A lightweight hybrid vision transformer network for radar-based human activity recognition.

Radar-based human activity recognition (HAR) offers a non-contact technique with privacy protection and lighting robustness for many advanced applications. Complex deep neural networks demonstrate significant performance advantages when classifying the radar micro-Doppler signals that have unique correspondences with human behavior. However, in embedded applications, the demand for lightweight and low latency poses challenges to the radar-based HAR network construction. In this paper, an efficient network based on a lightweight hybrid Vision Transformer (LH-ViT) is proposed to address the HAR accuracy and network lightweight simultaneously. This network combines the efficient convolution operations with the strength of the self-attention mechanism in ViT. Feature Pyramid architecture is applied for the multi-scale feature extraction for the micro-Doppler map. Feature enhancement is executed by the stacked Radar-ViT subsequently, in which the fold and unfold operations are added to lower the computational load of the attention mechanism. The convolution operator in the LH-ViT is replaced by the RES-SE block, an efficient structure that combines the residual learning framework with the Squeeze-and-Excitation network. Experiments based on two human activity datasets indicate our method's advantages in terms of expressiveness and computing efficiency over traditional methods.

Dynamic contrast-enhanced magnetic resonance imaging-based radiomics for the prediction of progression-free survival in advanced nasopharyngeal carcinoma.

To establish a multidimensional nomogram model for predicting progression-free survival (PFS) and risk stratification in patients with advanced nasopharyngeal carcinoma (NPC). This retrospective cross-sectional study included 156 patients with advanced NPC who underwent dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Radiomic features were extracted from the efflux rate constant (Ktrans ) and extracellular extravascular volume (Ve ) mapping derived from DCE-MRI. Least absolute shrinkage and selection operator (LASSO) Cox regression analysis was applied for feature selection. The Radscore was constructed using the selected features with their respective weights in the LASSO Cox regression analysis. A nomogram model combining the Radscore and clinical factors was built using multivariate Cox regression analysis. The C-index was used to assess the discrimination power of the Radscore and nomogram. The Kaplan-Meier method was used for survival analysis. Of the 360 radiomic features, 28 were selected (7, 6, and 15 features extracted from Ktrans , Ve, and Ktrans +Ve images, respectively). The combined Radscore k trans +Ve (C-index, 0.703, 95% confidence interval [CI]: 0.571-0.836) showed higher efficacy in predicting the prognosis of advanced NPC than Radscore k trans (C-index, 0.693; 95% CI, 0.560-0.826) and Radscore Ve (C-index, 0.614; 95% CI, 0.481-0.746) did. Multivariable Cox regression analysis revealed clinical stage, T stage, and treatment with nimotuzumab as risk factors for PFS. The nomogram established by Radscore k trans +Ve and risk factors (C-index, 0.732; 95% CI: 0.599-0.864) was better than Radscore k trans +Ve in predicting PFS in patients with advanced NPC. A lower Radscore k trans +Ve (HR 3.5584, 95% CI 2.1341-5.933), lower clinical stage (hazard ratio [HR] 1.5982, 95% CI 0.5262-4.854), lower T stage (HR 1.4365, 95% CI 0.6745-3.060), and nimotuzumab (NTZ) treatment (HR 0.7879, 95% CI 0.4899-1.267) were associated with longer PFS. Kaplan-Meier analysis showed a lower PFS in the high-risk group than in the low-risk group (p<0.0001). The nomogram based on combined pretreatment DCE-MRI radiomics features, NTZ, and clinicopathological risk factors may be considered as a noninvasive imaging marker for predicting individual PFS in patients with advanced NPC.

A Nanoparticle-Conjugated Anti-TBK1 siRNA Induces Autophagy-Related Apoptosis and Enhances cGAS-STING Pathway in GBM Cells.

