A responsive disulfide bond switch aptamer prodrug exhibiting enhanced stability and anticancer efficacy.

Aptamers have shown significant potential in treating diverse diseases. However, challenges such as stability and drug delivery limited their clinical application. In this paper, the development of AS1411 prodrug-type aptamers for tumor treatment was introduced. A Short oligonucleotide was introduced at the end of the AS1411 sequence with a disulfide bond as responsive switch. The results indicated that the aptamer prodrugs not only enhanced the stability of the aptamer against nuclease activity but also facilitated binding to serum albumin. Furthermore, in the reducing microenvironment of tumor cells, disulfide bonds triggered drug release, resulting in superior therapeutic effects in vitro and in vivo compared to original drugs. This paper proposes a novel approach for optimizing the structure of nucleic acid drugs, that promises to protect other oligonucleotides or secondary structures, thus opening up new possibilities for nucleic acid drug design.

Anisamide-conjugated hairpin antisense oligonucleotides prodrug co-delivering doxorubicin exhibited enhanced anticancer efficacy.

Antisense oligonucleotides (ASONs)-based therapeutics offers tremendous promise for the treatment of diverse diseases. However, there is still a need to develop ASONs with enhanced stability against enzymes, improved drug delivery, and enhanced biological potency. In this study, we propose a novel anisamide (AA)-conjugated hairpin oligonucleotide prodrug loading with chemotherapeutic agent (doxorubicin, DOX) (AA-loop-ASON/DOX) for oncotherapy. Results indicated that the introduction of a hairpin conformation and AA ligand in prodrug significantly improved the stability against enzymatic hydrolysis, as well as the cellar uptake of ASONs and DOX. The incorporation of disulfide bonds could trigger mechanical opening, resulting in the release of ASON and DOX in response to the intracellular glutathione (GSH) in tumors. Moreover, the composite of DOX-loading ASONs prodrug exhibited a robust and selective inhibition of tumor cell proliferation. This paper introduces a novel design concept for nucleic acid-based therapeutics, aiming to enhance the delivery of drug and improve biological effectiveness.

A recently quenched galaxy 700 million years after the Big Bang.

Local and low-redshift (z < 3) galaxies are known to broadly follow a bimodal distribution: actively star-forming galaxies with relatively stable star-formation rates and passive systems. These two populations are connected by galaxies in relatively slow transition. By contrast, theory predicts that star formation was stochastic at early cosmic times and in low-mass systems1-4. These galaxies transitioned rapidly between starburst episodes and phases of suppressed star formation, potentially even causing temporary quiescence-so-called mini-quenching events5,6. However, the regime of star-formation burstiness is observationally highly unconstrained. Directly observing mini-quenched galaxies in the primordial Universe is therefore of utmost importance to constrain models of galaxy formation and transformation7,8. Early quenched galaxies have been identified out to redshift z < 5 (refs. 9-12) and these are all found to be massive (M⋆ > 1010 M⊙) and relatively old. Here we report a (mini-)quenched galaxy at z = 7.3, when the Universe was only 700 Myr old. The JWST/NIRSpec spectrum is very blue (U-V = 0.16 ± 0.03 mag) but exhibits a Balmer break and no nebular emission lines. The galaxy experienced a short starburst followed by rapid quenching; its stellar mass (4-6 × 108 M⊙) falls in a range that is sensitive to various feedback mechanisms, which can result in perhaps only temporary quenching.

Cervicovaginal microbiota long-term dynamics and prediction of different outcomes in persistent human papillomavirus infection.

