were selected on M9-salts medium plates supplemented with citrate (20 mM) and gentamicin (50 mg/liter). To select for recombinants which had lost the integrated plasmid, cell suspensions were streaked on M9-salts agar plates supplemented with 20 mM citrate and 10% sucrose. Sucrose-resistant and gentamicin-susceptible colonies were screened by PCR for the presence of the expected deletion in the corresponding target gene. Two independent clones for each mutant were selected for further analyses and frozen as glycerol stocks at −80 °C.The upstream and downstream flanking regions of the target locus were PCR amplified  using primers with HindIII/BamHI or BamHI/EcoRI restriction sites for the upstream and downstream flanking regions, respectively. DNA fragments were ligated and inserted into the suicide vector pEXG2  using HiP. aeruginosa strain PA14. All primers contained BamHI and HindIII restriction sites. DNA fragments were cloned into the expression vector pIApX2 (P. aeruginosa by electroporation and transformants were selected on LB agar plates supplemented with 200 mg/liter of carbenicillin. Two independent colonies were chosen for further analyses.The DNA regions with at least 60 nucleotides upstream of the ATG initiation codon and downstream of the STOP codon were amplified by PCR from genomic DNA of r pIApX2 . PlasmidGSE139257.The meta-data and raw data generated in this study are available at GEO under the following series record:


Enterovirus A71 (EV-A71) is a major causative agent of hand, foot, and mouth disease (HFMD) and herpangina. Moreover, EV-A71 infection can lead to neurological complications and death. Vaccination is the most efficient way to control virus infection. There are currently three inactivated, whole EV-A71 vaccines licensed by the China NMPA . Several other types of vaccines, such as virus-like particles and recombinant VP1 (capsid protein), are also under development. In this review, we discuss recent advances in the development of EV-A71 vaccines. Picornaviridae. EV-A71 consists of three distinct genotypes . Genotype B is divided into subgenotypes B1 to B5. Genotype C is divided into subgenotypes C1 to C5, and C4 is further divided into C4 a and C4 b. Subgenotypes B3, B4, C1, and C2 co-circulated in the Asia–Pacific region from 1999 to 2016. C4 is the most prevalent genotype circulating in China; C1 and C2 are most prevalent in Europe while B4 and B5 are most prevalent in other regions [Enterovirus A71 (EV-A71) is one of the most important neurotropic viruses and poses a serious threat to public health worldwide. EV-A71 is a major causative agent of hand, foot, and mouth disease (HFMD) which is highly contagious. EV-A71 infection in young children can also lead to neurological complications, rapid fatal pulmonary edema, myocarditis, hemorrhage, and death ,3,4,5. T regions .EV-A71 is a non-enveloped virus containing a positive-stranded RNA genome ~7400 nucleotides in length. The viral genome within its icosahedral capsid is flanked by highly structured 5′ and 3′ untranslated regions (UTR) and is polyadenylated at its 3′ end. The large single polyprotein encoded by the viral genome is subsequently processed by viral proteases 2 A and 3 C into P1, P2, and P3.P1 is further processed to produce four capsid proteins—VP1 to 4. P2 and P3 are processed into seven nonstructural proteins—2 A−2 C and 3 A−3 D—for viral replication . The VP1Currently, there is no US FDA-approved antiviral agent or vaccine against EV-A71. However, there are three inactivated, EV-A71 whole-virus vaccines approved by the China National Medical Products Administration (NMPA) and are commercially available in China. Large-scale clinical trials showed that vaccine efficacy against EV-A71-associated Hand, foot, and mouth disease (HFMD) was at least 90%. Vaccine effectiveness in HFMD patients under five years old was 100% for severe cases and ~80% for mild cases, suggesting that the vaccine performs well in practice . VaccinaInactivation of whole virus is the conventional and safest way to produce viral vaccine . There aThe safety and efficacy of inactivated EV-A71 vaccines had been confirmed in large phase III clinical trials . VaccineCAMS, Vigoo, and Sinovac  initiated the phase I trials between December 2010 and Feburary 2011. The phase III trials were completed in 2013. CAMS recruited 12,000 infants and children of 6 to 71 months of age for phase III clinical trials . Children randomly received two doses of 100 U (EV-A71 antigen unit) CAMS vaccine or placebo four weeks apart and were followed for two years. The EV-A71 antigen contents were measured by a quantitative ELISA. Based on the EV-A71 antigen reference standard (1600 U/mL) established by the China NMPA, the specific activity of EV-A71 antigen is 421.1 U/μg ,23.The eIn the Sinovac  phase III trial (NCT01507857), 10,077 children aged 6 to 35 months were given 400 U of Sinovac vaccine or placebo at days 1 and 28 and were followed for two years. At the end of the first year, the vaccine efficacy against EV-A71-induced HFMD and herpangina was 94.8%. The efficacy against EV-A71-associated HFMD that manifested to neurological complications was 100%. At the end of the second year, the vaccine efficacy against EV-A71-associated HFMD was 95.1% . SinovacThe National Health Research Institutes (NHRI) in Taiwan used a subgenotype B4 strain to produce the inactivated whole virus EV-A71 vaccine ,27. For The Korea National Research Institute of Health  developed an inactivated whole virus vaccine against