BACKGROUND: Gene therapy shows considerable clinical benefit in cancer therapy, in which single-stranded ribonucleic acid (siRNA) is a promising strategy in the treatment of glioblastoma (GBM). TANK-binding kinase 1 (TBK1) is critical in tumorigenesis and development, which lays a foundation for an ideal target for tumor therapy. However, the practical application of free siRNA is limited. It is urgent to develop novel strategies to deliver TBK1 siRNA to activate apoptosis and cGAS-STING pathway as a therapeutic strategy for GBM. METHODS: The expression and prognostic value of TBK1 were evaluated in the TCGA, CGGA, and GTEx databases. A novel gene delivery system was designed here via PEGylated reduced graphene oxide (rGO-PEG) to targeted delivery of anti-TBK1 siRNA efficiently. The efficacy of TBK1si/rGO-PEG was evaluated in GBM cells. The underlying pathways were explored by Western blot. RESULTS: TBK1 was highly expressed in glioma samples, and its high expression indicated poor prognoses in glioma patients. The rGO-PEG presented great efficiency in targeted delivery of TBK1si RNA into GBM cells with up to 97.1% transfection efficiency. TBK1si/rGO-PEG exhibited anti-GBM activities by inhibiting TBK1 and autophagy, as well as activating apoptosis and cGAS-STING pathway. CONCLUSION: The rGO-PEG could be an efficient system facilitating the delivery of specific siRNA. TBK1si/rGO-PEG could be a novel strategy for the treatment of GBM.

A Novel Mice Model for Studying the Efficacy and IRAEs of Anti-CTLA4 Targeted Immunotherapy.

BACKGROUND: Patient-derived orthotopic xenograft (PDOX) is a popular animal model for translational cancer research. Immunotherapy is a promising therapy against glioblastoma (GBM). However, the PDOX model is limited to evaluating immune-related events. Our study aims to establish GBM humanized PDOX (HPDOX) mice models to study the mechanism of anti-CTLA4 immunotherapy and immune-related adverse events (IRAEs). METHODS: HPDOX models were established by culturing GBM tissues and intracranially implanting them in NSG mice. Meanwhile, peripheral blood mononuclear cells (PBMCs) were separated from peripheral blood and of GBM patients and administrated in corresponding mice. The population of CD45+, CD3+, CD4+, CD8+, and regulatory T (Treg) cells was estimated in the peripheral blood or tumor. RESULTS: T cells derived from GBM patients were detected in HPDOX mice models. The application of anti-CTLA4 antibodies (ipilimumab and tremelimumab) significantly inhibited the growth of GBM xenografts in mice. Moreover, residual patient T cells were detected in the tumor microenvironment and peripheral blood of HPDOX mice and were significantly elevated by ipilimumab and tremelimumab. Additionally, Treg cells were decreased in mice with IRAEs. Lastly, the proportion of CD4+/CD8+ T cells dramatically increased after the administration of ipilimumab. And the degree of IRAEs may be related to CD56+ expression in HPDOX. CONCLUSIONS: Our study established HPDOX mice models for investigating the mechanism and IRAEs of immunotherapies in GBM, which would offer a promising platform for evaluating the efficacy and IRAEs of novel therapies and exploring personalized therapeutic strategies.

Immune-related genes with APA in microenvironment indicate risk stratification and clinical prognosis in grade II/III gliomas.

Tumor microenvironment and alternative polyadenylation (APA) have drawn more attention in cancer research. However, their roles in grade II and III gliomas, termed as lower-grade glioma (LGG) in this study, remain to be fully elucidated. Here, we conducted this study and found that stromal and immune scores were elevated in higher grade and isocitrate dehydrogenase (IDH) wild-type glioma. Besides, higher stromal and immune scores indicated a poor prognosis in patients with LGG. APA events in immune-related genes were associated with overall survival, RNA expression, IDH mutation, and disease-free survival. Patients in the high-risk group had poor prognoses, and the risk score could be used to predict the survival rate. The risk score was positively correlated with the expression of immune checkpoints, inflammatory cytokines, and infiltrated immune cells. Moreover, risk stratification could predict the efficacy of radiotherapy and provide a reference for the treatment of grade III glioma. Our study revealed that immune-related genes with APA events in the microenvironment could predict risk stratification and clinical prognosis in patients with LGG.

Expression of m6A Regulators Correlated With Immune Microenvironment Predicts Therapeutic Efficacy and Prognosis in Gliomas.

