STAT3 regulates 5-Fu resistance in human colorectal cancer cells by promoting Mcl-1-dependent cytoprotective autophagy.

Chemoresistance to 5-fluorouracil (5-Fu)-based chemotherapy is one of the primary reasons for the failure of colorectal cancer (CRC) management. STAT3 can mediate tumor drug resistance through a variety of diverse mechanisms. Nonetheless, the underlying mechanisms of STAT3-induced 5-Fu resistance in CRC are still poorly understood. Here, we aimed to investigate the potential mechanism(s) of STAT3-induced 5-Fu resistance in CRC. Quantitative RT-PCR and Western blot were used to test the expression of STAT3 and Mcl-1 in chemosensitive and chemoresistant CRC tissues and cell lines. After overexpression or knockdown of STAT3 or Mcl-1, and/or treatment with or without 5-Fu or chloroquine (CQ), we tested cell viability, inhibitory concentration 50% (IC50 ) value of 5-FU, cell apoptosis, proliferation, migration, and autophagy. STAT3 and Mcl-1 were significantly upregulated in the chemoresistant CRC tissues and cell lines, and STAT3 positively regulated Mcl-1. Functional studies demonstrated that STAT3 promoted 5-Fu resistance in CRC. Mechanistically, STAT3 triggered autophagy via Mcl-1 to induce cancer chemoresistance. Our results show that STAT3 regulates 5-Fu resistance in CRC by promoting Mcl-1-dependent cytoprotective autophagy. Our results provide a novel role of STAT3 and may offer a new approach for managing CRC 5-Fu resistance.

Photo-Thermal Superhydrophobic Sponge for Highly Efficient Anti-Icing and De-Icing.

Ice accretion always brings much inconvenience in the field of production and life. How to anti-ice or de-ice easily on solid surfaces becomes research focus in the engineering material fields. In this work, a kind of photo-thermal superhydrophobic polyurethane sponge (PSP-SPONGE) was developed by depositing Fe3O4 nanoparticles and polydopamine and simple fluorination treatment to realize anti-icing and de-icing fast under faint sunlight irradiation. Utilizing the thermal insulation of porous PSP-SPONGE, the photo-thermal energy was located at the sunlight irradiation area, which heated PSP-SPONGE surface rapidly under sunlight irradiation in cold surroundings. Water droplets on PSP-SPONGE surface would never freeze under faint 0.3 kW/m2 ("0.3 sun") sunlight illumination in -30 °C damp surroundings, and the ice melts entirely within 18 min under "1 sun" illumination. Furthermore, PSP-SPONGE has excellent self-cleaning and self-healing properties that can cope with the complex and volatile natural environment to guarantee durable anti-icing and de-icing performances. The simulated outdoor snow removal test also proved that snow on PSP-SPONGE surface could melt under "0.5 sun" sunlight illumination in -30 °C damp surroundings. The PSP-SPONGE fabricated with simple preparation and easy access has wide application prospects in anti-icing and de-icing.

Polymorphism of E6 and E7 Genes in Human Papillomavirus Types 31 and 33 in Northeast China.

Persistent infection with human papillomavirus (HPV) types 31 and 33 is an important causative factor for cervical cancer. The E6/E7 genes are key oncogenes involved in the immortalization and transformation of human epithelial cells. Genetic polymorphism may lead to differences in the virus' carcinogenic potential, the immune reaction of the host, and the potencies of vaccines. Few studies on HPV31/33 E6/E7 genetic polymorphism have been carried out. To study the genetic polymorphism of HPV31 and HPV33 E6/E7 genes in northeast China, these genes (HPV31 E6/E7, n = 151; HPV33 E6/E7, n = 136) were sequenced and compared to reference sequences (J04353.1, M12732.1) using BioEdit. Phylogenetic trees were constructed by the neighbor-joining method using MegaX. The diversity of the secondary structure was estimated using the PSIPred server. The positively selected sites were analyzed using PAML4.9. The major histocompatibility complex (MHC) class I and MHCII epitopes were predicted using the ProPred-I server and ProPredserver. B-cell epitopes were predicted using the ABCpred server. In the 151 HPV31E6 sequences, 25 (25/450) single-nucleotide mutations were found, 14 of which were synonymous mutations and 11 were nonsynonymous. In the 151 HPV31E7 sequences, 8 (8/297) nucleotide mutations were found, 3 of which were synonymous mutations and 5 were nonsynonymous. In the 136 HPV33E6 sequences, 17 (17/450) nucleotide mutations were observed, 7 of which were synonymous mutations and 10 were nonsynonymous. C14T/G (T5I/S) was a triallelic mutation. Finally, in the 136 HPV33E7 sequences, 9 (9/294) nucleotide mutations were observed, 3 of which were synonymous mutations and 6 were nonsynonymous. C134T/A (A45V/E) and C278G/A (T93S/N) were triallelic mutations. Lineage A was the most common lineage in both HPV31 and HPV33. In all of the sequences, we only identified one positively selected site, HPV33 E6 (K93N). Most nonsynonymous mutations were localized at sites belonging to MHC and/or B-cell predicted epitopes. Data obtained in this study should contribute to the development and application of detection probes, targeted drugs, and vaccines.