EV-A71 using an EV-A71 strain (genotype C4 a) isolated from a Korean patient. . The vacIn Thailand, there was an early development of an inactivated EV-A71 vaccine. A genotype C4 EV71-A71 strain was grown in Vero cells in roller bottles. The purified, inactivated virus was able to induce neutralizing antibodies against EV-A71 in BALB/C mice. This prototype product will be further developed into an EV-A71 vaccine candidate .Live-attenuated vaccines have the advantages of long-lasting immunity and cost-effective production; however, the molecular pathogenic mechanism of EV-A71 is not fully understood. The EV-A71 virulence determinants have not been completely elucidated. However, several EV-A71 virulence determinants have been identified in the 5′UTR, VP1, VP2, 2 A, 3 C, and 3 D . One of The vaccine candidate EV7-A71 (S1–3′), a temperature-sensitive mutant of the prototype BrCr, demonstrated induction of an efficient immune response to a lethal dose of EV-A71 (BrCr-TR) (a temperature-resistant mutant of BrCr) in cynomolgus monkeys. However, inoculation of EV-A71 (S1–3′) caused neurological symptoms in monkeys. Four and 10 days after inoculation, infectious virus was isolated from the monkeys’ lumbar spinal cords. This raised serious safety concerns about this vaccine candidate .Increasing the fidelity of the 3 D RNA-dependent RNA polymerase enhances the safety and stability of live-attenuated vaccines. G64 R, G64 T, and S264 L mutations in 3 D enhanced the polymerase fidelity. G64 R and L123 F mutations in 3 D polymerase attenuated EV-A71 virulence in mice ,40. BaseVLP (virus-like particle) is a promising vaccine candidate because it resembles the authentic virus in appearance and antigenicity. VLP can elicit innate and adaptive immunity and is safer because the lack of a viral genome makes them incapable of replicating in the host. Several VLP-based vaccines, such as Recombivax HB and Engerix-B for hepatitis B virus, and Gardasil, Cervarix, and Gardasil−9 for human papillomavirus, have been licensed ,44.Several strategies have been employed to produce EV-A71 VLPs. The baculovirus expression system is widely used for the production of VLPs. To produce EV-A71 VLPs, insect cells were infected with a recombinant baculovirus co-expressing the EV-A71 P1 region and the viral protease 3 CD from various promoters ,46. The Pichia pastoris. The EV-A71 VLP yield was high and potently induced neutralizing antibodies against various strains of EV-A71 in a mouse model [Zhang et al.  improvedse model . More imse model ,51.Tsou et al. and Yan et al. used Adenovirus and Vesicular stomatitis virus-based vaccines on expressed EV-A71 VLPs in mammalian cells. Both VLPs elicited antibodies against EV-A71 infection in mice ,53.Hand, foot, and mouth disease (HFMD) is highly contagious and has led to significant morbidity and mortality worldwide . EV-A71,Escherichia coli (E. coli), baculovirus, and HIV gag-based VLP carrier were used to express recombinant VP1 protein. The purified recombinant VP1 protein protected mice from EV-A71 infection; however, the protection was lower than that of inactivated EV-A71 vaccine [The EV-A71 VP1 capsid protein contains neutralization epitopes. Several expression strategies, such as  vaccine ,57,58,59Pichia pastoris as a vaccine candidate. P1 protein induced persistent, high cross-neutralization antibodies for different EV-A71 subtypes in rabbits. Vaccination of pregnant mice cross-protected neonatal mice against different subtypes of EV-A71. EV-A71 antibody production elicited by P1 was one week later than that of inactivated EV-A71 virus. However, the level and duration of EV-A71 antibody production was stronger than that of inactivated EV-A71 virus. This could be related to antigen glycosylation [P. pastoris-expressed P1 protein is a promising EV-A71 vaccine candidate.Han et al.  expressesylation  as studisylation ,62. WithSynthetic peptides are safe and efficacious for multivalent vaccine development. Several groups have focused on mapping the antigen epitopes in EV-A71 capsid proteins (VP1–4).Foo et al.  used synLiu et al.  selectedAw-Yong et al.  synthesiE. coli. The recombinant protein elicited neutralizing antibodies against EV-A71 in a mouse model.Several groups used a fusion protein strategy to express EV-A71 epitopes. Xu et al.  fused EVEV-A71 is a major causative agent of HFMD, and EV-A71 infection has also led to neurological complications and death in young children worldwide. Vaccines are the most effective way to prevent EV-A71 infection.The primary strategy to develop EV-A71 vaccines is to use viral structural proteins as immunogens. The inactivated whole-virus vaccine, live-attenuated virus vaccine, VLP vaccine, recombinant VP1 vaccine, and synthetic peptide vaccine all deliver wholly or partially expressed viral proteins to the host to elicit host immunogenicity and produce neutralizing antibodies. The inactivated, whole-virus vaccines yield high immunogenicity levels with high neutralization titers and induce cross-genotype neutralizing antibody responses more effectively. Currently, there are three inactivated, whole-virus vaccines against EV-A71 approved by the China NMPA, and WHO recommendations believe the Sinovac vaccine could be used worldwide . HoweverThe advantages of recombinant VP1 and synthetic peptide vaccines are that they are safer and more cost-effective. These vaccines reduce the risk of unwanted immune responses such as antibody-dependent enhancement (ADE). ADE is a general concern for vaccine design because the mechanisms underlying antibody protection against any pathogen infection have a theoretical potential to amplify the infection or trigger harmful immune responses . HowevermRNA vaccines represent a promising alternative vaccine platform technology because of their high potency, rapid development, completely synthetic nature, low-cost manufacturing process, and safe administration. Nine months after the COVID−19 pandemic began, the US government granted emergency-use authorization to two COVID−19 mRNA vaccines—Pfizer-BioNTech  and Mode