BACKGROUND: N6-methyladenosine (m6A) RNA methylation and tumor immune microenvironment played crucial roles in cancer development. However, their association in gliomas remains to be fully elucidated. METHODS: A total of 2144 glioma patients from CGGA, TCGA, and Rembrandt databases were extracted in our study, in which 325 were set as the training cohort and 1819 were defined as the validation cohort. Survival differences evaluated by Kaplan-Meier analysis between groups. Patients were clustered into subgroups by consensus clustering. ESTIMATE algorithm was applied to calculate immune and stroma scores. The infiltration of immune cells was characterized by TIMER algorithm. The risk signature was constructed by multivariate Cox regression analysis. RESULTS: Nineteen m6A regulators were highly expressed in glioma tissues. The expression of m6A regulators was associated with prognoses, grade, isocitrate dehydrogenase (IDH) status, and 1p19q status of gliomas. Two subgroups were identified by consensus clustering, in which cluster 1 was associated with favorable prognosis, high stroma and immune scores, and high immune infiltration. When the patients were divided into high risk and low risk groups based on their risk scores, we found that patients in the high risk group had poor prognoses. Besides, patients in the high risk group had a higher stroma and immune scores, and higher abundance of immune infiltration. These results were further verified in the validation cohort, which contained three independent datasets. Moreover, patients in the low risk group enjoyed better prognoses without chemoradiotherapy or single chemotherapy. CONCLUSION: Our study revealed that m6A regulators could predict the prognosis and therapeutic efficacy, and were also associated with the immune microenvironment in gliomas.

Early Stage Markers of Late Delayed Neurocognitive Decline Using Diffusion Kurtosis Imaging of Temporal Lobe in Nasopharyngeal Carcinoma Patients.

Purpose: To determine whether the early assessment of temporal lobe microstructural changes using diffusion kurtosis imaging (DKI) can predict late delayed neurocognitive decline after radiotherapy in nasopharyngeal carcinoma (NPC) patients. Methods and Materials: Fifty-four NPC patients undergoing intensity-modulated radiotherapy (IMRT) participated in a prospective DKI magnetic resonance (MR) imaging study. MR imaging was acquired prior to IMRT (-0), 1 month (-1), and 3 (-3) months after IMRT. Kurtosis (Kmean, Kax, Krad) and Diffusivity (Dmean, Dax, Drad) variables in the temporal lobe gray and white matter were computed. Neurocognitive function tests (MoCA) were administered pre-radiotherapy and at 2 years post-IMRT follow-up. All the patients were divided into neurocognitive function decline (NFD group) and neurocognitive function non-decline groups (NFND group) according to whether the MoCA score declined ≥3 2 years after IMRT. All the DKI metrics were compared between the two groups, and the best imaging marker was chosen for predicting a late delayed neurocognitive decline. Results: Kurtosis (Kmean-1, Kmean-3, Kax-1, Kax-3, Krad-1, and Krad-3) and Diffusivity (Dmean-1 and Dmean-3) of white matter were significantly different between the two groups (p<0.05). Axial Kurtosis (Kax-1, Kax-3) of gray matter was significantly different between the two groups (p<0.05). By receiver operating characteristic (ROC) curves, Kmean-1 of white matter performed best in predicting of MoCA scores delayed decline (p<0.05). The radiation dose was also significantly different between NFD and NFND group (p=0.031). Conclusions: Temporal lobe white matter is more vulnerable to microstructural changes and injury following IMRT in NPC. Metrics derived from DKI should be considered as imaging markers for predicting a late delayed neurocognitive decline. Both temporal lobe white and gray matter show microstructural changes detectable by DKI. The Kmean early after radiotherapy has the best prediction performance.

Quantitative Proteomics Reveals the Mechanism of Silver Nanoparticles against Multidrug-Resistant Pseudomonas aeruginosa Biofilms.

The decline of clinically effective antibiotics has made it necessary to develop more effective antimicrobial agents, especially for refractory biofilm-related infections. Silver nanoparticles (AgNPs) are a new type of antimicrobial agent that can eradicate biofilms and reduce bacterial resistance, but its anti-biofilm mechanism has not been elucidated. In this study, we investigated the molecular mechanism of AgNPs against multidrug-resistant Pseudomonas aeruginosa by means of anti-biofilm tests, scanning electron microscopy (SEM), and tandem mass tag (TMT)-labeled quantitative proteomics. The results of anti-biofilm tests demonstrated that AgNPs inhibited the formation of P. aeruginosa biofilm and disrupted its preformed biofilm. SEM showed that when exposed to AgNPs, the structure of the P. aeruginosa biofilm was destroyed, along with significant reduction of its biomass. TMT-labeled quantitative proteomic analysis revealed that AgNPs could defeat the P. aeruginosa biofilm in multiple ways by inhibiting its adhesion and motility, stimulating strong oxidative stress response, destroying iron homeostasis, blocking aerobic and anaerobic respiration, and affecting quorum sensing systems. Our findings offer a new insight into clarifying the mechanism of AgNPs against biofilms, thus providing a theoretical basis for its clinical application.