Persistent human papillomavirus (HPV) infection can lead to cervical intraepithelial neoplasia (CIN) and cervical cancer, posing serious threats to the health of women. Although the cervicovaginal microbiota is strongly associated with CIN, the dynamics of the microbiota during CIN development are unknown. In this retrospective cohort study, we analyzed 3-year longitudinal data from 72 patients diagnosed with a persistent HPV infection almost all caused by high-risk HPV types. Patients were categorized into groups with HPV persistent infection (n = 37), progression to CIN (n = 16), and CIN regression (n = 19) based on infection outcome during the follow-up period. Furthermore, 16S rRNA gene sequencing was performed on consecutively collected cervical samples to explore the composition and dynamics of the cervicovaginal microbiota during the development and regression of CIN. Our results showed that the composition of the cervicovaginal microbiota varied among women with different HPV infection outcomes and remained relatively stable during the follow-up period. Notably, the serial follow-up data showed that these microbial alterations were present for at least 1-2 years and occurred before pathologic changes. In addition, microbial markers that were highly discriminatory for CIN progression or regression were identified. This study provides evidence for a temporal relationship between changes in the cervicovaginal microbiota and the development of CIN, and our findings provide support for future microbial intervention strategies for CIN.

Whole genome sequence of carbapenem-resistant hypermucoviscous Klebsiella pneumoniae K2-ST375 with blaNDM-harbouring conjugative IncX3 and pLVPK-like virulence plasmids from a patient in China.

OBJECTIVES: Carbapenem-resistant hypermucoviscous Klebsiella pneumoniae (CR-HMKP) poses unprecedented public health challenges. However, genomic information regarding the CR-HMKP K2-ST375 strain is scarce. The aim of this study was to characterize the whole genome sequence of the CR-HMKP K2-ST375 strain Kp0179 isolated from a male patient in China. METHODS: The whole genome of Kp0179 was sequenced using the DNBSEQ and Pacific Biosciences RSII platforms. The capsular serotype, multilocus sequence typing (MLST), antimicrobial resistance genes, and virulence factors were determined using available databases and bioinformatics tools. Conjugation experiments were performed using rifampicin-resistant Escherichia coli C600 as the recipient. RESULTS: The Kp0179 strain with hypermucoviscous phenotype was resistant to almost all ß-lactams, including ertapenem and imipenem. Whole genome sequencing revealed that Kp0179 belonged to K2-ST375 and contained blaNDM-IncX3 and a virulence plasmid ca. 121 kb. Kp0179 contained 5146 coding genes, 88 tRNAs, 25 rRNAs and 38 non-coding RNA genes. Among the six acquired antibiotic resistance genes, blaSHV-99, fosA, oqxAB were located on the chromosome, whereas blaNDM-1, qnrS1 and blaSHV-12 were located on the conjugative plasmid pNDM-Kp0179 (IncX3 type). Virulence gene analysis indicated that pLVPK-Kp0179 carried multiple virulence-encoding genes, such as iroBCDN, iucABCDiutA, rmpA and rmpA2. In addition to carrying a virulence plasmid, capsule formation (kvgA) and the type 3 fimbriae operon (mrkABCDFHIJ) were located on the chromosome of Kp0179. CONCLUSION: To our knowledge, this is the first report of a CR-HMKP K2-ST375 strain with a blaNDM-harboured conjugative IncX3 plasmid and a pLVPK-like virulence plasmid from a patient in China. Therefore, the spread of CR-HMKP K2-ST375 isolates in China should be closely monitored.

Design, characterization and biological evaluation of a new chimeric 4A2-5-antisense prodrug combined with chemotherapy.

Issues surrounding rapid degradation and limited therapeutic efficacy still exist in the development of native antisense oligonucleotides (ASONs). In this paper, a novel strategy of chimeric 4A2-5-ASON prodrug combined with chemotherapy for oncotherapy was proposed. The self-assembled hairpin-end prodrug structure provided a DOX loading site, while enhancing stability against nuclease degradation. The disulfide led responsive drug release, and excellent therapeutic effects were achieved by the combined action of RNase H and RNase L recruitment, along with chemotherapy drug Doxorubicin (DOX), both in vitro and in vivo. This work provides evidence for the development of designing nucleic acid drugs with combined mechanisms.

Volatile Methylsiloxanes in Complex Samples: Recent Updates on Pretreatment and Analysis Methods.