Neurotrophic factor levels in the serum and cerebrospinal fluid of neonates infected with human cytomegalovirus.

Human cytomegalovirus (HCMV) is most likely to damage the central nervous system (CNS) during early embryonic development; however, the early neurodevelopmental abnormalities caused by HCMV infection and the regulation of cytokines remain unclear. Therefore, we investigated neuronal factors in the serum and cerebrospinal fluid (CSF) of newborns infected with HCMV using protein microarray technology with a view to elucidating the changes in specific neuronal factors for use in the development of a reliable index for predicting CNS injury caused by HCMV infection. Serum and CSF were collected from four newborns with HCMV infection and CNS injury (HCMV-infected group) and from four newborns without CNS infection (control group). A protein microarray containing 29 kinds of CNS-related cytokines was used to identify differentially expressed neuronal factors in the serum and CSF of the HCMV-infected and control groups. The levels of the differentially expressed proteins were verified further in 30 CSF samples from an HCMV-infected group using enzyme-linkedimmunosorbent assay (ELISA). Between newborns in the HCMV-infected and control groups, the protein microarray analysis identified three differentially expressed neurotrophic factors in the CSF samples: Acrp30, MMP-3, and interleukin-1 alpha (IL-1α). No differential cytokine expression was seen in the serum. ELISA showed significantly higher expression levels of Acrp30 and MMP-3 in the CSF of the 30 newborns with HCMV infection and CNS injury than in those in the control group, whereas the expression of IL-1α was significantly lower. Our results demonstrate that changes in the expression levels of Acrp30, MMP-3, and IL-1α in the CSF of newborns infected with HCMV may be related to the pathogenesis of CNS infection.

Sertad1 promotes prostate cancer progression through binding androgen receptor ligand binding domain.

Androgen receptor (AR) signaling is involved in the initiation and progression of prostate cancer (PCa), which is the most frequently diagnosed nonskin cancer and remains a leading cause of cancer-related death in men. Further investigation of the involvement of AR signaling in PCa progression is urgently needed. In the present study, we performed a yeast two-hybrid screen and demonstrated that SERTA domain-containing protein 1 (Sertad1) is a novel AR-binding protein that binds to the AR ligand binding domain (LBD). The binding between AR-LBD and Sertad1 was confirmed by glutathione S-transferase (GST) pull-down assays and immunoprecipitation (IP) and confocal immunofluorescence co-localization experiments. Furthermore, we demonstrated that DHT inhibited Sertad1 protein degradation in prostate cancer cell lines and that Sertad1 knockdown inhibited the proliferation of prostate cancer cells in vitro. In human PCa tumor tissues, Sertad1 expression is positively correlated with AR expression and the Gleason score. Taken together, this report is the first to show that Sertad1 is a novel AR-LBD-binding protein, and DHT-liganded AR-LBD inhibits Sertad1 degradation. Thus, Sertad1 may represent a novel therapeutic target for the treatment of AR-positive PCa.

Viral load and integration status of HPV58 associated with cervical lesion severity in women of Northeast China.