Tumor treating fields for high-grade gliomas.

High-grade gliomas (HGG) are the most common malignant intracranial tumors with poor prognosis. Current treatments have not yielded optimal remission rates; there are no standard treatments for recurrent and drug-resistant gliomas. Tumor treating fields, which was recently approved by the Food and Drug Administration (FDA), could significantly improve progression free survival and the overall survival of glioma patients. In this review, we elaborate on the mechanism of tumor treating fields in tumor cells and detail various preclinical and clinical studies on gliomas. Tumor treating fields could be a promising option for patients with malignant tumors for which there are no standard treatment plans. Moreover, we identify several potential problems for the practical application of tumor treating fields and predict future directions for further studies. Tumor treating fields may be a potential therapy with high efficacy, fewer adverse effects, and high cost-effectiveness.

Prevalence and characteristics of Epstein-Barr virus associated gastric carcinoma in Gansu Province, Northwest China with mRNA expression of glycoprotein BMRF2.

Gansu province is a region with the highest gastric cancer incidence and mortality in Northwest China. Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is an important subtype of gastric cancer which shows specific clinicopathological features such as older-age bias, male predominance, lower lymph-node-metastasis, and a better cancer-related survival comparing to EBV-negative gastric cancers. However, the prevalence of EBVaGC has never been studied in Gansu Province, Northwest China. The present study investigated the incidence, characteristics, and EBV messenger RNA (mRNA) profile of EBVaGC in this area. We have collected 270 stomach samples from gastric cancer patients and analyzed the presence of EBV DNA and EBV-encoded small RNAs (EBERs) by nested polymerase chain reaction (PCR) and in situ hybridization, respectively. The EBV mRNA profiling was investigated by quantitative reverse transcription PCR (qRT-PCR). EBV DNA was detected in 51/95 patients (53.7%), while EBER transcripts were detected in 18/270 patients (6.7%). EBER positivity was significantly associated with older age and less lymph node metastasis, but no obvious association with gender or histological type of tumors. The expression of EBV genes was observed with different patterns, and the mRNA of glycoprotein BMRF2 was detected in EBVaGC. The present study showed unique clinicopathological features and mRNA expression patterns of EBVaGC in Gansu Province, Northwest China, suggesting that geographic variation can contribute to new epidemiological features in EBVaGC. The transcript of glycoprotein BMRF2 was observed consistently in EBVaGC, which may serve as a biomarker and play a role in the pathogenesis of EBVaGC in Gansu Province, Northwest China.

Antibacterial activity and mechanism of silver nanoparticles against multidrug-resistant Pseudomonas aeruginosa.

BACKGROUND: The threat of drug-resistant Pseudomonas aeruginosa requires great efforts to develop highly effective and safe bactericide. OBJECTIVE: This study aimed to investigate the antibacterial activity and mechanism of silver nanoparticles (AgNPs) against multidrug-resistant P. aeruginosa. METHODS: The antimicrobial effect of AgNPs on clinical isolates of resistant P. aeruginosa was assessed by minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). In multidrug-resistant P. aeruginosa, the alterations of morphology and structure were observed by the transmission electron microscopy (TEM); the differentially expressed proteins were analyzed by quantitative proteomics; the production of reactive oxygen species (ROS) was assayed by H2DCF-DA staining; the activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) was chemically measured and the apoptosis-like effect was determined by flow cytometry. RESULTS: Antimicrobial tests revealed that AgNPs had highly bactericidal effect on the drug-resistant or multidrug-resistant P. aeruginosa with the MIC range of 1.406-5.625 µg/mL and the MBC range of 2.813-5.625 µg/mL. TEM showed that AgNPs could enter the multidrug-resistant bacteria and impair their morphology and structure. The proteomics quantified that, in the AgNP-treated bacteria, the levels of SOD, CAT, and POD, such as alkyl hydroperoxide reductase and organic hydroperoxide resistance protein, were obviously high, as well as the significant upregulation of low oxygen regulatory oxidases, including cbb3-type cytochrome c oxidase subunit P2, N2, and O2. Further results confirmed the excessive production of ROS. The antioxidants, reduced glutathione and ascorbic acid, partially antagonized the antibacterial action of AgNPs. The apoptosis-like rate of AgNP-treated bacteria was remarkably higher than that of the untreated bacteria (P<0.01). CONCLUSION: This study proved that AgNPs could play antimicrobial roles on the multidrug-resistant P. aeruginosa in a concentration- and time-dependent manner. The main mechanism involves the disequilibrium of oxidation and antioxidation processes and the failure to eliminate the excessive ROS.