Volatile methylsiloxanes (VMSs) are massively produced chemicals having applications in industry and home because of their physical and chemical characteristics. They are used in personal care products such as cosmetics, household coatings, cleaners, skin care products, and others. Resultantly, large number of VMSs are discharged into air where they can be subjected to atmospheric migrations over long distances causing toxic and estrogenic effects, persistence, and bioaccumulations. Many institutions have taken measures to control VMSs. They require accurate, rapid, and sensitive pretreatment and analysis methods for diverse samples. Herein, the pretreatment and determination methods of VMSs as reported in recent years are reviewed and summarized. Pretreatments include commonly methods such as membrane-assisted solvent extraction, liquid-liquid extraction, and others, while novel methods are solid phase extraction, solid phase microextraction, diverse liquid phase microextraction and others. Analyses are made through gas chromatography-based methods. In addition, the advantages, and disadvantages of techniques are compared, and the prospects of pretreatment and analysis methods are discussed.

Pros and cons of different pretreatment and detection methods for VMSs were discussed.Different novel pretreatment techniques for VMSs were discussed.High-resolution mass spectrometry (HRMS) for the determination of VMSs was discussed.

Impact of alirocumab/evolocumab on lipoprotein (a) concentrations in patients with familial hypercholesterolaemia: a systematic review and meta-analysis of randomized controlled trials.

INTRODUCTION: Familial hypercholesterolaemia (FH) is a common hereditary genetic disorder, characterized by elevated circulating low-density lipoprotein cholesterol (LDL-C) and lipoprotein (a) [Lp(a)] concentrations, leading to atherosclerotic cardiovascular disease (ASCVD). Two types of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors- alirocumab and evolocumab- are efficient drugs in the treatment of FH, which can effectively reduce Lp(a) levels. MATERIAL AND METHODS: Embase, MEDLINE, and PubMed up to November 2022 were searched for randomized clinical trials (RCTs) evaluating the effect of alirocumab/evolocumab and placebo treatment on plasma Lp(a) levels in FH. Statistics were analysed by Review Manager (RevMan 5.3) and Stata 15.1. RESULTS: Eleven RCTs involved a total of 2408 participants. Alirocumab/evolocumab showed a significant efficacy in reducing Lp(a) [weighted mean difference (WMD): -20.10%, 95% confidence interval (CI): -25.59% to -14.61%] compared with placebo. In the drug type subgroup analyses, although the efficacy of evolocumab was slightly low (WMD: -19.98%, 95% CI: -25.23% to -14.73%), there was no difference with alirocumab (WMD: -20.54%, 95% CI: -30.07% to -11.02%). In the treatment duration subgroup analyses, the efficacy of the 12-week duration group (WMD: -17.61%, 95% CI: -23.84% to -11.38%) was lower than in the group of ≥ 24 weeks' duration (WMD: -22.81%, 95% CI: -31.56% to -14.07%). In the participants' characteristics subgroup analyses, the results showed that no differential effect of alirocumab/evolocumab therapy on plasma Lp(a) concentrations was observed (heterozygous FH [HeFH] WMD: -20.07%, 95% CI: -26.07% to -14.08%; homozygous FH [HoFH] WMD: -20.04%, 95% CI: -36.31% to -3.77%). Evaluation of all-cause adverse events (AEs) between alirocumab/evolocumab groups and placebo groups [relative risk (RR): 1.05, 95% CI: 0.98-1.12] implied no obvious difference between the 2 groups. CONCLUSIONS: Anti-PCSK9 drugs (alirocumab and evolocumab) may be effective as therapy for reducing serum Lp(a) levels in FH, and no differences were observed in treatment durations, participant characteristics, and other aspects of the 2 types of PCSk9 inhibitors. However, further experimental studies and RCTs are warranted to clarify the mechanism of PSCK9 inhibitors to lowering Lp(a) concentrations in FH.

