Prevalence of high-risk human papillomavirus genotypes in two regions of Peru.

OBJECTIVE: This study aims to discover the most prevalent high-risk (hr) HPV genotypes in the regions of Loreto and La Libertad, Peru. METHODS: In 2015, cervical cell samples were collected during pelvic examinations from women in the geographically distinct regions of Loreto and La Libertad, Peru. In 2017, additional samples were collected in La Libertad. A total of 429 women between the ages of 18 and 65 years living in these regions were enrolled in the study. All samples were analyzed by polymerase chain reaction for the hrHPV genotypes 16, 18, and 35. RESULTS: Sample collection from 126 women in 2015 in Loreto revealed an hrHPV incidence of 15.9% (20 of 126), with 60% (12 of 126) of HPV infections due to hrHPV 16. Samples from La Libertad revealed an hrHPV incidence of 14.5% incidence (44 of 303) (among 303 women). Of these infections, 38% (17) were attributable to hrHPV type 35 and 20% (9) were due to hrHPV type 16. CONCLUSION: The prevalence of hrHPV genotypes in Peru may differ from those observed in North America and Europe. Loreto appears to follow the prevalence trend observed in North America, with hrHPV type 16 accounting for the majority of cases. However, hrHPV type 35 may account for a greater contribution to the cervical cancer burden in La Libertad. Further research, specifically on cervical tumor specimens, is needed.

Down-regulation of 14-3-3zeta reduces proliferation and increases apoptosis in human glioblastoma.

Many efforts have been taken to develop molecule target for cancer therapy. 14-3-3zeta protein has emerged as a critical regulator of diverse cellular pathways in multiple cancers. Furthermore, 14-3-3zeta expression was elevated and a predictor of poor prognosis in glioblastoma. However, there is no information to evaluate the potential effects of 14-3-3zeta RNAi in glioblastoma. The relationship between 14-3-3zeta expression and cell proliferation and apoptosis was tested in primary glioblastoma samples. Through an RNAi approach using human glioblastoma cells as a model system, we demonstrated the role of 14-3-3zeta in glioblastoma proliferation, apoptosis, invasion and tumor growth. The expression of 14-3-3zeta in glioblastoma stem cells was also investigated by immunostaining. The apoptosis was significantly higher in 14-3-3zeta-negative group than in positive group. 14-3-3zeta immunoreactivity score was negatively correlated with the apoptosis, and positively with proliferation in human specimens. 14-3-3zeta RNAi reduced cell proliferation, induced apoptosis, decreased the invasive capability and colony-formation, and impaired the growth of glioblastoma xenografts in nude mice. Moreover, 14-3-3zeta was positively expressed in glioblastoma stem cells. Our data highlight the importance of 14-3-3zeta in glioblastoma and identify 14-3-3zeta as a potential molecular target for glioblastoma treatment.

Author Correction: Whole-exome SNP array identifies 15 new susceptibility loci for psoriasis.

This corrects the article DOI: 10.1038/ncomms7793.

The OncoPPi network of cancer-focused protein-protein interactions to inform biological insights and therapeutic strategies.

As genomics advances reveal the cancer gene landscape, a daunting task is to understand how these genes contribute to dysregulated oncogenic pathways. Integration of cancer genes into networks offers opportunities to reveal protein-protein interactions (PPIs) with functional and therapeutic significance. Here, we report the generation of a cancer-focused PPI network, termed OncoPPi, and identification of >260 cancer-associated PPIs not in other large-scale interactomes. PPI hubs reveal new regulatory mechanisms for cancer genes like MYC, STK11, RASSF1 and CDK4. As example, the NSD3 (WHSC1L1)-MYC interaction suggests a new mechanism for NSD3/BRD4 chromatin complex regulation of MYC-driven tumours. Association of undruggable tumour suppressors with drug targets informs therapeutic options. Based on OncoPPi-derived STK11-CDK4 connectivity, we observe enhanced sensitivity of STK11-silenced lung cancer cells to the FDA-approved CDK4 inhibitor palbociclib. OncoPPi is a focused PPI resource that links cancer genes into a signalling network for discovery of PPI targets and network-implicated tumour vulnerabilities for therapeutic interrogation.

Evaluation of Nucleic Acid Isothermal Amplification Methods for Human Clinical Microbial Infection Detection.

Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70-120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99.3%, and a PPV of 84% and NPV of 98% as compared to clinical microbial detection methods. Results indicate the LAMP-based In-Dx panel allows rapid and precise diagnosis of clinical infections by targeted pathogens across multiple culture types for point-of-care utilization.

Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform.

Large-scale genomics studies have generated vast resources for in-depth understanding of vital biological and pathological processes. A rising challenge is to leverage such enormous information to rapidly decipher the intricate protein-protein interactions (PPIs) for functional characterization and therapeutic interventions. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform with both high sensitivity and robustness in a mammalian cell environment remains to be established. Here we describe the development and integration of a highly sensitive NanoLuc luciferase-based bioluminescence resonance energy transfer technology, termed BRET(n), which enables ultra-high-throughput (uHTS) PPI detection in live cells with streamlined co-expression of biosensors in a miniaturized format. We further demonstrate the application of BRET(n) in uHTS format in chemical biology research, including the discovery of chemical probes that disrupt PRAS40 dimerization and pathway connectivity profiling among core members of the Hippo signaling pathway. Such hippo pathway profiling not only confirmed previously reported PPIs, but also revealed two novel interactions, suggesting new mechanisms for regulation of Hippo signaling. Our BRET(n) biosensor platform with uHTS capability is expected to accelerate systematic PPI network mapping and PPI modulator-based drug discovery.

Downregulation of urea transporter UT-A1 activity by 14-3-3 protein.

Urea transporter (UT)-A1 in the kidney inner medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the kidney inner medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione-S-transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.

Whole-exome SNP array identifies 15 new susceptibility loci for psoriasis.

Genome-wide association studies (GWASs) have reproducibly associated ∼40 susceptibility loci with psoriasis. However, the missing heritability is evident and the contributions of coding variants have not yet been systematically evaluated. Here, we present a large-scale whole-exome array analysis for psoriasis consisting of 42,760 individuals. We discover 16 SNPs within 15 new genes/loci associated with psoriasis, including C1orf141, ZNF683, TMC6, AIM2, IL1RL1, CASR, SON, ZFYVE16, MTHFR, CCDC129, ZNF143, AP5B1, SYNE2, IFNGR2 and 3q26.2-q27 (P<5.00 × 10(-08)). In addition, we also replicate four known susceptibility loci TNIP1, NFKBIA, IL12B and LCE3D-LCE3E. These susceptibility variants identified in the current study collectively account for 1.9% of the psoriasis heritability. The variant within AIM2 is predicted to impact protein structure. Our findings increase the number of genetic risk factors for psoriasis and highlight new and plausible biological pathways in psoriasis.

Cables1 complex couples survival signaling to the cell death machinery.

Cables1 is a candidate tumor suppressor that negatively regulates cell growth by inhibiting cyclin-dependent kinases. Cables1 expression is lost frequently in human cancer but little is known about its regulation. Here, we report that Cables1 levels are controlled by a phosphorylation and 14-3-3-dependent mechanism. Mutagenic analyses identified two residues, T44 and T150, that are specifically critical for 14-3-3 binding and that serve as substrates for phosphorylation by the cell survival kinase Akt, which by binding directly to Cables1 recruits 14-3-3 to the complex. In cells, Cables1 overexpression induced apoptosis and inhibited cell growth in part by stabilizing p21 and decreasing Cdk2 kinase activity. Ectopic expression of activated Akt (AKT1) prevented Cables1-induced apoptosis. Clinically, levels of phosphorylated Cables1 and phosphorylated Akt correlated with each other in human lung cancer specimens, consistent with pathophysiologic significance. Together, our results illuminated a dynamic regulatory system through which activated Akt and 14-3-3 work directly together to neutralize a potent tumor suppressor function of Cables1.

Pro-oncogenic function of HIP-55/Drebrin-like (DBNL) through Ser269/Thr291-phospho-sensor motifs.

HIP-55 (HPK1-interacting protein of 55 kDa, also named DBNL, SH3P7, and mAbp1) is a multidomain adaptor protein that is critical for organ development and the immune response. Here, we report the coupling of HIP-55 to cell growth control through its 14-3-3-binding phospho-Ser/Thr-sensor sites. Using affinity chromatography, we found HIP-55 formed a complex with 14-3-3 proteins, revealing a new node in phospho-Ser/Thr-mediated signaling networks. In addition, we demonstrated that HIP-55 is required for proper cell growth control. Enforced HIP-55 expression promoted proliferation, colony formation, migration, and invasion of lung cancer cells while silencing of HIP-55 reversed these effects. Importantly, HIP-55 was found to be upregulated in lung cancer cell lines and in tumor tissues of lung cancer patients. Upregulated HIP-55 was required to promote the growth of tumors in a xenograft animal model. However, tumors with S269A/T291A-mutated HIP-55, which ablates 14-3-3 binding, exhibited significantly reduced sizes, supporting a vital role of the HIP-55/14-3-3 protein interaction node in transmitting oncogenic signals. Mechanistically, HIP-55-mediated tumorigenesis activity appears to be in part mediated by antagonizing the tumor suppressor function of HPK1. Thus, the HIP-55-mediated oncogenic pathway, through S269/T291, may be exploited for the development of new therapeutic strategies.