Celastrol Alleviates Chronic Obstructive Pulmonary Disease by Inhibiting Cellular Inflammation Induced by Cigarette Smoke via the Ednrb/Kng1 Signaling Pathway.

Chronic obstructive pulmonary disease (COPD) is a debilitating disease caused by chronic exposure to cigarette smoke (CS). Celastrol is a pentacyclic triterpenoid compound exhibits potent antioxidant and anti-inflammatory activities. Also it is presently known to protect against liver damage induced by type II diabetes. However, its role in COPD is unclear. In this study, we investigated the effects of Celastrol on cellular inflammation in mice exposed to CS and Beas-2B cells treated with CS extract (CSE). C57BL/6 mice and Beas-2B cells were randomly divided into three groups: control group, COPD or CSE group, and Celastrol treatment group. The COPD mice models were subjected to smoke exposure and cell models were treated with CSE. Bioinformatics analysis was performed to identify differentially expressed genes following treatment with Celastrol in COPD, the molecular networks was mapped by Cytoscape. The levels of inflammatory cytokinesinterleukin-8, tumor necrosis factor α, monocyte chemoattractant protein-1, and oxidative stress factors superoxide dismutase and catalase were measured by enzyme-linked immunosorbent assay. Hematoxylin and eosin staining to detect the injury of mouse lung tissue. mRNA and protein levels of Ednrb and Kng1 in the tissues and cells were measured by quantitative polymerase chain reaction (PCR) and western blotting, respectively. Apoptosis was measured by flow cytometry and TUNEL staining. Compared to mice in the COPD group, mice treated with Celastrol had significantly reduced levels of inflammatory cytokines interleukin-8, tumor necrosis factor α and monocyte chemoattractant protein-1 in the serum and bronchoalveolar lavage fluid, and significantly increased levels of oxidative stress factors superoxide dismutase and catalase. The same results were obtained at the cellular level using Beas-2B cells. Compared to the model groups, Celastrol reduced lung injury in mice and significantly reduced cellular apoptosis. Bioinformatics analysis showed that Ednrb is a target gene of Celastrol and differentially expressed in COPD. Quantitative PCR analysis showed that Ednrb expression in patients with COPD was significantly increased compared to that in healthy controls. Additionally, Celastrol effectively reduced Ednrb/Kng1 expression in both cell and animal models. Celastrol has a therapeutic effect on COPD and may alleviate COPD by inhibiting inflammation development by suppressing the Ednrb/Kng1 signaling pathway.

Hsa-miR-27b is up-regulated in cytomegalovirus-infected human glioma cells, targets engrailed-2 and inhibits its expression.