Objective: We aimed to evaluate the viral load and integration status of human papillomavirus 58 in women with different grades of cervical lesions to determine whether viral load and integration status are related to malignant transformation in HPV58-infected women. Methods: A total of 212 cervical specimens were collected from women in Northeast China who had undergone human papillomavirus genotyping and were HPV58-positive. The HPV58 viral load was determined using real-time polymerase chain reaction, and the integration status was discriminated using the ratio of HPV58 E2 gene copy number to E6 gene copy number. Results: The median HPV58 viral load in women with normal cervix or cervicitis, low-grade squamous cell intraepithelial lesion, high-grade squamous cell intraepithelial lesion and cervical cancer was 352.12, 864.21, 1199.75 and 693.04 copies/genome, respectively. High significance was obtained when comparing the viral load of infected women presenting normal/cervicitis with that of the women either with precancerous cervical lesions or cervical cancer (P < 0.05). The HPV58 genome was in the episomal form in 35 samples (16.5%), mixed episomal and integrated forms in 165 (77.8%) samples, and completely integrated into the host genome in 12 (5.7%) samples. The HPV58 E2/E6 copy number ratio in the cervical cancer group was significantly lower than that in the other groups (P < 0.01). Conclusions: The HPV58 viral load in patients with precancerous cervical lesions or cervical cancer increases significantly with disease progression. The HPV58 E2/E6 copy number ratio in patients with cervical cancer is lower than that for less severe cervical lesions, suggesting a high degree of viral integration may be a considerable risk factor for cervical cancer.

Multiple human papillomavirus infections and type-competition in women from a clinic attendee population in China.

To investigate the multi-infection patterns and type competition for human papillomavirus (HPV) in Chinese clinic attending women and evaluate the association between the infection status and cancer risk. Three hundred and thirty-two HPV-DNA-positive samples were genotyped for 21 HPVs and tested for 13 types of HPV neutralizing antibody using pseudoviron-based assay. Odds ratios (ORs) were calculated to evaluate the coinfection patterns for both DNA and neutralizing antibody (NAb), and the associations of HSIL+ with HPV DNA and NAb. Of the 332 HPV-DNA-positive subjects, 279 (84.0%) were detected as NAb positive. Multi-positive results were identified in 23.2% (77/332) HPV DNA tests and 60.2% (168/279) HPV NAb assays. The NAb titers for the multiple-positive samples (geometric mean 214) were significantly higher than the single-positive samples (geometric mean 114) (P < 0.01). HPV16-HPV52 was identified as a type-competition pair for both HPV DNA (OR = 0.09; 95%CI = 0.02-0.42) and NAb (OR = 0.27; 95%CI = 0.11-0.69) data. Compared to other types, HPV16 DNA was associated significantly with high risk of HSIL+ (OR = 2.57; 95%CI = 1.23-5.38). HPV16-HPV52 was identified as a potential type-competition pair, which might result in the type modulation with the implementation of the approved bi- or quadri-valent vaccines. J. Med. Virol. 88:1989-1998, 2016. © 2016 Wiley Periodicals, Inc.

Human Cytomegalovirus Increases the Risk of Future Hemorrhagic But Not Ischemic Stroke　- A Nested Case-Control Study.

BACKGROUND: The cause-and-effect relationship between human cytomegalovirus (HCMV) and stroke has not been widely elucidated. We aimed to determine if HCMV infection has an increased risk of future stroke in hypertensive patients in rural areas of China. METHODS AND RESULTS: This was a nested case-control study from a prospective cohort study. A total of 300 newly diagnosed stroke cases with a median follow-up period of 8.4 years and 300 matched controls were selected for the present analysis. Adjusted odds ratio (OR) for stroke associated with HCMV DNA seropositivity was calculated by conditional logistic regression. HCMV DNA was detected in 38 of 300 samples from stroke patients and in 17 of 300 control samples (12.7% vs. 5.7%; P=0.023). Seropositivity for HCMV DNA increased the risk of incident stroke (unadjusted OR, 1.437; 95% confidence interval (CI), 1.023-2.020, P=0.037) and adjustment for other potential cardiovascular confounders only slightly changed the OR (1.464; 95% CI, 1.003-2.137, P=0.048). After controlling for potential cardiovascular confounders, the OR for hemorrhagic stroke associated with HCMV DNA was 1.718 (95% CI, 1.042-2.832), whereas the OR for ischemic stroke was 0.450 (95% CI, 0.142-1.428). CONCLUSIONS: Seropositivity for HCMV DNA was positively associated with total and hemorrhagic but not ischemic stroke, which persisted after controlling for other cardiovascular factors. (Circ J 2016; 80: 2235-2239).