The Rho family of GTPases consists of 20 members including RhoE. Here, we discover the existence of a short isoform of RhoE designated as RhoEα, the first Rho GTPase isoform generated from alternative translation. Translation of this new isoform is initiated from an alternative start site downstream of and in-frame with the coding region of the canonical RhoE. RhoEα exhibits a similar subcellular distribution while its protein stability is higher than RhoE. RhoEα contains binding capability to RhoE effectors ROCK1, p190RhoGAP and Syx. The distinct transcriptomes of cells with the expression of RhoE and RhoEα, respectively, are demonstrated. The data propose distinctive and overlapping biological functions of RhoEα compared to RhoE. In conclusion, this study reveals a new Rho GTPase isoform generated from alternative translation. The discovery provides a new scope of understanding the versatile functions of small GTPases and underlines the complexity and diverse roles of small GTPases. Dai et al. report the identification and characterization of a new isoform of RhoE (RhoEα), a member of the Rho GTPase family, which is generated from the same gene by alternative translation initiation at the downstream ATG codon 46. Compared to RhoE, RhoEα does not differ in the subcellular localization but has increased protein stability and distinct molecular signalling profile. Rho GTPases have shown to regulate many aspects of intracellular actin dynamics, such as cytoskeleton rearrangement, cell migration, vesicular trafficking, cell polarity, cell cycle, and transcriptional dynamics5. In mammals, the Rho family contains 20 members1. Though the majority of them are respectively activated and deactivated by the GTP-bound and GDP-bound cycling, “atypical” GTPases in the Rnd subgroup are unusual as they are constitutively GTP bound7. Therefore, it is not regulated by GTP/GDP cycling, but by the expression level and by protein modifications, such as prenylation and phosphorylation4. There are three members, Rnd1, Rnd2, and Rnd3/RhoE, in the Rnd subgroup and Rnd3/RhoE is the most extensively studied member. The current paradigm, derived from the majority of the published studies on RhoE, is that RhoE functions as a repressor that directly inhibits the RhoA effector Rho-associated coiled-coil kinase 1 (ROCK1)9. While the regulatory roles of RhoE have been almost entirely linked to cell actin cytoskeleton dynamics, cell migration, and apoptosis in its early studies13, we have uncovered versatile functions of RhoE in intracellular Ca2+ homeostasis regulation14, apoptosis15, angiogenesis16, inflammation17, brain development18, and glioblastoma genesis20 along with other groups24. All of these studies suggest a broad spectrum of biological and pathological functions of RhoE.The Rho proteins belong to the Ras superfamily and these small GTPases function as G signaling proteins that convert and amplify external signals into cellular effects26, we report here that RhoEα is the first Rho GTPase isoform generated from alternative translation, demonstrating the diversity of the GTPase family. The study opens a new perspective for future studies in the exploration of Rho GTPase multiplicity and the interpretation of their diverse functions.In this study, we discovered the existence of a new short isoform of RhoE, which we designated as RhoEα. Using multiple approaches, we identified a new alternative translation start site responsible for the expression of RhoEα protein, and this site is located downstream of the classical RhoE translation start site. The RhoEα protein characterizations, including subcellular distribution and protein stability were defined. Interactions of RhoEα to RhoE effectors were determined as well. Finally, the potential differential functions of the two RhoE isoforms were investigated and compared using RNA-seq approaches. While new isoforms derived from alternative splicing were identified in Rho GTPase family before17. Figure RhoE in these cells  along with the sgRNA specifically targeting ted Fig. , indicatTo provide definitive evidence, mass spectrometry analysis was performed in two protein bands ~27 kDa immunoprecipitated by RhoE antibody Fig. . The mas30. To determine whether these mechanisms are responsible for existence of the additional RhoE protein band, we first generated RhoE knockout cell line by CRISPR technique in HeLa cells, and then an expression vector containing human RhoE 5′ untranslated region (UTR) and coding sequence was transiently transfected into this cell line. We detected the expression of both RhoE and the additional band in the transient transfection cells  in RhoE mRNA coding region and found five additional ATG codons in-frame with ATG1. They were ATG46, ATG388, ATG493, ATG658, and ATG730 , which were involved in the regulation of cell protrusion and migrationors Fig. , suggest36, we further compared gene expression profiles and the associated pathways in two proteins, using