Identification of multiomics map and key biomarkers in uveal melanoma with chromosome 3 loss.

Purpose: Chromosome 3 loss is an independent risk factor for uveal melanoma (UM), but its exact molecular mechanisms remain unclear. This study was designed to investigate the relationship between chromosome 3 loss and molecular alterations at multiple levels to construct a prognostic model. Methods: Forty-four UM cases with chromosome 3 loss (chr3 del group) and 36 UM cases without copy number variation on chromosome 3 (chr3 wt group) were collected from the Cancer Genome Atlas (TCGA). The TCGA dataset was subjected to a univariate Cox regression analysis to identify different expressed genes, and a subsequent random forest algorithm analysis revealed significant changes in different expressed genes, which were used to develop key biomarkers for UM. Following that, the immune cell infiltration analysis and drug sensitivity analyses were carried out. The UM cell line was then utilized to investigate the potential functions of the key biomarker via cell apoptosis, proliferation, cycle assays, WB, and RT-qPCR. Results: By analyzing the 80 cases data in TCGA, the authors unveiled molecular changes relevant to loss of chromosome 3 in UM as well as their poor survival. In addition, machine learning analysis identified three hub genes (GRIN2A, ACAN, and MMP9) as potential therapeutic targets. The differentially enriched pathways between the two groups were mainly about immune-system activity, and hub genes expression was also highly correlated with immune infiltration levels. Conclusion: Chromosome 3 loss has considerable clinical significance for UM, and GRIN2A may be useful in diagnosing, treating, and prognosticating the condition.

Isolation, characterization, evaluation of pathogenicity, and immunomodulation through interferon production of duck adenovirus type-3 (DAdV-3).

Duck adenovirus type-3 (DAdV-3) is a poorly characterized duck virus. A comprehensive analysis of the DAdV-3 pathogenicity and host immune response could be a valuable addition. Herein, DAdV-3 was isolated from Muscovy duck and virus-specific genes were confirmed by polymerase chain reaction (PCR). The obtained gene fragments were sequenced and compared with the reference sequence. Results confirmed that the clinically isolated virus was DAdV-3, named as HF-AN-2020. To evaluate DAdV-3 host immune response, the expression levels of MDA5, STING, IRF7, MAVS, and NF-κB, and inflammatory cytokines (IFN-ß, IFN-γ, and IL-1ß) were determined by quantitative reverse transcriptase PCR (qRT-PCR). The expression levels of IFN-ß and IFN-γ were 32.6- and 28.6-fold, respectively, higher (P < 0.01) than the control group. It was found that the upregulation of STING and NF-κB pathways was directly involved in the regulation of inflammatory cytokines (IFN-ß, IFN-γ, and IL-1ß). Furthermore, the gene regulation pathways consecutively upregulated the expression levels of MDA5, STING, IRF7, MAVS, and NF-κB up to 31.6, 10.5, 31.4, 2.2, and 2.6-fold, respectively, higher (P < 0.01) than the control group. The TCID50 of DAdV-3 for Muscovy duck and chicken was 10-3.24/0.1 mL with 0% mortality, indicating low pathogenicity in both Muscovy ducks and chickens, but DAdV-3 can induce higher expression of interferons. Genome analysis showed mutations in 4 amino acids located in ORF19B (Ser to Thr), ORF66 (Leu to Phe, Ile to Leu), and ORF67 (Gly to stop codon). This study provides essential and basic information for further research on the mechanism of the cellular immune responses against adenoviruses.

Natural recombination of the torque teno canis virus within the ORF1, -2, and -3 genes and the untranslated region.

