Multi-omics analysis uncovered systemic lupus erythematosus and COVID-19 crosstalk.

BACKGROUND: Studies have highlighted a possible crosstalk between the pathogeneses of COVID-19 and systemic lupus erythematosus (SLE); however, the interactive mechanisms remain unclear. We aimed to elucidate the impact of COVID-19 on SLE using clinical information and the underlying mechanisms of both diseases. METHODS: RNA-seq datasets were used to identify shared hub gene signatures between COVID-19 and SLE, while genome-wide association study datasets were used to delineate the interaction mechanisms of the key signaling pathways. Finally, single-cell RNA-seq datasets were used to determine the primary target cells expressing the shared hub genes and key signaling pathways. RESULTS: COVID-19 may affect patients with SLE through hematologic involvement and exacerbated inflammatory responses. We identified 14 shared hub genes between COVID-19 and SLE that were significantly associated with interferon (IFN)-I/II. We also screened and obtained four core transcription factors related to these hub genes, confirming the regulatory role of the IFN-I/II-mediated Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling pathway on these hub genes. Further, SLE and COVID-19 can interact via IFN-I/II and IFN-I/II receptors, promoting the levels of monokines, including interleukin (IL)-6/10, tumor necrosis factor-α, and IFN-γ, and elevating the incidence rate and risk of cytokine release syndrome. Therefore, in SLE and COVID-19, both hub genes and core TFs are enriched within monocytes/macrophages. CONCLUSIONS: The interaction between SLE and COVID-19 promotes the activation of the IFN-I/II-triggered JAK-STAT signaling pathway in monocytes/macrophages. These findings provide a new direction and rationale for diagnosing and treating patients with SLE-COVID-19 comorbidity.

RNA epigenetic modifications in digestive tract cancers: Friends or foes.

Digestive tract cancers are among the most common malignancies worldwide and have high incidence and mortality rates. Thus, the discovery of more effective diagnostic and therapeutic targets is urgently required. The development of technologies to accurately detect RNA modification has led to the identification of numerous RNA chemical modifications in humans (epitranscriptomics) that are involved in the occurrence and development of digestive tract cancers. RNA modifications can cooperatively regulate gene expression to facilitate normal physiological functions of the digestive system. However, the dysfunction of relevant RNA-modifying enzymes ("writers," "erasers," and "readers") can lead to the development of digestive tract cancers. Consequently, targeting dysregulated enzyme activity could represent a potent therapeutic strategy for the treatment of digestive tract cancers. In this review, we summarize the most widely studied roles and mechanisms of RNA modifications (m6A, m1A, m5C, m7G, A-to-I editing, pseudouridine [Ψ]) in relation to digestive tract cancers, highlight the crosstalk between RNA modifications, and discuss their roles in the interactions between the digestive system and microbiota during carcinogenesis. The clinical significance of novel therapeutic methods based on RNA-modifying enzymes is also discussed. This review will help guide future research into digestive tract cancers that are resistant to current therapeutics.

Preliminary clinical study of personalized neoantigen vaccine therapy for microsatellite stability (MSS)-advanced colorectal cancer.

Immunotherapy based on immune checkpoint inhibitors (ICIs) has provided revolutionary results in treating various cancers. However, its efficacy in colorectal cancer (CRC), especially in microsatellite stability-CRC, is limited. This study aimed to observe the efficacy of personalized neoantigen vaccine in treating MSS-CRC patients with recurrence or metastasis after surgery and chemotherapy. Candidate neoantigens were analyzed from whole-exome and RNA sequencing of tumor tissues. The safety and immune response were assessed through adverse events and ELISpot. The clinical response was evaluated by progression-free survival (PFS), imaging examination, clinical tumor marker detection, circulating tumor DNA (ctDNA) sequencing. Changes in health-related quality of life were measured by the FACT-C scale. A total of six MSS-CRC patients with recurrence or metastasis after surgery and chemotherapy were administered with personalized neoantigen vaccines. Neoantigen-specific immune response was observed in 66.67% of the vaccinated patients. Four patients remained progression-free up to the completion of clinical trial. They also had a significantly longer progression-free survival time than the other two patients without neoantigen-specific immune response (19 vs. 11 months). Changes in health-related quality of life improved for almost all patients after the vaccine treatment. Our results shown that personalized neoantigen vaccine therapy is likely to be a safe, feasible and effective strategy for MSS-CRC patients with postoperative recurrence or metastasis.