unbiased approaches. We first established two cell lines with a RhoE null background in HEK 293 and HeLa cells, respectively, via CRISPR-mediated RhoE gene knockout. The two cell lines were chosen due to their wide applications in a broad spectrum of cell biology and cancer research. We then reintroduced RhoE and RhoEα individually into the RhoE null cell lines, by transient transfection followed by RNA-seq analysis 24 h after transient transfection. RNA-seq data from RhoE null cells were used as a control for the analysis. The efficacy and fidelity of expression of RhoE and RhoEα were validated by western blot. RhoEα protein was exclusively expressed by flag-RhoEα, but not flag-RhoE expression vector  and homogentisate 1,2-dioxygenase (HGD) were chosen for the assessment , respectively, in the HEK 293 cell line  mice14. All animal experiments were approved by the Institutional Animal Care and Use Committee of Texas A&M University College of Medicine, Institute of Biosciences and Technology.Tissues were collected from male C57BL/6 mice at the age of 10 weeks. RhoE siRNA (L-007794-00-0005), and mouse SMARTpool RhoE siRNA (L-064484-01-00005) were from GE Dharmacon. Restriction enzyme-based cloning was used for plasmid construction. For 5′ UTR-RhoE and 5′ UTR-RhoE-flag constructs, 5′ UTR and coding sequence of RhoE cDNA (ENST00000263895.8) was amplified by PCR from HeLa cell cDNA library, and inserted into pcDNA3.1 and CMV-COOH-3xflag vector. For flag-RhoE and GFP-RhoE constructs, human RhoE coding sequence was inserted into CMV-NH2-3xflag and pEGFP-C3 vector. For flag-RhoEα and mCherry-RhoEα, human RhoE coding region starting from ATG46 was inserted into CMV-NH2-3xflag and pmCherry-C1 vector. Plasmids with point mutation were constructed using the Q5 site-directed mutagenesis kit . Oligos used for RT-qPCR included: PLA2G10: GGTTGCTTTTGTGGCTTGGGAG and GATTGACGCACTGCCAGGAGTA; HGD: CATCTTGGAGGTCTATGGTGTCC and GACCGTGTAACCACCTGGTACT; Casp5: ACAACCGCAACTGCCTCAGTCT and GAATCTGCCTCCAGGTTCTCAG; and CYP4F2: GACAGCCATTGTCAGGAGAAACC and TGCAGGAGGATCTCATGGTGTC. The antibodies used in the study included: RhoE , GAPDH , flag , ROCK1 , Syx , p190RhoGAP , anti-mouse IgG HRP-linked secondary antibody , anti-rabbit IgG HRP-linked secondary antibody , conformation-specific anti-mouse IgG HRP-linked secondary antibody , and conformation-specific anti-rabbit IgG HRP-linked secondary antibody .Nontargeting siRNA (D-001810-01-05), human SMARTpool HEK 293 cell lysate was subjected to anti-RhoE antibody pull-down followed by SDS–PAGE gel separation. The gel was stained with 0.1% Coomassie brilliant blue R250 and two bands ~27 kDa were excised. The samples were digested by trypsin. The mass spectrometry analysis was performed by the Mass Spectrometry Facility at the University of Texas Medical Branch at Galveston using SCIEX TOF/TOF 5800 system.57. For RhoE deletion in HeLa and HEK 293 cells, guide RNA (GGGCGGACATTGTCATAGTA) specifically targeting on RhoE exon 4 was cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 . Cells were transfected with the fused plasmid by lipofectamine 2000. Two days after transfection, the cells were treated with puromycin at 2.5 µg/ml for 3 days. Puromycin-resistant cells were harvested and further seeded into a 96-well plate, using a serial dilution for single-cell clones. Cell clones were validated by immunoblot. For CRISPR-mediated activation of RhoE promoter, HeLa cells were transfected with the Cas9-SAM components, including dCas9-VP64-GFP , MS2-P65-HSF1-GFP , and sgRNA(MS2) cloning vector  fused with guide RNA (ATCTGCCTCCTCCCCTTTTA) targeting on human RhoE promoter region. For CRISPR-mediated genomic point mutation, guide RNA (TTTGCTGGATAATTTCTGGC) targeting on RhoE exon 2 was cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0. The single-stranded donor oligonucleotide (ssODN) designed as repair template to create ATG1 deletion mutation is CACACTGACTGTCTCCCACCACAACTATCT TGCATTTCACGTTCTGATTAGGATCCATGATAGATTTGCTGGATAATTTCTGGCTTGCTCTTCTCTCCTTTGATGTTGCCTTATTTTCTCTTGGAACAGGAATTTTCTCTTAAGAAG. The ssODN as repair template to create ATG46 deletion mutation is CACACTGACTGTCT CCCACCACAACTATCTTGCATTTCACGTTCTGATTAGGATCGATAGATTTGCTGGATAATTTCTGGCTTGCTCTTCTCTCCTTCATTGATGTTGCCTTATTTTCTCTTGGAACAGGAATTTTCTCTTAAGAAG. HeLa cells were co-transfected with the sgRNA/Cas9 containing plasmid and the ssODN. Two days after transfection, the cells were treated with puromycin at 2.5 µg/ml for three days. Puromycin-resistant cells were subjected to immunoblot assay.The CRISPR-mediated genomic deletion was performed as described previouslyNIH-3T3 cells were cultured in a 35-mm culture dish  for overnight and then transfected with three expression vectors control, flag-RhoE and flag-RhoEα individually. Twenty four hours after the transfection, cells were washed and fixed by 4% paraformaldehyde for 30 min. The cells were probed by the anti-flag antibody  followed by the second antibody, Alexa Fluor 594 goat anti-mouse IgG . Alexa Fluor 488 Phalloidin  was used for cytoskeleton actin staining. For fluorescent imaging analysis, HeLa cells were co-transfected with GFP-RhoE and mCherry-RhoEα plasmids. Imaging was taken by Nikon Confocal A1 laser microscope.The degradation of RhoE and RhoEα was analyzed by CHX chase assay. HeLa cells were seeded in a six-well plate, incubated overnight, and subsequently treated with 20 μg/ml CHX for 0, 3, and 6 h, respectively. Cell lysates were then collected and subjected to immunoblotting assay for detection of RhoE and