Regulation of cell survival by the HIP-55 signaling network.

HIP-55 (hematopoietic progenitor kinase 1 [HPK1]-interacting protein of 55 kDa) is the mammalian homologue of the yeast Abp1p. It contains a C-terminal Src homology 3 domain and an N-terminal actin depolymerization factor (ADF-H/C) domain. HIP-55 appears to be critical for organ development and immune response and is important for the regulation of the actin cytoskeleton through its interactions with F-actin and various cytoskeletal and cell signaling proteins. However, the function of HIP-55 in tumors remains unknown. Here, we found that HIP-55 is up-regulated or down-regulated in several types of tumor tissues in patients. Of these, lung cancer tissues had the highest expression of HIP-55. To gain full insight into the function of HIP-55 in lung cancer, microarray assay was performed using Affymetrix U133 Plus 2.0 expression arrays in both HIP-55 knockdown and scramble control A549 cells. The ingenuity pathway analysis tool was utilized to construct biological networks and analyze functions that might be associated with HIP-55. Functional analysis strongly suggested that HIP-55 may be involved in cancer cell survival and cell death, which was then confirmed by further experimentation. Experimental results showed that downregulation of HIP-55 decreased the viability and increased the apoptosis of A549 cells treated with the anticancer agent etoposide. Our data suggested that HIP-55 may be a newly discovered regulatory node in the growth signaling network and a new target for therapeutic interventions in proliferative disorders.

14-3-3 adaptor proteins recruit AID to 5'-AGCT-3'-rich switch regions for class switch recombination.

Class switch DNA recombination (CSR) is the mechanism that diversifies the biological effector functions of antibodies. Activation-induced cytidine deaminase (AID), a key protein in CSR, targets immunoglobulin H (IgH) switch regions, which contain 5'-AGCT-3' repeats in their core. How AID is recruited to switch regions remains unclear. Here we show that 14-3-3 adaptor proteins have an important role in CSR. 14-3-3 proteins specifically bound 5'-AGCT-3' repeats, were upregulated in B cells undergoing CSR and were recruited with AID to the switch regions that are involved in CSR events (Smu-->Sgamma1, Smu-->Sgamma3 or Smu-->Salpha). Moreover, blocking 14-3-3 by difopein, 14-3-3gamma deficiency or expression of a dominant-negative 14-3-3sigma mutant impaired recruitment of AID to switch regions and decreased CSR. Finally, 14-3-3 proteins interacted directly with AID and enhanced AID-mediated in vitro DNA deamination, further emphasizing the important role of these adaptors in CSR.

14-3-3zeta mediates resistance of diffuse large B cell lymphoma to an anthracycline-based chemotherapeutic regimen.

Patients presenting with diffuse large B cell lymphoma (DLBCL) are treated with a standard anthracycline-based chemotherapeutic mixture consisting of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). Half of DLBCL patients will develop chemo-refractory tumors due to the emergence of CHOP-resistant DLBCL cells. We isolated DLBCL cells that were resistant to CHOP as a model system to investigate the molecular basis of CHOP resistance. Resistant cells emerged from CHOP-sensitive DLBCL populations after repeated cycles of on-off exposure to stepwise increased dosages of CHOP. A proteomic analysis of CHOP-sensitive and -resistant DLBCL cells identified the zeta isoform of the 14-3-3 family as a differentially expressed protein. CHOP-sensitive cells showed reduced expression of 14-3-3zeta protein in the presence of high-dose CHOP relative to control cells. In contrast, CHOP-resistant cells expressed markedly higher levels of 14-3-3zeta regardless the presence of high-dose CHOP. Because 14-3-3zeta is known to exert anti-apoptotic influences and chemoresistance in lung, colon, and prostate carcinoma, we hypothesized that 14-3-3zeta promotes survival of DLBCL cells in CHOP. In support of our hypothesis, knockdown of 14-3-3zeta by small interfering RNA restored the sensitivity of resistant DLBCL to CHOP-induce apoptosis. In addition, 14-3-3zeta expression was highly up-regulated in a resected DLBCL lymph node relative to a normal lymph node by Western blot analysis. Furthermore, more than half of 35 DLBCL tissues showed elevated 14-3-3zeta expression relative to normal lymph tissue by immunohistochemical analysis. Our study implicates 14-3-3zeta in the pathogenesis of DLBCL and suggests a promising combination strategy with a 14-3-3 inhibitor for the treatment of refractory DLBCL.