A cluster of 3' coterminal transcripts from US12-US17 locus of human cytomegalovirus.

Among all the human cytomegalovirus (HCMV) gene families, US12 family is relatively undefined in their transcriptional profile and biological functions. In this study, the transcription pattern and characteristics of HCMV US12-US17 gene region were studied extensively. Twenty-three clones harboring US12 cDNA sequence were screened out from a late cDNA library of an HCMV clinical isolate, Han. Using a set of US12-US17 gene-specific probes, six transcripts from US12-US17 locus were detected by northern blot at late kinetics of the clinical isolate. One additional transcript was found in late RNA of HCMV strain AD169. No evidence showing these transcripts contain introns by reverse transcription PCR. 3' and 5' termini of these transcripts were confirmed by Rapid Amplification of cDNA Ends. A novel protein-coding region was predicted in the shorter US14 transcript with an alternative in-frame 5' translation initiation site compared to that of the previously predicted US14 ORF. Our findings demonstrate the existence of a cluster of 3' coterminal unspliced transcripts with distinct 5' transcriptional initiation sites originated from US12-US17 gene region in the late infection phase of an HCMV clinical strain.

Prevalence of microorganisms co-infections in human papillomaviruses infected women in Northern China.

PURPOSE: Due to scarce data on the prevalence of genital microorganism infections among individuals with human papillomavirus (HPV) infections, the present study aimed to evaluate microorganism co-infections and associated risk factors in HPV-infected women in Northern China. METHODS: Cervical samples of 4290 enrolled female patients were collected to detect HPV, bacterial and yeast infections in gynecologic outpatients. Serum samples collected were analyzed for the presence of serological markers for human immunodeficiency virus (HIV), hepatitis B (HBV) and hepatitis C (HCV) and Treponema pallidum infections. HPV typing was carried out by polymerase chain reaction and flow-through hybridization on cervical brush specimens. Chlamydia trachomatis was identified by strand displacement amplification, and other microorganisms were detected by conventional methods. Odds ratio of co-infection was assessed between HPV-positive and -negative patients. RESULTS: Overall, co-infections among HPV-infected patients showed a trend for risk compared with HPV-negative patients. In this cross-sectional study on 4290 female outpatients, statistical analyses revealed a significant association between HPV and detection of anti-HBV antibodies, bacterial vaginosis, C. trachomatis and Ureaplasma urealyticum; no correlation was found between HPV infection and anti-HIV, anti-HCV, T. pallidum, Trichomonas vaginalis and Neisseria gonorrhoeae, which were detected only in few cases among either HPV-positive or -negative patients. CONCLUSION: The study demonstrated a significant association between HPV and HBV, bacterial vaginosis, C. trachomatis and U. urealyticum. The study results suggest that it may be important to screen for the simultaneous presence of different microorganism co-infections with HPV that may have synergistic pathological effects.

Development of a fluorescence-based multiplex genotyping method for simultaneous determination of human papillomavirus infections and viral loads.

BACKGROUND: Persistent high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing intraepithelial lesion or malignancy (NILM). The aims of the current study is to establish a method named BioPerfectus Multiplex Real Time (BMRT) HPV assay for simultaneous typing and quantifying HPVs, and to evaluate it by comparison with HPV GenoArray test and PCR-sequencing method, as well as histological status. METHODS: A total of 817 cervical specimens were evaluated by BMRT method and HPV GenoArray test, using PCR-sequencing method as the reference standard; simultaneously, high-risk HPV-16 and -18 DNA loads were assessed in 443 specimens to investigate the correlation with infection outcomes. RESULTS: The overall detection coincidence rate between BMRT assay and HPV GenoArray test is 96.6 % and the Kappa value is 0.760. In addition, the sensitivity and positive predictive value of BMRT is 98.4 % and 95.7 % compared with the results detected by PCR-sequencing method, respectively. HPV-16 viral load has a correlation with CINs or worse lesions. By comparing with infected women presenting NILM /cervicitis, the cutoff value for HPV-16 from patients with CINs was 0.827. With this cutoff value, 74.6 % sensitivity and 72.5 % specificity for prediction of HPV-16 infected patients with CINI and higher CIN were achieved. High significance was obtained when comparing the infected women presenting NILM/cervicitis with women either with CIN and cervical carcinomas (p < 0.001). CONCLUSIONS: The BMRT assay seemed to be a good alternative approach for HR-HPV testing, due to its high level of automation and ability to quantify HPV-16, HPV-18 and other HR-HPVs.