RhoEα levels. Immunoblots were qualified by densitometry of the film using ImageJ software. Three independent experiments were performed, and the data were analyzed by the associated statistical analysis.www.encodeproject.org/software/fastqc/). The sequencing reads were aligned to hg38 using Tophat and the reads mapping to each of the 56,269 genes/locations were counted using HTSeq. R package DESeq was used to normalize and generate the variance of stabilizing transformation data. The fold changes were calculated for differential gene expression analysis. IPA was used to analyze the differentially expressed genes with |fold change| ≥ 2. The raw RNA-seq data were deposited in the Gene Expression Omnibus (GEO) with accession number GSE132718.RNA was isolated from HEK 293 cells and HeLa cells using TRIzol reagent. For each sample, a total of 10 μg RNA was used for mRNA purification and cDNA library preparation according to the Ultra Directional RNA Library Prep Kit for Illumina . The quality control of the library quality control was assessed by Qubit (Thermo Fisher) and qPCR. The libraries were sequenced using an Illumina Novaseq 6000 platform with paired-end 150 bp strategy. The quality of raw RNA-sequencing data was validated by the FastQC software (t test was used. A value of p < 0.05 was considered statistically significant. For protein stability assay, the experiment was independently replicated three times and the results were qualified together. Experiments and data assessment were conducted double blinded by investigators.For two-group comparisons, unpaired student’s Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review File


Four types of complexes exist on the potential energy surfaces. Isomers A form at the P atom near the end of a P-C bond, B at a C-C bond, C at the centroid of the C-C-C ring along the C3 symmetry axis, and D at the P atom along the C3 symmetry axis. Complexes A and B are stabilized by hydrogen bonds when FH and ClH are the acids, and by halogen bonds when ClF is the acid. In isomers C, the dipole moments of the two monomers are favorably aligned but in D the alignment is unfavorable. For each of the monomers, the binding energies of the complexes decrease in the order A > B > C > D. The most stabilizing Symmetry Adapted Perturbation Theory (SAPT) binding energy component for the A and B isomers is the electrostatic interaction, while the dispersion interaction is the most stabilizing term for C and D. The barriers to converting one isomer to another are significantly higher for the A isomers compared to B. Equation of motion coupled cluster singles and doubles (EOM-CCSD) intermolecular coupling constants J(X-C) are small for both B and C isomers. J(X-P) values are larger and positive in the A isomers, negative in the B isomers, and have their largest positive values in the D isomers. Intramolecular coupling constants 1J(P-C) experience little change upon complex formation, except in the halogen-bonded complex FCl:P(CH3) A.Ab initio MP2/aug’-cc-pVTZ calculations have been performed to determine the structures and binding energies of complexes formed by phosphatetrahedrane, P(CH) Archimedean structures mainly known thanks to the fullerenes, the five Platonic structures which do not have atoms in the center, but only on the periphery, are also intrinsically beautiful. The Platonic structures are illustrated in Symmetry in Chemistry is one of the cornerstones of chemistry, both in terms of the beauty it bestows on molecules, and its importance for life. There is no question that highly symmetrical molecules are intrinsically beautiful and fascinating. Moreover, symmetry in chemistry, particularly chirality, is important for life itself, as first demonstrated by Pasteur ,2,3,4. Icloso-carboranes  which range from 10 kJ mol−1 for the B isomers of ClH:P(CH)3 and FCl:P(CH)3 to 35 kJ mol−1 for the A isomer of FCl:P(CH)3. The electrostatic interaction term is the most stabilizing term for all of these isomers except ClH:P(CH)3 isomer B, in which case the dispersion term is the leading stabilizing term. For FCl:P(CH)3 isomer B, the dispersion term is about 1 kJ mol−1 less stabilizing than the electrostatic term. The SAPT binding energies are less than the MP2 binding energies except for FCl:P(CH)3 isomer A which has a SAPT binding energy that is 3 kJ mol−1 greater than the MP2 energy.The DFT-SAPT data for the B isomers are also reported in 2hJ(F-C) for FH:P(CH)3 with a value of only −1.8 Hz. It is interesting to note from 2hJ(F-Cl) is the PSO term followed by the SD term. That the FC term is negligible is quite unusual for a hydrogen-bonded complex, but the hydrogen bonds in these B complexes are not typical hydrogen bonds.Spin–spin coupling constants across intermolecular bonds for the C and D isomers on the XY:P(CH)3 surfaces. In C the dipole moment vectors of XY and P(CH)3 are favorably aligned head-to-tail, while in D there is an unfavorable alignment, as illustrated in D which would lead to favorable dipole alignments are not the equilibrium structures. This and related questions will be addressed below.There are two additional equilibrium C isomers are reported in 3 in the FCl:P(CH)3 complex. The structure in which Cl interacts with P(CH)3 is not an equilibrium structure. C isomers. It is not surprising that the binding energies no longer follow the order of binding energies found for the hydrogen-bonded A and B isomers, but decrease instead in the order ClH > ClF > HF. The binding energies range from 4.5 kJ mol−1 