Human cytomegalovirus (HCMV) dormant infection can alter the expression of the hosts' microRNAs (miRNAs) and impact on the regulation of target genes. To investigate the differentially expressed miRNAs induced by HCMV in human glioma U251 cells, a comprehensive miRNA screen was performed. As a result, 19 up-regulated and 14 down-regulated miRNAs were determined. Of these, hsa-miR-27b (miR-27b) attracted our attention. MiR-27b levels in U251 cells increased 7.70-fold, 8.64-fold, and 4.78-fold, respectively, post 24 h, 48 h, and 72 h HCMV infection, compared to those in the mimic-infected cells, and this up-regulation was further confirmed by quantitative RT-PCR. The bioinformatic analyses show that miR-27b targets engrailed-2 (EN2) gene; however, the effect of miR-27b on EN2 is rarely encountered. In this study, we initially conducted dual luciferase assay to validate the target function of miR-27b on EN2. The results manifested that EN2 is a novel target of miR-27b, which could directly target the 3' untranslated region (3'-UTR) of the gene. We further found that the miR-27b transfected glioma U251 cells exhibited longer cell bodies with more synapses and multiple-angle shapes; moreover, Western blot detection revealed that the EN2 protein levels in these cells were significantly low. In conclusion, our study originally reports the up-regulation of miR-27b in HCMV-infected glioma cells. Our study also provides the first experimental evidence that miR-27b could affect glioma cells' growth, target EN2 and inhibit its expression in glioma cells. Our data indicate that miR-27b may be related to the development of neurological disorders with HCMV infection. The newly identified miR-27b/EN2 signal pathway may provide new insights into the glioma pathogenesis and a novel target for glioma therapy. Impact statement Our study is the first to demonstrate that the HCMV infection could alter the expression of cellular microRNAs of the host glioma cells, which may develop an understanding of the pathogenesis of the HCMV infection in the microRNA level. Recently, HCMV infection and engrailed-2 have been reported to be related to the autism spectrum disorder (ASD). In this study, we confirmed that engrailed-2 is the target of hsa-miR-27b. As far as we know, our findings of the hsa-miR-27b up-regulation in the HCMV-infected glioma cells, targeting engrailed-2 and inhibiting its expression have never been reported or documented. Our data indicate that miR-27b may be related to the development of neurological disorders with the HCMV infection. The newly identified miR-27b/EN2 signal pathway may provide new insights into the glioma pathogenesis and a novel target for glioma therapy.

Tissue expression and developmental regulation of chicken cathelicidin antimicrobial peptides.

Cathelicidins are a major family of antimicrobial peptides present in vertebrate animals with potent microbicidal and immunomodulatory activities. Four cathelicidins, namely fowlicidins 1 to 3 and cathelicidin B1, have been identified in chickens. As a first step to understand their role in early innate host defense of chickens, we examined the tissue and developmental expression patterns of all four cathelicidins. Real-time PCR revealed an abundant expression of four cathelicidins throughout the gastrointestinal, respiratory, and urogenital tracts as well as in all primary and secondary immune organs of chickens. Fowlicidins 1 to 3 exhibited a similar tissue expression pattern with the highest expression in the bone marrow and lung, while cathelicidin B1 was synthesized most abundantly in the bursa of Fabricius. Additionally, a tissue-specific regulatory pattern was evident for all four cathelicidins during the first 28 days after hatching. The expression of fowlicidins 1 to 3 showed an age-dependent increase both in the cecal tonsil and lung, whereas all four cathelicidins were peaked in the bursa on day 4 after hatching, with a gradual decline by day 28. An abrupt augmentation in the expression of fowlicidins 1 to 3 was also observed in the cecum on day 28, while the highest expression of cathelicidin B1 was seen in both the lung and cecal tonsil on day 14. Collectively, the presence of cathelicidins in a broad range of tissues and their largely enhanced expression during development are suggestive of their potential important role in early host defense and disease resistance of chickens.

Development of a bacterial artificial chromosome (BAC) recombineering procedure using galK-untranslated region (UTR) for the mutation of diploid genes.

Bacterial artificial chromosome (BAC) recombineering using galK selection allows DNA cloned in Escherichia coli to be modified without introducing an unwanted selectable marker at the modification site. Genomes of some herpesviruses have a pair of inverted repeat sequences that makes it very difficult to introduce mutations into diploid (duplicate) genes using the galK selection method. To mutate diploid genes, we developed a galK-UTR BAC recombineering procedure that blocks one copy of the target diploid gene by insertion of a galK untranslated region (UTR), which enables the simple mutation of the other copy. The blocked copy can then be replaced with an UTR-specific primer pair. The IR2 gene of equine herpesvirus 1 (EHV-1) maps within both the internal (IR) and terminal repeat (TR) of the genomic short region and is expressed at low levels because its promoter is TATA-less. Both IR2 promoters in EHV-1 BAC were replaced with a mutant IR2 promoter containing three Sp1-binding motifs and a consensus TATA box by galK-UTR BAC recombineering. The expression level of the IR2 protein controlled by the modified promoter increased approximately 4-fold as compared to that of wild-type EHV-1. The galK-UTR method will provide a useful tool in studies of herpesviruses.