DEMoS: a deep learning-based ensemble approach for predicting the molecular subtypes of gastric adenocarcinomas from histopathological images.

MOTIVATION: The molecular subtyping of gastric cancer (adenocarcinoma) into four main subtypes based on integrated multiomics profiles, as proposed by The Cancer Genome Atlas (TCGA) initiative, represents an effective strategy for patient stratification. However, this approach requires the use of multiple technological platforms, and is quite expensive and time-consuming to perform. A computational approach that uses histopathological image data to infer molecular subtypes could be a practical, cost- and time-efficient complementary tool for prognostic and clinical management purposes. RESULTS: Here, we propose a deep learning ensemble approach (called DEMoS) capable of predicting the four recognized molecular subtypes of gastric cancer directly from histopathological images. DEMoS achieved tile-level area under the receiver-operating characteristic curve (AUROC) values of 0.785, 0.668, 0.762 and 0.811 for the prediction of these four subtypes of gastric cancer [i.e. (i) Epstein-Barr (EBV)-infected, (ii) microsatellite instability (MSI), (iii) genomically stable (GS) and (iv) chromosomally unstable tumors (CIN)] using an independent test dataset, respectively. At the patient-level, it achieved AUROC values of 0.897, 0.764, 0.890 and 0.898, respectively. Thus, these four subtypes are well-predicted by DEMoS. Benchmarking experiments further suggest that DEMoS is able to achieve an improved classification performance for image-based subtyping and prevent model overfitting. This study highlights the feasibility of using a deep learning ensemble-based method to rapidly and reliably subtype gastric cancer (adenocarcinoma) solely using features from histopathological images. AVAILABILITY AND IMPLEMENTATION: All whole slide images used in this study was collected from the TCGA database. This study builds upon our previously published HEAL framework, with related documentation and tutorials available at http://heal.erc.monash.edu.au. The source code and related models are freely accessible at https://github.com/Docurdt/DEMoS.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mouse papillomavirus type 1 (MmuPV1) DNA is frequently integrated in benign tumors by microhomology-mediated end-joining.

MmuPV1 is a useful model for studying papillomavirus-induced tumorigenesis. We used RNA-seq to look for chimeric RNAs that map to both MmuPV1 and host genomes. In tumor tissues, a higher proportion of total viral reads were virus-host chimeric junction reads (CJRs) (1.9‰ - 7‰) than in tumor-free tissues (0.6‰ - 1.3‰): most CJRs mapped to the viral E2/E4 region. Although most of the MmuPV1 integration sites were mapped to intergenic regions and introns throughout the mouse genome, integrations were seen more than once in several genes: Malat1, Krt1, Krt10, Fabp5, Pard3, and Grip1; these data were confirmed by rapid amplification of cDNA ends (RACE)-Single Molecule Real-Time (SMRT)-seq or targeted DNA-seq. Microhomology sequences were frequently seen at host-virus DNA junctions. MmuPV1 infection and integration affected the expression of host genes. We found that factors for DNA double-stranded break repair and microhomology-mediated end-joining (MMEJ), such as H2ax, Fen1, DNA polymerase Polθ, Cdk1, and Plk1, exhibited a step-wise increase and Mdc1 a decrease in expression in MmuPV1-infected tissues and MmuPV1 tumors relative to normal tissues. Increased expression of mitotic kinases CDK1 and PLK1 appears to be correlated with CtIP phosphorylation in MmuPV1 tumors, suggesting a role for MMEJ-mediated DNA joining in the MmuPV1 integration events that are associated with MmuPV1-induced progression of tumors.

Scalp hair loss is not random across follicular units: A new insight into human hair ageing.