for FH:P(CH)3 to 9.2 kJ mol−1 for ClH:P(CH)3. The X-C and X-P distances decrease in the order ClF > ClH > FH. That the mostly weakly bound complex with FH has the shortest X-C and X-P distances may be attributed at least in part to the smaller van der Waals radius of F.The structures, total energies, and molecular graphs of the 3 at very long distances. J(F-P) is −15.8 Hz at a distance of 6.22 Å in the complex with ClF and −12.1 Hz in the complex with FH. When the acid is ClH, J(Cl-P) has a value of −3.2 Hz.D isomers are reported in D isomers are reported in C isomers. The D isomers have binding energies that range from 0.8 kJ mol−1 for HF:P(CH)3 at a F-P distance of 3.49 Å, to 3.9 kJ mol−1 for HCl:P(CH)3 at a Cl-P distance of 3.63 Å. The binding energy of the FCl:P(CH)3 complex is slightly less at 3.6 kJ mol−1, but the Cl-P distance of 3.22 Å is much shorter.The structures, total energies, and molecular graphs of the D isomers have large spin–spin coupling constants J(X-P), and 3. HF:P(CH)3 has a value of J(F-P) equal to 118 Hz. The largest coupling constant among the D isomers is J(F-P) for FCl:P(CH)3 with a value of 299 Hz.The C and D isomers are given in 3 isomer D is unbound with an unfavorable electrostatic interaction energy of 0.5 kJ mol−1. As usual, the first-order SAPT exchange term is the destabilizing term. However, the electrostatic term is not the primary stabilizing term in these isomers as it is in the A and B isomers, but rather, it is the second-order dispersion term. In the FH:P(CH)3 isomer C, the dispersion term cancels the exchange term. In the remaining complexes, it has an absolute value that is no more than 2 kJ mol−1 less than the exchange term. All of the other terms are stabilizing, except for the induction term for the C and D isomers of ClF:P(CH)3. The sum of the stabilizing terms leads to binding energies between 3 and 5 kJ mol−1 for the C isomers, and between 1 and 2 kJ mol−1 for the bound D isomers.The SAPT energies for the 1J(P-C) for P(CH)3 changes, depending on whether the P-C bond interacts or does not interact with FH, ClH, or ClF. The data required for this analysis are given in B, C, and D isomers, 1J(P-C) varies by about ±1 Hz relative to isolated P(CH)3. For these, 1J(P-C) is essentially independent of bond formation by P(CH)3. Bond formation in these isomers occurs at relatively long distances, and does not produce a large enough change in the electron distributions of P and C to produce changes in 1J(P-C). This is also evident from the small values of the charge-transfer energies in these isomers.Up to this point, the discussion of coupling constants has focused on coupling across intermolecular bonds. However, it is also of interest to examine how A isomers, 1J(P-C) coupling constants in complexes with FH and ClH span a slightly greater range compared to the isomers B, C, and D. However, it is the A isomer with ClF that exhibits dramatic changes. This is consistent with the stronger halogen bonding interaction between P(CH)3 and ClF which leads to a significant lengthening of the P-C bond to 1.946 Å, and changes in the electron density of that bond. The electron density changes are evident from the charge-transfer energies, and from the coupling constant for the interacting P-C bond, which decreases in absolute value from −40 Hz in isolated P(CH)3 to −33 Hz in this complex. Although the noninteracting P-C bonds decrease in length by only 0.007 Å, 1J(P-C) for these bonds increases in absolute value to −45 Hz in response to the changes in the electron density of this isomer.For the A-tr and B-tr are given in 3 and FH:P(CH)3-A-tr, and ClF:P(CH)3 and ClF:P(CH)3B-tr, are illustrated in 3v symmetry and are not equilibrium structures on their potential surfaces, since they have two degenerate imaginary frequencies. Rather, complexes labeled A-tr are the transition structures for converting one isomer A to another one of the A isomers, and B-tr are the transition structures for converting one isomer B to another one of the isomers B.The structures, total energies, and molecular graphs of the complexes 3A-tr and B-tr. The intermolecular distances in these transition structures are longer than the X-P and X-C distances in the corresponding complexes A and B, respectively. The A-tr structures are bound with stabilization energies ranging from 8 kJ mol−1 for FH:P(CH)3A-tr and ClH:P(CH)3A-tr to 13 kJ mol−1 for ClF:P(CH)3A-tr. The B-tr isomers have binding energies that range from 10 kJ mol−1 for FH:P(CH)3B-tr to 13 kJ mol−1 for ClF:P(CH)3B-tr. From these energies of corresponding equilibrium complexes and transition structures, it is possible to evaluate the barrier for converting one A or B complex to another equivalent complex. These barriers are relatively high for the A isomers, and vary from 9 kJ mol−1 for ClH:P(CH)3 to 19 kJ mol−1 for ClF:P(CH)3. Given the structure of the B isomers, it is not surprising that the barriers to converting one isomer B to another B are much lower. These barriers vary from 2 kJ mol−1 for ClH:P(CH)3 to 4 kJ mol−1 for FH:P(CH)3 and FCl:P(CH)3.3, the monomers HF, HCl, and ClF, and the complexes formed by phosphatetrahedrane and the monomers were optimized at second-order Møller–Plesset perturbation theory (MP2) [The structures of the isolated phosphatetrahedrane molecule, P(CH)ry (MP2) ,30,31,32ry (MP2) . This bary (MP2) ,35 by rery (MP2) . Complex3 using the Topmod program [The electron density properties at bond critical points (BCPs) of complexes have been analyzed using the