Characterization of cis-acting elements required for autorepression of the equine herpesvirus 1 IE gene.

The immediate-early protein (IEP), the major regulatory protein encoded by the IE gene of equine herpesvirus 1 (EHV-1), plays a crucial role as both transcription activator and repressor during a productive lytic infection. To investigate the mechanism by which the EHV-1 IEP inhibits its own promoter, IE promoter-luciferase reporter plasmids containing wild-type and mutant IEP-binding site (IEBS) were constructed and used for luciferase reporter assays. The IEP inhibited transcription from its own promoter in the presence of a consensus IEBS (5'-ATCGT-3') located near the transcription initiation site but did not inhibit when the consensus sequence was deleted. To determine whether the distance between the TATA box and the IEBS affects transcriptional repression, the IEBS was displaced from the original site by the insertion of synthetic DNA sequences. Luciferase reporter assays revealed that the IEP is able to repress its own promoter when the IEBS is located within 26-bp from the TATA box. We also found that the proper orientation and position of the IEBS were required for the repression by the IEP. Interestingly, the level of repression was significantly reduced when a consensus TATA sequence was deleted from the promoter region, indicating that the IEP efficiently inhibits its own promoter in a TATA box-dependent manner. Taken together, these results suggest that the EHV-1 IEP delicately modulates autoregulation of its gene through the consensus IEBS that is near the transcription initiation site and the TATA box.

Butyrate enhances disease resistance of chickens by inducing antimicrobial host defense peptide gene expression.

Host defense peptides (HDPs) constitute a large group of natural broad-spectrum antimicrobials and an important first line of immunity in virtually all forms of life. Specific augmentation of synthesis of endogenous HDPs may represent a promising antibiotic-alternative approach to disease control. In this study, we tested the hypothesis that exogenous administration of butyrate, a major type of short-chain fatty acids derived from bacterial fermentation of undigested dietary fiber, is capable of inducing HDPs and enhancing disease resistance in chickens. We have found that butyrate is a potent inducer of several, but not all, chicken HDPs in HD11 macrophages as well as in primary monocytes, bone marrow cells, and jejuna and cecal explants. In addition, butyrate treatment enhanced the antibacterial activity of chicken monocytes against Salmonella enteritidis, with a minimum impact on inflammatory cytokine production, phagocytosis, and oxidative burst capacities of the cells. Furthermore, feed supplementation with 0.1% butyrate led to a significant increase in HDP gene expression in the intestinal tract of chickens. More importantly, such a feeding strategy resulted in a nearly 10-fold reduction in the bacterial titer in the cecum following experimental infections with S. enteritidis. Collectively, the results indicated that butyrate-induced synthesis of endogenous HDPs is a phylogenetically conserved mechanism of innate host defense shared by mammals and aves, and that dietary supplementation of butyrate has potential for further development as a convenient antibiotic-alternative strategy to enhance host innate immunity and disease resistance.

Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication.

The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain.

[Temporal expression of HCMV IE1 and pp65 in human glioma U(251) cells].

OBJECTIVE: To determine whether U(251) cells are permissive for human cytomegalovirus (HCMV), and to investigate the characteristics of temporal expression of proteins IE1 and pp65. METHODS: U(251) cells were infected with HCMV, and then the cells were observed under the transmission electronic microscope, and the viral nucleic acid was detected by PCR, and the expression levels of IE1 and pp65 were analyzed by immunohistochemical assay with anti-IE1 monoclonal antibody and anti-pp65 monoclonal antibody at various time spost infection. RESULTS: Morphological changes of the infected cells appeared under the transmission electron microscope. The viral nucleic acid was detected successfully by PCR. The expression of IE1 was detected firstly at 4h post infection, and reached a peak within 14h, and then decreased. The incoming pp65 was detected at 1h, the low expression levels of pp65 were detected firstly at 4h, and they could remain relatively constant through 96 h, but the maximum expression occurred at 120 h. CONCLUSION: Human glioma U(251) cells are permissive for HCMV, the temporal cascade of HCMV gene expression can be observed in the infected U(251) cells, but it is delayed obviously in the human fibroblast.