Synthesis, Biophysical Properties, and Antitumor Activity of Antisense Oligonucleotides Conjugated with Anisamide.

Antisense oligonucleotides (ASONs) have proven potential for the treatment of various diseases. However, their limited bioavailability restricts their clinical application. New structures with improved enzyme resistance stability and efficient drug delivery are needed. In this work, we propose a novel category of ASONs bearing anisamide conjugation at phosphorothioate sites for oncotherapy. ASONs can be conjugated with the ligand anisamide very efficiently and flexibly in a solution. The conjugation sites and the ligand amount both influence anti-enzymatic stability and cellular uptake, resulting in changes in antitumor activity that are detectable by cytotoxicity assay. The conjugate with double anisamide (T6) was identified as the optimal conjugate, and its antitumor activity and the underlying mechanism were examined further in vitro and in vivo. This paper presents a new strategy for the design of nucleic acid-based therapeutics with improved drug delivery and biophysical and biological efficacy.

Dumbbell-Shaped Antisense Oligonucleotide Prodrugs Showed Improved Antinuclease Stability and Anticancer Efficacy.

Antisense oligonucleotides (ASONs) have generated widespread interest as antitumor agents. Nevertheless, the utility of natural ASONs is limited due to their rapid degradation by intracellular and extracellular nucleases. In this work, we proposed a novel prodrug-type ASON with a dumbbell conformation and a responsive disulfide switch. A degradation assay showed that the dumbbell-shaped ASON (DS-ASON) exhibited stronger stability against enzymatic degradation compared with that of the linear or single-end looped ASON. The native ASON could dissociate via breakage of the disulfide switch when in the reductive microenvironment of a tumor. In addition, an optimal DS-ASON, L2, displayed robust antitumor activity both in vitro and in vivo. This paper presents a new design of nucleic acid-based therapeutics featuring a conformational change that provides improved stability and biological efficacy.

Comparative analysis of the main haematological indexes and RNA detection for the diagnosis of SARS-CoV-2 infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has become a public health emergency of international concern. SARS-CoV-2 RNA detection is the diagnostic criterion for coronavirus disease 2019 (COVID-19). Nevertheless, RNA detection has many limitations, such as being time-consuming and cost-prohibitive, and it must be performed in specialized laboratories. Virus antibody detection is a routine method for screening for multiple viruses, but data about SARS-CoV-2 antibody detection are limited. METHOD: Throat swabs and blood were collected from 67 suspected SARS-CoV-2 infection patients at the Affiliated Hospital of Zunyi Medical University and Zunyi Fourth People's Hospital isolated observation departments. Throat swab samples were subjected to SARS-CoV-2 RNA detection by real-time PCR. Blood was used subjected to SARS-CoV-2 IgG/IgM detection by an enzyme-linked immunosorbent assay (ELISA) and gold immunochromatography assay (GICA). Blood underwent C-reactive protein detection by immunoturbidimetry, and white blood cells, neutrophil percentages and lymphocyte percentages were counted and calculated, respectively. Clinical symptoms, age and lifestyle habits (smoking and drinking) in all patients were recorded. Data were analysed using SPSS version 19. The results were confirmed by T and χ2 tests; correlations with detection results were analysed by kappa coefficients. Odds ratio (OR) and corrected OR values were analysed by logistic regression. P < 0.05 was considered statistically significant. RESULTS: Of the 67 patients included in this study, 26 were SARS-CoV-2 RNA-positive. GICA IgM sensitivity was 50.9% (13/26), and specificity was 90.2% (37/41). ELISA IgM sensitivity was 76.9% (20/26), and specificity was 90.2% (37/41). ELISA IgG sensitivity was 76.9% (20/26), and specificity was 95.1% (39/41). The kappa coefficients between RNA detection and ELISA IgG, ELISA IgM, and GICA IgM results were 0.741 (P < 0.01), 0.681 (P < 0.01) and 0.430 (P < 0.01), respectively. CONCLUSION: Among the candidate blood indicators, serum IgG and IgM detected by ELISA had the best consistency and validity when compared with standard RNA detection; these indicators can be used as potential preliminary screening tools to identify those who should undergo nucleic acid detection in laboratories without RNA detection abilities or as a supplement to RNA detection.