Atoms in Molecules (AIM) methodology ,38,39,40 program  to furthel(1) is the first-order electrostatic interaction energy of the monomers each with its unperturbed electron distribution; Eex(1) is the first-order exchange energy term; Eind(2) denotes the second-order induction energy arising from the interaction of permanent multipoles with induced multipole moments and charge-transfer contributions, plus the change in the repulsion energy induced by the deformation of the electron clouds of the monomers; Edisp(2) is the second-order dispersion energy, which is related to the instantaneous multipole-induced multipole moment interactions plus the second-order correction for coupling between the exchange repulsion and the dispersion interactions. In addition to the terms listed in Equation (1), a Hartree–Fock correction term δHF, which includes higher-order induction and exchange corrections, is also included [Density Functional Theory–Symmetry Adapted Perturbation Theory (DFT-SAPT) has been used to investigate interaction energies. In this approach, the energies of interacting monomers are expressed in terms of orbital energies obtained from Kohn–Sham density functional theory ,47 The Sincluded . The DFTincluded  at the Pincluded .13C and 19F, and the qz2p basis on 31P, 35Cl, and the 1H atom of FH and ClH. The Dunning cc-pVDZ basis set was placed on the 1H atoms bonded to C atoms. All terms that contribute to the total coupling constant, namely, the paramagnetic spin orbit (PSO), diamagnetic spin orbit (DSO), Fermi contact (FC), and spin dipole (SD) have been evaluated. Coupling constant calculations were performed using ACES II [Equation of motion coupled cluster singles and doubles (EOM-CCSD) spin–spin coupling constants were evaluated in the CI (configuration interaction)-like approximation ,53 with  ACES II  on the H3, and the monomers HF, HCl, and ClF. The following statements are supported by the results of this study.A, B, C, and D, have been found on the potential energy surfaces. Isomers A form at the P atom near the end of a P-C bond, B at a C-C bond, C at the centroid of the C-C-C ring along the C3 symmetry axis, and D at the P atom along the C3 symmetry axis.Four types of complexes, designated A and B are stabilized by hydrogen bonds when FH and ClH are the acids, and by halogen bonds when ClF is the acid. In isomers C, the dipole moments of the two monomers are favorably aligned, and there exists a long-range interaction between the atom at the negative end of the dipole moment vector and the P atom. In the D isomers, the dipole moment vectors are unfavorably aligned.Complexes A and B isomers decrease in the order ClF > FH > ClH, while the binding energies of the C and D isomers decrease in the order ClH > ClF > HF. For fixed FH, ClH, or ClF, binding energies of the isomers decrease in the order A > B > C > D.The binding energies of the A, but the charge-transfer interactions are small in isomers B and D, and nonexistent in C.Charge-transfer stabilizes isomers A and B isomers are smaller than the corresponding MP2 binding energies. The most stabilizing SAPT component for these isomers is the electrostatic interaction, except for ClH:P(CH)3 isomer B for which the dispersion interaction is the most stabilizing. These two terms are similar for the B isomer of FCl:P(CH)3. In contrast, the dispersion interaction is the most stabilizing term for the C and D isomers.The SAPT binding energies for the A to another A, and one isomer B to another B, have been found on the potential surfaces. The barriers for converting one isomer to another are significantly higher for the A isomers.Transition structures for converting one isomer B and C isomers. J(X-P) values are larger and positive for the A isomers, negative for the B isomers, and have their largest values for the D isomers.EOM-CCSD coupling constants J(X-C) are small for both the 1J(P-C) in the isomers change very little from their value in isolated (PH)3, except for the complex FCl:P(CH)3 isomer A. 1J(P-C) decreases in absolute value for the P-C bond that interacts with ClF as the P-C bond elongates significantly. 1J(P-C) increases in absolute value for the other two P-C bonds, even though there is little change in the P-C distances relative to isolated P(CH)3.Coupling constants Ab initio MP2/aug’-cc-pVTZ calculations have been performed to determine the structures and binding energies of complexes formed by phosphatetrahedrane, P(CH)


Staphylococcus aureus (MRSA) in Taiwan. Previously, we reported that ST59 MRSA harbors enterococcal IS1216V-mediated multidrug-resistant composite transposons MESPM1 or MES6272-2. The MES were found to have a mosaic structure, largely originating in enterococci and partly native to S. aureus. The current study aimed to track the origin of the MES and how they disseminated from enterococci to ST59 S. aureus. A total of 270 enterococcal isolates were analyzed, showing that two ST64 Enterococcus faecalis isolated in 1992 and 11 clonal complex 17 Enterococcus faecium harbored MESPM1-like and MES6272-2-like structures, respectively. Sequence analysis revealed that ST64 E. faecalis strain N48 acquired the MESPM1-like structure on the plasmid pEflis48. The pEflis48 harbored the enterococci-originated region  and the S.aureus-originated region (chloramphenicol resistance) of MESPM1 but was separated by the replication region of the plasmid. Homologous recombination between the two direct repeats of IS1216V resulted in excision of the replication region of the plasmid