OBJECTIVES: Scalp hair has the greatest number of hairs (typically 1-5) per follicular unit but is also the most susceptible body site to hair loss with age. Hence, we set-out to determine the degree to which scalp hair parameters change with age in women and men, any sex differences thereof and whether hair loss is random across follicular units. METHODS: A retrospective cross-sectional study of 200 Chinese men and 200 Chinese women (30-69 years). Image analysis and manual counting methods were used to measure occipital located hair parameters from 6 × 8 mm shaved scalp photographs and plucked hair microscopy images. RESULTS: Of the five hair parameters, the number of hairs per follicular unit had the greatest (negative) correlation with age in both men and women. Men had a greater number of hairs and follicular units than women on average but had a greater decrease in the number of hairs per follicular unit with age, particularly for the loss of multi-hair (3+) follicular units. The loss of hairs with age was significantly different to that expected by a random loss of hairs across follicular units and better described by a model of increased hair loss risk the greater number of hairs per follicular unit. CONCLUSIONS: We have found evidence of hair loss preferentially occurring in multi-hair follicular units, which was more pronounced in men. These data suggest that part of the reason scalp hair is more susceptible to hair loss than on other body sites is due to the greater presence of multi-hair follicular units on the scalp.

OBJECTIFS: Le cuir chevelu possède le plus grand nombre de cheveux (généralement de 1 à 5) par unité folliculaire, mais c'est aussi le site le plus sensible à la perte de cheveux avec l'âge. Nous avons donc entrepris de déterminer dans quelle mesure les paramètres des cheveux du cuir chevelu changent avec l'âge chez les femmes et les hommes, quelles sont les différences entre les sexes et si la perte de cheveux est aléatoire entre les unités folliculaires. MÉTHODES: Étude transversale rétrospective portant sur 200 hommes et 200 femmes chinois (30-69 ans). Des méthodes d'analyse d'image et de comptage manuel ont été utilisées pour mesurer les paramètres des cheveux situés dans la région occipitale à partir de photographies du cuir chevelu rasé de 6x8 mm et d'images microscopiques de cheveux arrachés. RÉSULTATS: Parmi les 5 paramètres capillaires, le nombre de cheveux par unité folliculaire présentait la corrélation la plus forte (négative) avec l'âge, tant chez les hommes que chez les femmes. Les hommes avaient en moyenne un plus grand nombre de cheveux et d'unités folliculaires que les femmes, mais le nombre de cheveux par unité folliculaire diminuait davantage avec l'âge, en particulier pour la perte d'unités folliculaires à plusieurs cheveux (3+). La perte de cheveux avec l'âge était significativement différente de celle attendue par une perte aléatoire de cheveux dans les unités folliculaires, et mieux décrite par un modèle d'augmentation du risque de perte de cheveux plus le nombre de cheveux par unité folliculaire est élevé. CONCLUSIONS: Nous avons trouvé des preuves que la perte de cheveux se produit préférentiellement dans les unités folliculaires à plusieurs cheveux, ce qui était plus prononcé chez les hommes. Ces données suggèrent qu'une partie de la raison pour laquelle les cheveux du cuir chevelu sont plus sensibles à la perte de cheveux que sur d'autres sites du corps est due à la plus grande présence d'unités folliculaires à cheveux multiples sur le cuir chevelu.

Sustained expression of HPV16 E7 oncoprotein promotes p-AKT(Ser473)/p-Src(Tyr527) signaling to drive precancerous lesions to invasive cervical cancer.