SLC16A1-AS1 enhances radiosensitivity and represses cell proliferation and invasion by regulating the miR-301b-3p/CHD5 axis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), a common type of human malignancies, leads to increasing incidence and fairly high mortality. An increasing number of studies have verified that long noncoding RNAs (lncRNAs) played key roles in the development of multiple human cancers. As a biomarker, SLC16A1-AS1 has been reported in non-small cell lung cancer (NSCLC) and oral squamous cell carcinoma (OSCC). Thus, we decided to investigate whether SLC16A1-AS1 exerts its biological function in HCC. In this study, we discovered that SLC16A1-AS1 was obviously downregulated in HCC tissues and cells. Overexpression of SLC16A1-AS1 inhibited HCC cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) process as well as promoted cell apoptosis. Moreover, SLC16A1-AS1 was confirmed to enhance the radiosensitivity of HCC cells. Molecular mechanism exploration suggested that SLC16A1-AS1 served as a sponge for miR-301b-3p and CHD5 was the downstream target gene of miR-301b-3p in HCC cells. Rescue assays implied that CHD5 knockdown could recover the effects of SLC16A1-AS1 overexpression on HCC cellular processes. In brief, our study clarified that SLC16A1-AS1 acted as a tumor suppressor in HCC by targeting the miR-301b-3p/CHD5 axis, which may be a promising diagnostic biomarker and provide promising treatment for HCC patients.

Neisseria gonorrhoeae NGO2105 Is an Autotransporter Protein Involved in Adhesion to Human Cervical Epithelial Cells and in vivo Colonization.

Autotransporters are important virulence factors in the outer membrane of gram-negative bacteria. Although several autotransporters have been identified in Neisseria meningitidis, only IgA1 protease has been identified in Neisseria gonorrhoeae. A sequence analysis showed a marked difference in the distribution of autotransporters between the two strains. It has been speculated that only two autotransporters, the IgA1 protease and the NGO2105 protein, might be encoded by N. gonorrhoeae. Here, we describe the identification of NGO2105, a new autotransporter in N. gonorrhoeae. A sequence alignment showed that NGO2105 is highly similar to the adhesion and penetration protein (App) in N. meningitidis. We found that NGO2105 is exported to the outer membrane, cleaved and released into the culture supernatant by endogenous serine protease activity in N. gonorrhoeae and E. coli. The site-directed mutagenesis of S267A in the predicted enzyme catalytic triad abolished autoproteolytic cleavage to allow secretion. The NGO2105 ß-barrel shows the ability to translocate the heterologous Hbp passenger domain. NGO2105 is involved in gonococcal adherence to and invasion into human cervical epithelial cells. Furthermore, antibodies raised against NGO2105 are able to block gonococcal adherence to human cervical epithelial cells. The Δngo2105 mutant and anti-NGO2105 antiserum significantly attenuated the colonization of N. gonorrhoeae in mice. Collectively, our results suggest that the newly identified serine protease autotransporter NGO2105 represents a novel virulence factor of gonococcus and a potential vaccine target.

Characteristics of human papillomaviruses distribution in Guizhou Province, China.