Human cytomegalovirus miR-UL36-5p inhibits apoptosis via downregulation of adenine nucleotide translocator 3 in cultured cells.

Human cytomegalovirus (HCMV) encodes at least 26 microRNAs (miRNA). These miRNAs are utilized by HCMV to regulate its own genes as well as the genes of the host cell during infection. It has been reported that a cellular gene, solute carrier family 25, member 6 (SLC25A6), which is also designated adenine nucleotide translocator 3 (ANT3), was identified as a candidate target of hcmv-miR-UL36-5p by hybrid PCR. In this study, ANT3 was further demonstrated to be a direct target of hcmv-miR-UL36-5p by luciferase reporter assays. The expression level of ANT3 protein was confirmed, by western blotting, to be directly downregulated by overexpression of hcmv-miR-UL36-5p in HEK293 cells, U373 cells and HELF cells. Moreover, HCMV-infected cells showed a decrease in the ANT3 protein level. Using ANT3-specific small interfering RNA (siRNA) and an inhibitor for hcmv-miR-UL36-5p, it was shown that inhibition of apoptosis by hcmv-miR-UL36-5p in these cells specifically occurred via inhibition of ANT3 expression. These results imply that hcmv-miR-UL36-5 may play the same role during actual HCMV infection in order to establish a balance between the host cell and the virus.

Genetic variations of E6 and long control region of human papillomavirus type 16 from patients with cervical lesion in Liaoning, China.

BACKGROUND: High-risk human papillomavirus type 16 (HPV16) is a risk factor for cervical cancer. Previous studies suggest that polymorphisms in the E6 gene or the long control region(LCR)of HPV16 may alter the oncogenic potential of the virus. The aims of this study were to investigate the genetic variations of HPV16 E6 gene and LCR in isolates from Chinese population and correlation of the E6 and LCR polymorphisms with disease status of infected patients. METHODS: HPV16 positive endocervical specimens were collected from 304 women living in Northeast of China. Sequences of E6 gene and LCR were analyzed by PCR-sequencing. RESULTS: Two lineages were found in the populations, including EUR lineage and As lineage. Based on the HPV16 prototype, the most frequent variation in the E6 gene was T178A/G (48.7%), followed by mutations of G94A (12.2%) and T350G (9.9%). The rank orders of incidence of E6 variations in amino acid were as follows: D25E (46.3%), L83V (9.9%) and H78Y (4.3%). Nucleotide variations in LCR were found in all the 304 isolates from HPV16 positive cervical samples. The most commonly observed LCR variations were the transition replacement G7193T, 7434CIns, G7521A and 7863ADel (100%). The As lineage was associated with HPV persistent infections and with disease status of ≥CIN2,3. The EUR lineage variants showed a negative trend of association with the severity of ≥CIN2,3. Among 41 variations found in LCR, 25 (61.0%) were located at the binding sites for transcription factors. Occurrence of ≥CIN2,3 was significantly associated with the mutations of R10G/L83V in E6 and the C7294T co-variation in LCR, after adjusting for ages of infected patients. CONCLUSIONS: Associations between As lineage and HPV persistent infections, and with disease status of ≥CIN2,3, and an association between the EUR lineage and negative trend of association with the severity of ≥CIN2,3 were found in this study. An association between a co-variation of R10G/L83V in E6 and C7294T in LCR and an increased risk for developing CIN-2,3 was found in a HPV16 infected population of Chinese women. These findings indicate that HPV16 polymorphism influences development of CIN-2,3.

The expression of interleukin-32 is activated by human cytomegalovirus infection and down regulated by hcmv-miR-UL112-1.