Human papillomavirus (HPV) E7 oncogene plays the most important role in cervical cancer. However, whether E7 oncoprotein is continuously expressed, associated with AKT(Ser473)/p-Src(Tyr527) signaling to trigger cervical carcinogenesis remains unclear. Here, we explored first if HPV16 E7 oncoprotein could be detected in clinical biopsies and is sustainedly expressed, and then investigated how this oncoprotein interacted with AKT(Ser473)/p-Src(Tyr527) signaling in cancer progression. We used ZHPV16E7384 affibody to detect E7 expression in HPV16-positive cervical cancer biopsies and animal tumors by immunohistochemistry (IHC). Results showed that ZHPV16E7384 affibody had intense and specific staining for E7 oncoprotein in the detected specimen. The E7 oncoprotein was continuously expressed to correspond with the development of precancerous lesions to invasive cervical cancer. IHC staining also revealed that AKT, p-AKT(Ser473), Src and p-Src(Tyr527) proteins were expressed in both patient biopsies and animal tumors, with the highest levels of p-AKT(Ser473)/p-Src(Tyr527) present in invasive cancer. Furthermore, siRNA experiments revealed that HPV16 E7 knockdown significantly impaired expression of p-AKT(Ser473)/p-Src(Tyr527) in both HPV16 E7-positive cancer cells and transformed cells. In addition, transient expression of HPV16 E7 protein promoted significantly expression of p-AKT(Ser473)/p-Src(Tyr527) in primary human keratinocytes. Finally, co-immunoprecipitation analysis proved that HPV 16 E7 protein interacted reciprocally with p-AKT(Ser473)/p-Src(Tyr527). In conclusion, we demonstrate that HPV16 E7 oncoprotein is continuously expressed to promote expression of p-AKT(Ser473)/p-Src(Tyr527) leading to drive the initiation and progression of cervical cancer. Our data provide a novel insight that HPV16 E7 activates p-AKT(Ser473)/p-Src(Tyr527) to establish a mechanistic link between the oncogene and the AKT/Src signaling to trigger cervical carcinogenesis.

Novel EBV LMP-2-affibody and affitoxin in molecular imaging and targeted therapy of nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) infection is closely linked to several human malignancies including endemic Burkitt's lymphoma, Hodgkin's lymphoma and nasopharyngeal carcinomas (NPC). Latent membrane protein 2 (LMP-2) of EBV plays a pivotal role in pathogenesis of EBV-related tumors and thus, is a potential target for diagnosis and targeted therapy of EBV LMP-2+ malignant cancers. Affibody molecules are developing as imaging probes and tumor-targeted delivery of small molecules. In this study, four EBV LMP-2-binding affibodies (ZEBV LMP-212, ZEBV LMP-2132, ZEBV LMP-2137, and ZEBV LMP-2142) were identified by screening a phage-displayed LMP-2 peptide library for molecular imaging and targeted therapy in EBV xenograft mice model. ZEBV LMP-2 affibody has high binding affinity for EBV LMP-2 and accumulates in mouse tumor derived from EBV LMP-2+ xenografts for 24 h after intravenous (IV) injection. Subsequent fusion of Pseudomonas exotoxin PE38KDEL to the ZEBV LMP-2 142 affibody led to production of Z142X affitoxin. This fused Z142X affitoxin exhibits high cytotoxicity specific for EBV+ cells in vitro and significant antitumor effect in mice bearing EBV+ tumor xenografts by IV injection. The data provide the proof of principle that EBV LMP-2-speicifc affibody molecules are useful for molecular imaging diagnosis and have potentials for targeted therapy of LMP-2-expressing EBV malignancies.

Periphery-aware COVID-19 diagnosis with contrastive representation enhancement.

Computer-aided diagnosis has been extensively investigated for more rapid and accurate screening during the outbreak of COVID-19 epidemic. However, the challenge remains to distinguish COVID-19 in the complex scenario of multi-type pneumonia classification and improve the overall diagnostic performance. In this paper, we propose a novel periphery-aware COVID-19 diagnosis approach with contrastive representation enhancement to identify COVID-19 from influenza-A (H1N1) viral pneumonia, community acquired pneumonia (CAP), and healthy subjects using chest CT images. Our key contributions include: 1) an unsupervised Periphery-aware Spatial Prediction (PSP) task which is designed to introduce important spatial patterns into deep networks; 2) an adaptive Contrastive Representation Enhancement (CRE) mechanism which can effectively capture the intra-class similarity and inter-class difference of various types of pneumonia. We integrate PSP and CRE to obtain the representations which are highly discriminative in COVID-19 screening. We evaluate our approach comprehensively on our constructed large-scale dataset and two public datasets. Extensive experiments on both volume-level and slice-level CT images demonstrate the effectiveness of our proposed approach with PSP and CRE for COVID-19 diagnosis.