BACKGROUND: Human papillomavirus (HPV) is one of the most common sexually transmitted viruses. Data about HPV infection in Guizhou is limited. METHODS: 56,768 cervical samples were collected and genotyped for 15 main high risk and 6 main low risk HPV types. RESULTS: 16.95% (9623/56768) of samples were HPV positive; 90.70% (8728/9623) of HPV positive women were infected by high risk HPV. High risk and high risk mix infection (1458; 70.85%) was the most common mix HPV infection type. The highest HPV detection rate was found in age group 41-45 years old (detection rate = 17.89%) (χ2 = 204.77; P < 0.001); the highest within-group HPV infection rates were found in the ≤20 (25.62%) and ≥ 61 (24.67%) years old age groups, the lowest within-group HPV infection rate was found in the 31-35 years old age group (15.02%). The highest mix infection proportions were found in the ≥61 (36.06%) and ≤ 20 (33.63%) years old age groups (χ2 = 111.21; P < 0.001), the lowest mix infection proportion was found in the 41-45 (17.42%) years old age group. The highest high risk infection proportions were found in the 26-30 (92.98%), ≥61 (92.68%), and 36-40 (92.16%) years old age groups (χ2 = 31.72; P < 0.001), the lowest high risk infection proportion was found in the ≤20 (84.96%) years old age group. HPV infection rates varied with seasons in Guizhou. CONCLUSIONS: Characteristics of HPV distribution in Guizhou were identified. There were significant differences in HPV distribution among age groups, prevention strategies should be adjusted according to the characteristics.

E6 and E7 gene polymorphisms in human papillomavirus Type-6 identified in Southwest China.

BACKGROUND: Human papillomavirus type-6 (HPV6) is the major etiological agent of anogenital warts both men and women. The present study aimed to characterize the genetic diversity among HPV6 in Southwest China, and to investigate the origin of, selective pressure experienced by, and impact of the resultantly identified genetic variants on the HPV6 secondary structure. METHODS: Phylogenetic trees were constructed by Maximum-likelihood and the Kimura 2-parameters methods by Molecular Evolutionary Genetics Analysis version 6.0. The diversity of secondary structure was analyzed by PSIPred software. The selection pressures acting on the E6/E7 genes were estimated by Phylogenetic Analyses by Maximum Likelihood version 4.8 software. RESULTS: HPV6 was the most prevalent low risk HPV type in southwest China. In total, 143 E6 and E7 gene sequences of HPV6 isolated from patients were sequenced and compared to GenBank HPV6 reference sequence X00203. The results of these analyses revealed that both the HPV6 E6 and E7 were highly conserved within the analyzed patient samples, and comprised only 3 types of variant sequence, respectively. Furthermore, the analysis of HPV6 E6 and E7 sequences revealed seven/five single-nucleotide mutations, two/four and five/one of which were non-synonymous and synonymous, respectively. The phylogenetic analyses of the E6 and E7 sequences indicated that they belonged to sub-lineage A1 and sub-lineage B1, whereas the selective pressure analyses showed that only the E7 mutation sites 4R, 34E, and 52F were positive selection. CONCLUSIONS: HPV6 (detection rate = 13.10%) was very prevalent in southwest China, both the HPV6 E6 and E7 sequences were highly conserved within the analyzed patient samples in southwest China, indicating that the low risk HPV6 can adapt to the environment well without much evolution.

Identification of variants and therapeutic epitopes in HPV-33/HPV-58 E6 and E7 in Southwest China.