[Research of HOXD13 and FHL1 in idiopathic congenital talipes equinovarus].

To investigate the relationship of HOXD13 and FHL1 in idiopathic congenital talipes equinovarus(ICTEV), 84 samples from patients with ICTEV were used in the study. Mutation in the coding region of HOXD13 was detected by denaturing gradinent electrophoresis. The mRNA and protein levels of HOXD13 and FHL1 were evaluated by RT-PCR and immunohistochemistry, respectively. The binding site of FHL1 to HOXD13 predicted by PMATCH software was validated by EMSA( Electrophoretic mobility shift assay,EMSA).No mutation was found in the coding region of HOXD13 in 84 samples from patients with ICTEV. Both HOXD13(33.3%) and FHL1(46.6%) were down-regulated in ICTEV muscle tissue. The result of EMSA showed that the special binding band appeared when HOXD13 existed. The results shows that HOXD13 gene mutation was not involved in outbreak in idiopathic congenital talipes equinovarus, but changes of HOXD13 and FHL1 gene expression related to the development of talipes equinovarus malformation. HOXD13 might play an role in ICTEV through regulating FHL1 expression.

Down-regulation of 14-3-3zeta suppresses anchorage-independent growth of lung cancer cells through anoikis activation.

The family of 14-3-3 proteins has emerged as critical regulators of diverse cellular responses under both physiological and pathological conditions. Here, we report an important role of 14-3-3zeta in tumorigenesis through a mechanism that involves anoikis resistance. 14-3-3zeta is up-regulated in a number of cancer types, including lung cancer. Through an RNAi approach using human lung adenocarcinoma-derived A549 cells as a model system, we have found that knockdown of a single zeta isoform of 14-3-3 is sufficient to restore the sensitivity of cancer cells to anoikis and impair their anchorage-independent growth. Enhanced anoikis appears to be mediated in part by up-regulated BH3-only proteins, Bad and Bim, coupled with decreased Mcl-1, resulting in the subsequent activation of Bax. This study suggests a model in which anchorage-independent growth of lung cancer cells requires the presence of 14-3-3zeta. This work not only reveals a critical role of 14-3-3zeta in anoikis suppression in lung cancer cells, but also identifies and validates 14-3-3zeta as a potential molecular target for anticancer therapeutic development.

[Proteomic analysis of the ankle joint bone, ankle joint tissue and spinal cord of clubfoot-like deformity in rat fetuses].

OBJECTIVE: To explore the etiology of idiopathic talipes equinovarus (ITEV) in all-trans retinoic acid (ATRA) induced clubfoot-like deformity in rat fetuses with two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). METHODS: Clubfoot-like deformity model in rat fetuses was induced with ATRA (135 mg/kg) in gestation day (GD10) pregnant Wistar rats. 2-DE was applied to separate the total proteins of ankle joint tissue, ankle joint bone and spinal cord of the animal models. The Coomassie Brilliant Blue staining gels were analyzed by 2-DE software PDQuest 7.1.0. Selected differential protein spots were identified with peptide mass fingerprinting based on matrix-assisted laser adsorption/ionization time-of-flight mass spectrometry and database searching. xiap, tnnt1 and col2 alpha 1, three genes of the differential proteins, were identified furthermore. Apoptosis study was made in terminal deoxynucleotidyl transferase nick end labeling. RESULTS: There were many differential expressed proteins in the clubfoot-like deformity model. Out of the differentially expressed proteins,16 protein spots were identified to be differentially expressed in the clubfoot-like deformity model with MS. Three of the 16 protein spots, xiap, tnnt1 and col2 alpha 1 were confirmed to be significantly down-regulated by the RT-PCR, and Xiap was further confirmed to be significantly down-regulated with immunohistochemistry. Another randomly selected gene, ngfr, did not express differently in ATRA-induced clubfoot-like deformity in rat fetuses. The rates of the apoptosis in the spinal, bone of the clubfoot-like deformity fetuses was 5.4 and 10 times of those of the normal fetuses respectively. CONCLUSION: The results suggest that there are certain differently expressed proteins in ankle joint tissue, ankle joint bone and spinal cord of the ATRA-induced clubfoot-like deformity in rat fetuses, and Xiap, sTnT, and Col2 alpha 1 show a significant correlation with ITEV. Ngfr is not correlation with ITEV. Apoptosis plays a key role in the development of ITEV and related to the decreased expression of the Xiap.