BACKGROUND: Interleukin-32 (IL-32) is an important factor in innate and adaptive immune responses, which activates the p38MAPK, NF-kappa B and AP-1 signaling pathways. Recent reports have highlighted that IL-32 is regulated during viral infection in humans. METHODS: Enzyme-linked immunosorbent assays (ELISA) were carried out to detect IL-32 levels in serum samples. Detailed kinetics of the transcription of IL-32 mRNA and expression of IL-32 protein during human cytomegalovirus (HCMV) infection were determined by semi-quantitative RT-PCR and western blot, respectively. The expression levels of hcmv-miR-UL112-1 were detected using TaqMan® miRNA assays during a time course of 96 hours. The effects of hcmv-miR-UL112-1 on IL-32 expression were demonstrated by luciferase assay and western blot, respectively. RESULTS: Serum levels of IL-32 in HCMV-IgM positive patients (indicating an active HCMV infection) were significantly higher than those in HCMV-IgM negative controls. HCMV infection activated cellular IL-32 transcription mainly in the immediately early (IE) phase and elevated IL-32 protein levels between 6 and 72 hours post infection (hpi) in the human embryonic lung fibroblast cell line, MRC-5. The expression of hcmv-miR-UL112-1 was detected at 24 hpi and increased gradually as the HCMV-infection process was prolonged. In addition, it was demonstrated that hcmv-miR-UL112-1 targets a sequence in the IL-32 3'-UTR. The protein level of IL-32 in HEK293 cells could be functionally down-regulated by transfected hcmv-miR-UL112-1. CONCLUSIONS: IL-32 expression was induced by active HCMV infection and could be functionally down-regulated by ectopically expressed hcmv-miR-UL112-1. Our data may indicate a new strategy of immune evasion by HCMV through post-transcriptional regulation.

Analysis and mapping of a 3' coterminal transcription unit derived from human cytomegalovirus open reading frames UL30-UL32.

BACKGROUND: It has been predicted that the UL31 gene originates from the positive strand of the human cytomegalovirus (HCMV) genome, whereas the UL30 and UL32 genes originate from the complementary strand. Except for the UL32 gene, the transcription of this gene region has not been investigated extensively. METHODS: Northern blotting, cDNA library screening, RACE-PCR,and RT-PCR were used. RESULTS: At least eight transcripts of the antisense orientation of UL31 were transcribed from the UL30-UL32 region during the late phase of HCMV infection. The 3' coterminus of these transcripts was located within the predicted UL30 gene. The longest 6.0-kb transcript was initiated upstream of the predicted UL32 gene. Other transcripts were derived from the predicted UL30 and UL31 gene region. Except for the previously predicted UL32 open reading frame (ORF), three novel ORFs, named UL31anti-1, UL31anti-2 and UL31anti-3, were located in the transcripts from the UL31anti-UL32 transcription unit. No transcription was found in UL31. CONCLUSION: A family of novel 3' coterminal transcripts was transcribed from the UL30-UL32 gene region.

Transcription characteristics of the human cytomegalovirus UL13 gene.

The human cytomegalovirus (HCMV) UL13 gene is located in the unique long (UL) region of its genome. The transcript structure of UL13 gene has not been investigated to date. By using cDNA library screening, northern blot, and rapid amplification of cDNA ends (RACE), the HCMV UL13 gene was demonstrated to be transcribed from the immediate early (IE) to the late (L) phase of infection, and at least one 1602-nt unspliced transcript was identified in the present study from three clinical isolates.

Variant lineages of human papillomavirus type 18 in Northeast China populations characterized by sequence analysis of E6, E7, and L1 regions.