BACKGROUND: Human papillomavirus (HPV) E6 and E7 oncoproteins play a crucial role in HPV-related diseases, such as cervical cancer, and can be used as ideal targets for therapeutic vaccines. Human leukocyte antigen (HLA) participates in the immune response to block HPV infection and invasion by its target/recognition function. HPV-33 and HPV-58 are highly prevalent among Chinese women. Therefore, it is of great significance to study the E6 and E7 region-specific gene polymorphisms of HPV-33 and HPV-58 in Southwest China and to identify ideal epitopes for vaccine design. Both HPV-33 and HPV-58 belong to α-9 genus HPV and are highly homologous, so their correlations are included in our research. METHODS: To study the E6 and E7 variations and polymorphisms of HPV-33 and HPV-58 in Southwest China, we collected samples, extracted and sequenced DNA, and identified variants. Nucleotide sequences were translated into amino acids by Mega 6.0 software. The physical/chemical properties, amino acid-conserved sequences and secondary structure of protein sequences were analysed by the Protparam server, ConSurf server and PSIPRED software. The T and B cell epitopes of the E6/E7 reference and variant sequences in HPV-33 and HPV-58 were predicted by the Immune Epitope Database (IEDB) analysis server and the ABCpred server, respectively. RESULTS: Five and seven optimal HLA-I restricted T cell epitopes were selected from HPV-33 and HPV-58 E6, respectively, and these optimal epitopes are mainly located in 41-58EVYDFAFADLTVVYREGN of HPV-33 E6 and 40-60SEVYDFVFADLRIVYRDGNPF of HPV-58 E6. Six optimal HLA-I-restricted T cell epitopes were selected from HPV-33 and HPV-58 E7, and these epitopes are mainly located in 77-90RTIQQLLMGTVNIV of HPV-33 E7 and 78-91RTLQQLLMGTCTIV of HPV-58 E7. CONCLUSIONS: HPV-33/HPV-58 E6/E7 gene polymorphisms and T/B cell epitopes of their reference and variant sequences were studied, and candidate epitopes were selected by bioinformatics techniques for therapeutic vaccine design for people in Southwest China. This study was the first to investigate the correlation of epitopes between HPV-33 and HPV-58. After experimental validation, these selected epitopes will be employed to induce a wide range of immune responses in heterogeneous HLA populations.

Genetic variability of human papillomavirus type 51 E6, E7, L1 and L2 genes in Southwest China.

Genetic variations among HR-HPV types lead to altered biological functions with possible clinical significance in different geographical locations. To explore intratype genetic variations of HPV51 E6, E7, L1 and L2 genes originating from Southwest China, a total of 5204 cervical scraped cell samples were collected for DNA extraction and HPV typing. And then the E6, E7, L1 and L2 genes of HPV51 (n = 79) were sequenced and compared to the reference sequence (M62877). The ConSurf server was used for identification of conserved structural and functional amino acids of the E6 and E7 oncogenes, and the changes of the secondary structure were analyzed by PSIPred software. Phylogenetic trees were constructed by the maximum likelihood method implemented in IQ-TREE. The selection pressure acting on the E6, E7, L1 and L2 genes was estimated by Datamonkey web server. 13 nucleotide polymorphism sites were observed in E6-E7 gene and the most common mutation sites were C395T (S100L), C756T (S66L), C796T, A832G. 36 nucleotide polymorphism sites were identified in full length L1 gene and the non-synonymous mutations T6311G, A6312T (V264G), G6313A (G265S) A5674C (I52L), A6335C (N272T), A6586C (T354P) and synonymous mutations A5649T, C6147T, A6435G, G6570A, A6651G, T6774C, A6784C, A6882G, C6918A, and G6984A were the most common mutations. 53 nucleotide variation sites were identified in full-length L2 gene including four insertion sites (4418A, 4670G, 4693A, 4694C) and one deletion site (A4430). Besides, the non-synonymous mutations G4227A (V32I), A4407G (I92V), G4945A (D271N), C4985A (T284K), T5260G (L376V), A5335C (T401P) and the synonymous mutations A4166G, G4229A, G4283A, T4453C, C4566A, T4596C, C4695T, C4830T, G4839A, A5160C, and T5286G were the most common mutations. Specially, a triallelic mutation site (G4461C/A) in L2 was identified, with 26% G4461C (E109D) being non-synonymous mutation. Selective pressure analysis showed that only codon site 66 in E7 and 52 in L1 were the positively selected sites and codon sites 72, 107, 342, 412, 427 were negatively selected sites in L2 gene. Our investigation also suggests that A2 and A4 were the most frequent HPV51 lineage in Southwest China.

Genetic variability and lineage phylogeny of human papillomavirus type 45 based on E6 and E7 genes in Southwest China.