[Association of homozygous deletion of p15 and p16 gene and amplification of EGFR gene in laryngeal squamous cell carcinoma].

To assess the relationship of deletion of p15 and p16 gene and EGFR gene amplification in laryngeal squamous cell carcinoma (LSCC). DNA was extracted from fresh tumor. Deletion of p15 exon 2(p15E2) and p16 exon 2(p16E2) in 30 cases of LSCC was detected by polymerase chain reaction (PCR) technique. Amplification of EGFR gene in 30 cases of LSCC was detected by FISH. The rate of p15E2 deletion in 30 cases was 13.3(4/30), and that of p16E2 was 16.7% (5/30). p15E2 and p16E2 codeletion rate was 6.7% (2/30). The rate of EGFR gene amplification in 30 cases was 30% (9/30), and was amplified 2 to 8 fold. Homozygous deletion of p16E2 and p15E2 and codeletion is related with amplification of EGFR gene (P = 0.000018), and may play an important role to oncogenesis and malignant progression in LSCC.

Exposure of human lung cancer cells to 8-chloro-adenosine induces G2/M arrest and mitotic catastrophe.

8-Chloro-adenosine (8-Cl-Ado) is a potent chemotherapeutic agent whose cytotoxicity in a variety of tumor cell lines has been widely investigated. However, the molecular mechanisms are uncertain. In this study, we found that exposure of human lung cancer cell lines A549 (p53-wt) and H1299 (p53-depleted) to 8-Cl-Ado induced cell arrest in the G2/M phase, which was accompanied by accumulation of binucleated and polymorphonucleated cells resulting from aberrant mitosis and failed cytokinesis. Western blotting showed the loss of phosphorylated forms of Cdc2 and Cdc25C that allowed progression into mitosis. Furthermore, the increase in Ser10-phosphorylated histone H3-positive cells revealed by fluorescence-activated cell sorting suggested that the agent-targeted cells were able to exit the G2 phase and enter the M phase. Immunocytochemistry showed that microtubule and microfilament arrays were changed in exposed cells, indicating that the dynamic instability of microtubules and microfilaments was lost, which may correlate with mitotic dividing failure. Aberrant mitosis resulted in mitotic catastrophe followed by varying degrees of apoptosis, depending on the cell lines. Thus, 8-Cl-Ado appears to exert its cytotoxicity toward cells in culture by inducing mitotic catastrophe.

[Expression and promoter methylation of Apaf-1 gene in laryngeal squamous cell carcinoma].

Loss of tumor suppressor genes and apoptosis-associated genes was common event in laryngeal squamous cell carcinoma (LSCC). Apaf-1 (apoptotic protease activating factor-1) is a key factor in cytochrome C-dependent apoptotic pathway. To investigate the effect of Apaf-1 in progression of LSCC, we analyzed Apaf-1 from DNA and RNA levels. Semi-quantitative RT-PCR analysis of Apaf-1 mRNA showed that over 40 percent of LSCCs (11/27) were low expression compared to the para-neoplastic laryngeal tissues (PNTs). The results of CGH indicated loss in 2 of 18 cases but no amplification on chromosome 12q22-23. The LOH frequencies of D12S327, D12S1657, D12S393, D12S1706, D12S346 on 12q22-23 were 18.2%, 13.6%, 18.2%, 22.2% and 16.6%, respectively in 72 matched samples of LSCCs and PNTs. Methylation-specific PCR displayed that all of 11 LSCCs, whose expression of Apaf-1 mRNA down-regulated, were methylated in promoter regions. In contrast, only 1 of 16 LSCCs with no changes in Apaf-1 mRNA levels was methylated (chi2 test, P=0.0001). The results implied that abnormal expression of Apaf-1 participates in the genesis of LSCCs and decreased expression of Apaf-1 mRNA were not mainly due to the deletion of Apaf-1 gene. The Apaf-1 DNA methylation in promoter region might contribute to the decreased transcription of Apaf-1 in LSCCs.