CD2AP inhibits metastasis in gastric cancer by promoting cellular adhesion and cytoskeleton assembly.

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that the alter of the cellular cytoskeleton and intercellular adhesion. CD2-associated protein (CD2AP) is one of the critical proteins regulating cytoskeleton assembly and intercellular adhesion. However, no study has investigated the expression and biological significance of CD2AP in gastric cancer (GC) to date. Therefore, the aim of our study was to explore if the expression of CD2AP is associated with any clinical features of GC and to elucidate the underlying mechanism. Immunohistochemistry of 620 patient tissue samples indicated that the expression of CD2AP is downregulated in DGC. Moreover, a low CD2AP level was indicative of poor patient prognosis. In vitro, forced expression of CD2AP caused a significant decrease in the migration and invasion of GC cells, whereas depletion of CD2AP had the opposite effect. Immunofluorescence analysis indicated that CD2AP promoted cellular adhesion and influenced cell cytoskeleton assembly via interaction with the F-actin capping protein CAPZA1. Overall, the upregulation of CD2AP could attenuate GC metastasis, suggesting CD2AP as a novel biomarker for the prognosis and treatment of patients with GC.

HPV16 E7-impaired keratinocyte differentiation leads to tumorigenesis via cell cycle/pRb/involucrin/spectrin/adducin cascade.

Here, we used codon usage technology to generate two codon-modified human papillomavirus (HPV)16 E7 genes and, together with wild-type E7, to construct three HPV16 E7 gene plasmids: Wt-E7, HB1-E7, and HB2-E7. The three HPV 16 E7 plasmids were used to investigate how HPV16 E7 protein was expressed in different cells and how this oncoprotein deregulated cellular and molecular events in human keratinocytes to induce carcinogenesis. We discovered that codon usage of HPV16 E7 gene played a key role in determining expression of E7 oncoprotein in all tested cells. HPV16 E7 inhibited significantly expression of pRb to impair keratinocyte differentiation and disrupted development of skin epidermis in mice. HPV16 E7 increased substantially the number of G0/G1 cells associated with upregulation of cyclin D2 and downregulation of cyclin B1 in keratinocytes. HPV16 E7 not only inhibited expression of involucrin and α-spectrin but also disrupted the organization of involucrin filaments and spectrin cytoskeleton. Furthermore, HPV16 E7 inhibited expression of ß-adducin, destroyed its cytoskeletal structure and induced phosphorylation of ß-adducin(Ser662) in keratinocytes. Importantly, HPV16 E7 induced carcinogenesis in mice associated with expression of phosphorylated ß-adducin(Ser662) and its nucleus-translocation. In conclusion, we provided evidence that HPV16 E7 oncoprotein inhibited keratinocyte differentiation in vitro and in vivo leading to carcinogenesis through cell cycle arrest and disruption of pRb/involucrin/spectrin/adducin cascade.

The full transcription map of mouse papillomavirus type 1 (MmuPV1) in mouse wart tissues.

Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5' RACE, 3' RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P7503, P360 and P859 as "early" promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P7107 and P533 as "late" promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P7107) or nt 757 to 3139 (P533) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P533-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology.

Use of nested PCR for the detection of trichomonads in bronchoalveolar lavage fluid.

BACKGROUND: The methods routinely used to detect trichomonads in the lungs are not sensitive enough, and an effective method is urgently needed. METHOD: Primers were first designed to match the conserved area of the 18S rRNA gene of trichomonads. Then, nested PCR was carried out to detect trichomonads in bronchoalveolar lavage fluid (BALF). Finally, all positive specimens were subjected to DNA sequencing and phylogenetic analysis. RESULTS: Among 115 bronchoalveolar lavage fluid samples, ten samples tested positive in nested PCR (10/115), while no samples were positive in wet mount microscopy (0/115) (P < 0.01). Among the ten positive specimens, two were identified as Tetratrichomonas spp. and the other eight as Trichomonas tenax in phylogenetic analysis. CONCLUSIONS: Nested PCR is an effective way to detect trichomonads in bronchoalveolar lavage fluid.