The current study investigated nucleotide variability and phylogeny in high-risk HPV45 collected from Chinese women. Fifty-one samples positive for single infections of HPV45 were collected for DNA extraction and HPV typing. The E6 and E7 genes of HPV45 were sequenced, and then the phylogenetic tree was reconstructed by the maximum likelihood method implemented in IQ-TREE under the HKY nucleotide substitution model. The selection pressures of the E6 and E7 genes were estimated using PAML software. Eleven nucleotide polymorphism sites were observed in the HPV45 E6 sequences, with 6 synonymous (C134T, T163C, A284C, T341C, T482C, A497G) and 5 non-synonymous (A124C, C157T, T162A, G259T, G487A) mutations. Six nucleotide polymorphism sites were observed in the E7 sequences, with 5 non-synonymous (G600A, A603C, A769C, G808T, G832T) and 1 synonymous (A718C) mutation. Our investigation suggests that B2 was the most frequent HPV45 sublineage in Southwest China, followed by A2; no A1 or A3 variants were detected. Selective pressure analysis showed that these mutations could reflect positive selection in HPV45 E6 and E7 genes.

Phylogeny and polymorphism in the long control regions E6, E7, and L1 of HPV Type 56 in women from southwest China.

Globally, human papillomavirus (HPV)­56 accounts for a small proportion of all high­risk HPV types; however, HPV­56 is detected at a higher rate in Asia, particularly in southwest China. The present study analyzed polymorphisms, intratypic variants, and genetic variability in the long control regions (LCR), E6, E7, and L1 of HPV­56 (n=75). The LCRs, E6, E7 and L1 were sequenced using a polymerase chain reaction and the sequences were submitted to GenBank. Maximum­likelihood trees were constructed using Kimura's two­parameter model, followed by secondary structure analysis and protein damaging prediction. Additionally, in order to assess the effect of variations in the LCR on putative binding sites for cellular proteins, MATCH server was used. Finally, the selection pressures of the E6­E7 and L1 genes were estimated. A total of 18 point substitutions, a 42­bp deletion and a 19­bp deletion of LCR were identified. Some of those mutations are embedded in the putative binding sites for transcription factors. 18 single nucleotide changes occurred in the E6­E7 sequence, 11/18 were non­synonymous substitutions and 7/18 were synonymous mutations. A total 24 single nucleotide changes were identified in the L1 sequence, 6/24 being non­synonymous mutations and 18/24 synonymous mutations. Selective pressure analysis predicted that the majority of mutations of HPV­56 E6, E7 and L1 were of positive selection. The phylogenetic tree demonstrated that the isolates distributed in two lineages. Data on the prevalence and genetic variation of HPV­56 types in southwest China may aid future studies on viral molecular mechanisms and contribute to future investigations of diagnostic probes and therapeutic vaccines.

Genetic variability in E5, E6, E7 and L1 genes of human papillomavirus type 31.

Human papillomavirus (HPV) type 31 is an important pathogenic subtype associated with cervical cancer. The aims of the present study were to analyze E5, E6, E7 and L1 gene mutations of HPV­31 among females, and to elucidate the evolutionary associations between them. In total, 87 positive samples were collected. The E5, E6, E7 and L1 genes were amplified by polymerase chain reaction and sequenced. Subsequently, two phylogenetic trees were constructed from the nucleotide sequences of the E5, E6 and E7 and the L1 variants of HPV­31. In total, 31 mutation sites of E5, E6 and E7 genes were identified, of which 16 were non­synonymous. T4053A (F80I), C285T (H60Y), C520T (A138V) and A743G (K62E) were the most common non­synonymous mutations. A total of 30 mutation sites of L1 genes were identified, of which four were non­synonymous. The most common non­synonymous mutations of L1 genes were A6350G (T29A) and C6372A (T36N). By phylogenetic analysis, A and C variants were most frequently detected, while B variants were less frequently detected in this population. The sequence variation data obtained in the present study provides a foundation for future research regarding HPV­induced oncogenesis, and may prove valuable for developing diagnostic probes and in the design of HPV vaccines for targeted populations.