The torque teno canis virus (TTCaV) was first reported in 2001 and it shares similarities with the known Torque teno virus (TTV) in terms of genomic organization and putative transcriptional features. It is a single-stranded DNA virus characterized by high rates of recombination and nucleotide substitution, like RNA viruses. Studies reported recombination events in torque teno virus; however, there is limited reporting of TTCaV reorganization events. This study screened fecal samples from domestic dogs in Henan Province. There was a positivity rate of 16.5% (19/115) for TTCaV. Four nearly complete TTCaV genomes, namely Canine/HeNan/4, 5, 6, and 13/2019, were obtained from the 19 positive fecal samples, whose genome sequence was obtained using gap-filling PCR. Sequence analysis revealed two unique amino acid mutation sites in the TTCaV strains, K278Q (compared with the first isolate Cf-TTV10 in Japan) and V/L268I (compared with the TTCaV strain from southern China). Subsequently, 17 near full-length TTCaV genome sequences were subjected to phylogenetic and recombination detection program analyzes. We obtained evidence supporting recombination events in the Chinese TTCaV strains. These findings suggest that mutation and recombination occurred in the three individual gene segments (ORF1, ORF2, ORF3) and the untranslated region, an area of major recombination in the Chinese TTCaV strain GX265 genome. Interestingly, the TTCaV strain (Canine/HeNan/6/2019) was a major parent involved in the genetic recombination of the GX265 strain. This study provides insights into the genetic variability and evolution of TTCaV.

The Effect of Poria cocos Polysaccharide PCP-1C on M1 Macrophage Polarization via the Notch Signaling Pathway.

The homogeneous galactoglucan PCP-1C extracted from Poria cocos sclerotium has multiple biological activities. The present study demonstrated the effect of PCP-1C on the polarization of RAW 264.7 macrophages and the underlying molecular mechanism. Scanning electron microscopy showed that PCP-1C is a detrital-shaped polysaccharide with fish-scale patterns on the surface, with a high sugar content. The ELISA assay, qRT-PCR assay, and flow cytometry assay showed that the presence of PCP-1C could induce higher expression of M1 markers, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-12 (IL-12), when compared with the control and the LPS group, and it caused a decrease in the level of interleukin-10 (IL-10), which is the marker for M2 macrophages. At the same time, PCP-1C induces an increase in the CD86 (an M1 marker)/CD206 (an M2 marker) ratio. The results of the Western blot assay showed that PCP-1C induced activation of the Notch signaling pathway in macrophages. Notch1, ligand Jagged1, and Hes1 were all up-regulated with the incubation of PCP-1C. These results indicate that the homogeneous Poria cocos polysaccharide PCP-1C improves M1 macrophage polarization through the Notch signaling pathway.

Identification of Two Isoforms of Canine Tetherin in Domestic Dogs and Characterization of Their Antiviral Activity against Canine Influenza Virus.

Canine influenza virus (CIV) significantly threatens the canine population and public health. Tetherin, an innate immune factor, plays an important role in the defense against pathogen invasion and has been discovered to restrict the release of various enveloped viruses. Two isoforms of canine tetherin (tetherin-X1 and tetherin-X2) were identified in peripheral blood leukocytes of mixed-breed dogs using reverse transcription polymerase chain reaction (RT-PCR). Amino acid alignment revealed that relative to full-length tetherin (tetherin-X1) and truncated canine tetherin (tetherin-X2) exhibited deletion of 34 amino acids. The deletion occurred at the C-terminus of the coiled-coiled ectodomain and the N-terminus of the glycosylphosphatidylinositol (GPI)-anchor domain. Tetherin-X2 was localized subcellularly at the cell membrane, which was consistent with the localization of tetherin-X1. In addition, canine tetherin-X1 and tetherin-X2 restricted the release of H3N2 CIV. However, canine tetherin-X1 had higher antiviral activity than canine tetherin-X2, indicating that the C-terminus of the coiled-coiled ectodomain and the N-terminus of the GPI-anchor domain of canine tetherin (containing the amino acids deleted in tetherin-X2) are critical for its ability to restrict H3N2 CIV release. This study provides insights for understanding the key functional domains of tetherin that restrict CIV release.

Efficiency of intraoperative endoscopic inspection in reducing residuals in canal-wall-up surgery for pediatric cholesteatoma involving the mastoid.