NovelmiRNA-25 inhibits AMPD2 in peripheral blood mononuclear cells of patients with systemic lupus erythematosus and represents a promising novel biomarker.

BACKGROUND: Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease with various clinical manifestations. MicroRNAs (miRNAs) and immunometabolism are recognized as key elements in SLE pathogenesis; however, the relationship between miRNAs in peripheral blood mononuclear cells (PBMCs) and metabolism in SLE remains unclear. METHODS: We detected PBMC miRNA and mRNA profiles from 3 pooled SLE patients and 3 healthy controls (HCs) using next-generation sequencing, predicted miRNA targets in dysregulated mRNAs, predicted functions and interactions of differentially expressed genes using bioinformatics analysis, validated candidate miRNAs using qRT-PCR, and investigated the association between the expression of candidate miRNAs and SLE clinical characteristics. Moreover, we validated the direct and transcriptional regulatory effect of NovelmiRNA-25 on adenosine monophosphate deaminase 2 (AMPD2) using a dual-luciferase reporter assay and western blot and confirmed AMPD2 mRNA and protein expression in PBMCs using qRT-PCR and western blot, respectively. RESULTS: Multilayer integrative analysis of microRNA and mRNA regulation showed that 10 miRNAs were down-regulated and 19 miRNAs were up-regulated in SLE patient PBMCs compared with HCs. Bioinformatics analysis of regulatory networks between miRNAs and mRNAs showed that 19 miRNAs were related to metabolic processes. Two candidate miRNAs, NovelmiRNA-25 and miR-1273h-5p, which were significantly increased in the PBMCs of SLE patients (P < 0.05), represented diagnostic biomarkers with sensitivities of 94.74% and 89.47%, respectively (area under the curve = 0.574 and 0.788, respectively). NovelmiRNA-25 expression in PBMCs was associated with disease activity in SLE patients, in both active and stable groups (P < 0.05). NovelmiRNA-25 overexpression downregulated AMPD2 expression in HEK293T cells through direct targeting of the AMPD2 3'UTR (P < 0.01), while inhibition of NovelmiRNA-25 activity led to increased AMPD2 expression (P < 0.01). NovelmiRNA-25 overexpression also downregulated AMPD2 protein expression in HEK293T cells; AMPD2 protein expression in SLE patient PBMCs was decreased. Our results show that differentially expressed miRNAs play an important role in SLE. CONCLUSIONS: Our data demonstrate a novel mechanism in SLE development that involves the targeting of AMPD2 expression by NovelmiRNA-25. miRNAs may serve as novel biomarkers for the diagnosis and evaluation of disease activity of SLE and represent potential therapeutic targets for this disease.

Bispecific affibody molecule targeting HPV16 and HPV18E7 oncoproteins for enhanced molecular imaging of cervical cancer.

High-risk human papillomavirus (HPV16 and HPV18) are now widely recognized as responsible for cervical cancer, which remains to be the most common gynecologic malignancy in women worldwide. It is well known that viral oncoproteins E6/E7 play key roles in HPV-associated cervical carcinogenesis. Thus, in vivo detection of the two oncoproteins may provide important diagnostic information influencing patient management. More recently, affibody molecules have been demonstrated to be a promising candidate for development as molecular imaging probes. Based on the two monomeric affibody molecules (ZHPV16E7 and ZHPV18E7) generated in our laboratory, here, we used a peptide linker (Gly4Ser)3 to link ZHPV16E7 and ZHPV18E7 to develop a novel heterodimeric affibody ZHPV16E7-(Gly4Ser)3-ZHPV18E7. Both biosensor and immunofluorescence assays have proved that the heterodimeric affibody molecule targeted simultaneously HPV16 and HPV18E7 proteins by binding to the viral oncoproteins. In vivo tumor-imaging experiments using the Dylight755-labeled heterodimeric affibody revealed that strongly high-contrast tumor retention of the heterodimers occurred in both HPV16- and HPV18-derived tumors of nude mice 0.5 h post-injection. The accumulation of Dylight755-labeled heterodimers in tumors was achieved over 48 h. Therefore, we believe that this novel heterodimeric affibody molecule has great potential utility in molecular imaging in vivo and diagnosis of HPV-associated cervical cancers.