INTRODUCTION: In cervical cancer, human papillomavirus (HPV) 18 is predominantly related to adenocarcinomas. Variant lineages of HPV type 16 have been well characterized, whereas the knowledge about HPV 18 variants is limited in Northeast China. METHODS: To identify prevalent and novel HPV 18 variants in Northeast China, the E6, E7, and L1 genes of HPV 18 from patients with cervical lesion were amplified and sequenced, and intratypic variants were analyzed by comparing to the known phylogenetic branches. RESULTS: The HPV-18 E6 variants of our studied strains belong to 2 main branches: Asian-American (AA) variants in 81.5% and European (E) variants in 18.5%. Strains with variations of C287G, T482C, and C519A in E6 and C751T in E7 were novel variants. All the L1 genes of the analyzed HPV 18 strains had 4 C-G transversions at nucleotide positions of 5701, 6460, 6625, and 6842 and one G-A transition at position 5503. Moreover, strains with L1 nucleotide variations of A5920T, A6431T, and G6987A leading to amino acid substitutions of A164V, Q334P/H, and D520N are novel variants. CONCLUSIONS: Based on the E6 gene, the prevalent HPV 18 in Northeast China was AA and E variants. Besides some common variations reported before, some new variations in the E6, E7, and L1 genes were found. Data about the novel variations found in the L1 gene of HPV 18 variants may be helpful to design the diagnostic reagents and vaccine for naturally infected HPV 18 in Northeast China.

Genomic polymorphism of human papillomavirus type 52 in women from Northeast China.

Human papillomavirus (HPV) 52 is an oncogenic HPV type prevalent in Asia. The aim of the study was to analyze HPV 52 genetic variations in women from Northeast China. To explore the intratypic variants of HPV 52, the genomic regions of L1, E6, E7 and long control region (LCR) of HPV 52, which have been identified in women from Northeast China by HPV GenoArray test, were analyzed. Twenty-five mutations were identified in the regions examined. Of the mutations found in the L1 gene, three novel nonsynonymous mutations of C5640T, A5641T and G5642A were located within the region that encodes the binding domain of neutralizing antibodies against HPV 52. Although four variations were identified in HPV 52 E6 and E7 genes, no significant association was found between the mutations and the cytological lesion of the patients. Eight mutations, including a novel CTT7681−7683 deletion, found in the LCR of HPV 52 encompassed the known transcription binding sites, which may possibly affect the transcription of the oncogenic genes of E6 and E7. The most prevalent HPV 52 variant in women from northeastern China belongs to clade L1-LN-A. The genetic variations of HPV 52, including three novel nonsynonymous mutations of C5640T, A5641T and G5642A in the L1 gene and a novel CTT7681−7683 deletion in the LCR, were first documented in strains from women in Northeast China. The statistical result showed no associations between the variants and the severities of the infected women. These findings provide new data regarding gene variations of HPV 52.

Transcriptional features and transcript structure of UL145 in different strains of human cytomegalovirus.

The human cytomegalovirus UL145 gene is located between the highly variable genes UL144 and UL146 in the UL/b' region. However, unlike its neighboring genes, UL145 is relatively conserved among strains isolated from patients. The transcriptional features and transcript structure of UL145 has not yet been examined. In this study, the transcriptional features and structure of UL145 were characterized using RNA preparations from three HCMV strains isolated from patients, designated H, C, and X, respectively. Two transcripts were identified by cDNA library screening. Two main clusters of transcripts, one located between 500 and 700 nt, and the other between 1,400 and 1,700 nt, were confirmed by Northern blot analysis. Abundant transcripts were detected at 96 h post-infection by Northern blot, suggesting that UL145 was a late gene. The terminal sequences of transcripts obtained by 3' rapid amplification of cDNA ends demonstrated the presence of polyadenylation. Transcripts identified in the RNA of H, C, and X strains 96 h post-infection had the same 3' end but different 5' ends. In addition to polycistronic transcripts with upstream genes, UL145 could be transcribed as a single transcript during late strain infections.

A rapid method to screen putative mRNA targets of any known microRNA.

BACKGROUND: microRNAs (miRNAs) are a group of regulatory RNAs that regulate gene expression by binding to specific sequences on target mRNAs. However, functional identification of mRNA targets is usually difficult and time consuming. Here we report hybrid-PCR as a new and rapid approach to screen putative mRNA targets in vitro. RESULTS: Fifteen putative target mRNAs for human cytomegalovirus (HCMV) miR-UL112-1, including previously confirmed HCMV IE72, were identified from mRNA-derived cDNAs using hybrid-PCR. Moreover, we randomly validated six different target candidates by luciferase reporter assays, and confirmed that their luciferase activities were down-regulated with co-transfection of HCMV miR-UL112-1. CONCLUSIONS: Our study demonstrated that hybrid-PCR is an effective and rapid approach for screening putative miRNA targets, with much more advantage of simplicity, low cost, and ease of implementation.