to regenerate MESPM1. The p4780-1 and pV19 of E. faecium carried MES6272-2-like structures with IS1216V, albeit with multiple insertions by other insertion sequences. The findings show that IS1216V plays important roles in bidirectional gene transfer of multidrug resistance between enterococci and S. aureus.Sequence type 59 (ST59) is the dominant type of community-associated methicillin-resistant  Staphylococcus aureus (MRSA) is the dominant type of community-associated MRSA (CA-MRSA) in Taiwan and can be divided into two clones: Panton–Valentine leukocidin (PVL)-positive/staphylococcal cassette chromosome mec (SCCmec) V and PVL-negative/SCCmec IV -aadE cluster displays 100% DNA sequence similarity to the corresponding region in pLG2 of Enterococcus faecalis -sat-ΔaadE-[aacA-aphD] cluster of the MES6272-2-like structure in E. faecium 4780-1 showed >99.7% identity to the corresponding region in ST59/SCCmec IV MRSA 6272-2, except an ISEfm1 insertion into the sat gene -IIIa]-sat-ΔaadE-[aacA-aphD] cluster of MES6272-2-like structures in E. faecium V19 showed 99.9% identity to the corresponding region in ST59/SCCmec IV MRSA 6272-2. However, the 3′-end of transposase of Tn551 was absent in E. faecium V19, and the direction of IS1216V in front of ermB was different between E. faecium V19 and ST59/SCCmec IV MRSA 6272-2. The region between the aacA-aphD gene and the second IS1216V of E. faecium V19 displayed different structures compared with MES6272-2, harboring multiple resistance determinants of lnu(B) (lincosamide resistance), lsa(E) , spw (spectinomycin resistance), and an aadE gene.For the MESS. aureus was performed by filter mating. E. faecalis N48 or E. faecium V19 were used as donors, while E. faecalis JH2-2 or S. aureus RN2677 were used as recipients. The MES-related resistances were successfully transferred from E. faecalis N48 or E. faecium V19 to E. faecalis JH2-2, with transfer frequencies of 3.7 × 10−2 and 2.5 × 10−7, respectively. Resistances were transferred to transconjugants .Conjugative transfer of MES-like structures between enterococci and njugants . All of PM1 and MES6272-2 in ST59 MRSA, the novel multidrug-resistant composite transposons mediated by enterococcal IS1216V and mingled with S. aureus- and enterococci-originated sequences [PM1-like and MES6272-2-like structures, both surrounded by IS1216V, were found in E. faecalis and E. faecium isolates, respectively. Furthermore, the MESPM1-like structure of E. faecalis isolated in 1992 had already acquired the cat gene and its surrounding genetic environment that was native to S. aureus, suggesting that horizontal gene transfer from S. aureus to enterococci might have occurred before the emergence of multidrug-resistant ST59 MRSA.Previously, we described MESequences ,5. This equences . In the 1216 isoform groups, including IS1216, IS1216E, and IS1216V, are one of the most multiply represented insertion sequences in enterococci and play important roles in the dissemination of resistance determinants [5385 , Tn5482, and Tn5506 (both encoding vancomycin resistance) [PM1 and MES6272-2 are rare cases of composite transposons mediated by IS1216V found in S. aureus [PM1 would be excised, resulting in a single IS1216V remaining in MESPM1 [E. faecium with the pbp5 gene surrounded by two direct repeats of IS1216V [E. faecalis carried a MESPM1-like structure, within which a region included the rep gene and a truncated prgZ gene encoding pheromone-binding proteins corresponding to that in pTEF3 of vancomycin-resistant E. faecalis  ,28,29. M IS1216V . In the faecalis . PCR wit IS1216V . As a recat genes found in staphylococci and enterococci are frequently located on small multicopy plasmids and are seldom associated with large multiresistant plasmids [E. faecalis and MESPM1 of ST59/SCCmecV MRSA, the cat gene and its surrounding environments were both highly related to a small S. aureus plasmid SAP084A  from donor E. faecalis N48 to recipient E. faecalis JH2-2, it failed to transfer to S. aureus RN2677. The reason is unknown and needs to be investigated in the future. A previous study reported that a pSK41-like plasmid is necessary to contribute to successful transfer of the Inc18-like vanA plasmid from E. faecalis to MRSA [S. aureus.The MESylococci ,33. Inc1reported . In the  to MRSA . FurtherE. faecalis belongs to CC8. Although CC8 was not included in the major global clusters associated with healthcare-associated infections such as CC2 [E. faecalis has been reported to be associated with multidrug resistance to erythromycin, kanamycin, streptomycin, gentamicin, and tetracycline [E. faecalis, all of the E. faecium isolates characterized in this study were CC17, the most prevalent cluster worldwide, notably in the nosocomial setting [E. faecium is associated with its success to adapt to conditions of modern hospitals [6272-2-like structures of the CC17 E. faecium strains isolated in 2003, 2013, and 2014 were inserted by several insertion sequences such as ISEfm1, ISEf1, and ISL3, and an additional cluster of resistance determinants lnu(B), lsa(E), spw, and aadE was acquired  and the S. aureus-originated region (chloramphenicol resistance) found in ST64 E. faecalis and ST59 S. aureus indicates that bidirectional gene transfer mediated by IS1216V can occur between enterococci and S. aureus. Our study is of great importance, as it is the first to demonstrate the role of IS1216V in interspecies transfer of multidrug resistance genes between enterococci and ST59 S. aureus.In conclusion, the MES