Establishment and Application of a Method for High-Risk Human Papillomavirus Genotyping in Cervical Cancer Tissue.

BACKGROUND: Persistent high-risk HPV infection is a major cause of cervical cancer and E6/E7 genes and the Li gene in the HPV genome are key targets to detect high-risk HPV. This study aims to explore the relationship between cervical lesions and E6/7 by establishing a polymerase chain reaction (PCR) to detect multiplex genes based on HPV EE7 genes. It is hoped that such methods will provide a more reliable method for clinical screening and the prevention of cervical cancer. METHODS: Based on alignment, specific primers were designed for HPV E6/E7 genes, the sequences of which came from five5 high-risk papillomaviruses that are common in China. This enabled an E6/E7 gene detection method based on multiplex PCR to be established. E6/E7 and Li gene testing were then performed on 65 cervical cancer tissue samples. The gene copy number of HPV E6/E7 genes and the Li gene were detected from different classifications by real-time fluorescence quantitative PCR. RESULTS: Out of the 65 cervical cancer tissue samples, 47 (72.31%) showed positive results in E6/E7 multiplex PCR, 21 (32.31%) showed positive results in the Ll gene PCR, and out of the 219 cervical exfoliate cell samples, 56 (25.57%) showed positive results in E6/E7 multiplex PCR, 21 (13.24%) showed positive results in the L1 gene PCR. There were significant differences (p < 0.05) between these two test results. Fluorescent quantitative PCR showed that the ratio of gene copy number of L1 genes and E6/E7 genes was below 1 (p < 0.05) in cervical cancer tissue, in which both the Li and E6/E7 genes coexist. CONCLUSIONS: The established HPV multiplex PCR assay based on the design of E6/E7 gene is a specific and sensitive method for the detection and genotype of five high-risk HPVs.

Lipoxin A4 activates alveolar epithelial sodium channel gamma via the microRNA-21/PTEN/AKT pathway in lipopolysaccharide-induced inflammatory lung injury.

Lipoxin A4 (LXA4), as an endogenously produced lipid mediator, promotes the resolution of inflammation. Previously, we demonstrated that LXA4 stimulated alveolar fluid clearance through alveolar epithelial sodium channel gamma (ENaC-γ). In this study, we sought to investigate the mechanisms of LXA4 in modulation of ENaC-γ in lipopolysaccharide (LPS)-induced inflammatory lung injury. miR-21 was upregulated during an LPS challenge and downregulated by LXA4 administration in vivo and in vitro. Serum miR-21 concentration was also elevated in acute respiratory distress syndrome patients as compared with healthy volunteers. LPS increased miR-21 expression by activation of activator protein 1 (AP-1). In A549 cells, miR-21 upregulated phosphorylation of AKT activation via inhibition of phosphatase and tensin homolog (PTEN), and therefore reduced the expression of ENaC-γ. In contrast, LXA4 reversed LPS-inhibited ENaC-γ expression through inhibition of AP-1 and activation of PTEN. In addition, an miR-21 inhibitor mimicked the effects of LXA4; overexpression of miR-21 abolished the protective effects of LXA4. Finally, both AKT and ERK inhibitors (LY294002 and UO126) blocked effects of LPS on the depression of ENaC-γ. However, LXA4 only inhibited LPS-induced phosphorylation of AKT. In summary, LXA4 activates ENaC-γ in part via the miR-21/PTEN/AKT signaling pathway.

Identification of linear B-cell epitopes within Tarp of Chlamydia trachomatis.

Chlamydia trachomatis is one of the most prevalent sexually transmitted pathogens. There is currently no commercially available vaccine against C. trachomatis. Chlamydial translocated actin-recruiting phosphoprotein (Tarp) can induce cellular and humoral immune responses in murine models and has been regarded as a potential vaccine candidate. In this report, the amino acid sequence of Tarp was analyzed using computer-assisted techniques to scan B-cell epitopes, and six possible linear B-cell epitopes peptides (aa80-95, aa107-123, aa152-170, aa171-186, aa239-253 and aa497-513) with high predicted antigenicity and high conservation were investigated. Sera from mice immunized with these potential immunodominant peptides was analyzed by ELISA, which showed that epitope 152-170 elicited serum immunoglobulin G (IgG) response and epitope 171-186 elicited both serum IgG and mucosal secretory immunoglobulin A response. The response of immune sera of epitope 171-186 to endogenous Tarp antigen obtained from the Hela229 cells infected with C. trachomatis was confirmed by Western blot and indirect fluorescence assay. In addition, binding of the antibodies against epitope 171-186 to endogenous Tarp was further confirmed by competitive ELISA. Our results demonstrated that the putative epitope (aa171-186) was an immunodominant B-cell epitope of Tarp. If proven protective and safe, this epitope, in combination with other well-documented epitopes, might be included into a candidate epitope-based vaccine against C. trachomatis.

Hepatitis B virus surface antigen as delivery vector can enhance Chlamydia trachomatis MOMP multi-epitope immune response in mice.

Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. There is currently no commercially available vaccine against C. trachomatis. Major outer membrane protein (MOMP) of C. trachomatis is considered to be an ideal candidate for prophylactic vaccine. We designed a MOMP multi-epitope containing T- and B-cell epitope-rich peptides and developed hepatitis B surface antigen (HBsAg) as antigen delivery vehicle. In order to study the immunogenicity and efficacy of the candidate vaccine in a murine model of chlamydial genital infection, we engineered a recombinant plasmid expressing HBsAg and MOMP multi-epitope genes. Results of reverse transcription polymerase chain reaction and immunofluorescence assay revealed successful expression of the recombinant HBsAg/MOMP multi-epitope gene at both the transcription and translation levels. Intramuscular administration in mice was able to elicit not only antibodies against Chlamydia and HBsAg but also cytotoxic T lymphocyte activity against Chlamydia. In addition, mice inoculated with the rHBsAg were highly resistant to C. trachomatis genital infection. The rHBsAg DNA with MOMP multi-epitope appended at the C terminus of the HBsAg stimulated a stronger immune response and protective response than that appended at the N terminus. Together, our results suggested that use of a recombinant HBsAg encoding the MOMP multi-epitope could be a powerful approach to developing a safe and immunogenic C. trachomatis vaccine.

A multi-epitope vaccine based on Chlamydia trachomatis major outer membrane protein induces specific immunity in mice.

We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein (MOMP) multi-epitope of Chlamydia trachomatis. A short gene of multi-epitope derived from MOMP containing multiple T- and B-cell epitopes was artificially synthesized. The recombinant plasmid pET32a(+) containing codon optimized MOMP multi-epitope gene was constructed. Expression of the fusion protein Trx-His-MOMP multi-epitope in Escherichia coli was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Balb/c mice were inoculated with the purified fusion protein subcutaneously three times with 2-week intervals. Results showed that the MOMP multi-epitope elicited not only strong humoral immune responses to C. trachomatis by generating significantly high levels of specific antibodies (IgG1 and IgG2a), but also a cellular immune response by inducing robust cytotoxic T lymphocyte responses in mice. Furthermore, the MOMP multi-epitope substantially primed secretion of IFN-γ, revealing that this vaccine could induce a strong Th1 response. Finally, the mice vaccinated with the MOMP multi-epitope displayed a reduction of C. trachomatis shedding upon a chlamydial challenge and an accelerated clearance of the infected C. trachomatis. In conclusion, the MOMP multi-epitope vaccine may have the potentiality for the development of effective prophylactic and therapeutic vaccines against the C. trachomatis infection.