OBJECTIVE: To investigate the efficiency of additional intraoperative endoscopic inspection in reducing residual cholesteatoma in pediatric cholesteatoma involving the mastoid treated with classic canal-wall-up mastoidectomy and tympanoplasty. MATERIALS AND METHODS: 32 cases of pediatric cholesteatoma involving the mastoid were enrolled in this perspective study and treated with classic canal-wall-up mastoidectomy and tympanoplasty. Transmastoid posterior tympanotomy, atticotomy and transecting tendon of tympani tensor were conducted to achieve adequate visualization of hidden spaces in the middle ear. After complete removal of cholesteatoma, endoscopic inspection was additionally performed to check residual cholesteatoma. All cases had at least a 2-year follow-up by routine otoscopy examination, CT scan or MR imaging. Residual rates of both intraoperative and follow-up findings were used to evaluate the efficiency of the endoscopic inspection in reducing residual cholesteatoma and compared with published reports. RESULTS: The additional intraoperative endoscopic inspection did not find any residual in this case series. In the 2-year follow-up, 2 cases (2/32, 6.3%) with residual cholesteatoma and 3 cases with recurrence (3/32, 9.4%) were found. The mean duration of endoscopic inspection and microscopic procedure were 17.9 min and 93.6 min, respectively. CONCLUSIONS: This study suggested that the additional intraoperative endoscopic inspection in microscopic CWU surgery for pediatric cholesteatoma involving the mastoid had no obvious value in reducing residual cholesteatoma but took extra time.

Evaluation of Zuo-Gui Yin Decoction Effects on Six CYP450 Enzymes in Rats Using a Cocktail Method by UPLC-MS/MS.

Background: Zuo-Gui Yin Decoction (ZGYD), a traditional Chinese prescription, is mainly used in various kinds of andrology and gynecology diseases. However, the study on the interaction of ZGYD and drugs has not been reported. Therefore, evaluating the interaction between ZGYD and metabolic enzymes is helpful to guide rational drug use. Objective: This study was conducted to explore the effects of ZGYD on the activity and mRNA expressions of six Cytochrome P450 (CYP450) enzymes in rats and to provide a basis for its rational clinical use. Methods: Sprague-Dawley rats were randomly divided into control, ZGYD high, medium, and low-dose group (n = 6). The concentrations of six probe substrates in plasma of rats in each group were determined by UPLC-MS/MS. In addition, RT-PCR and Western blot were used to determine the effects of ZGYD on the expression of CYP450 isoforms in the liver. Results: Compared with the control group, the main pharmacokinetic parameters AUC(0-t), AUC (0~∞), of omeprazole, dextromethorphan, and midazolam in the high-dose group were significantly decreased, while the CL of these were significantly increased. The gene expressions of CYP2C11 and CYP3A1 were upregulated in the ZGYD medium, high-dose group. The protein expression of CYP2C11 was upregulated in the high-dose group, and the protein expression of CYP3A1 was upregulated in the medium, high-dose group. Conclusion: The results showed that ZGYD exhibited the induction effects on CYP2C11 and CYP3A1 (CYP2C19 and CYP3A4 in humans) in rats. However, no significant change in CYP1A2, CYP2B1, CYP2C7, and CYP2D2 activities was observed. It would be useful for the safe and effective usage of ZGYD in clinic.

Forward Electric Stimulation-Induced Interference in Intracochlear Electrocochleography of Acoustic Stimulation in the Cochlea of Guinea Pigs.

Electric-acoustic stimulation (EAS) uses amplified sound by a hearing aid to stimulate an apical low-frequency region of the cochlea and electrical current from a cochlear implant (CI) to stimulate the basal high-frequency region. EAS recipients had significantly improved speech perception, music appreciation, and hearing function in noise compared to those relying on CI electrical stimulation (ES) alone. However, the interaction between basal ES and apical acoustic stimulation (AS) in the cochlea potentially affects EAS advantages. To investigate ES-AS interaction, we designed a system that recorded the electrically evoked compound action potential (ECAP) and the auditory evoked potential (AEP). We used an intracochlear electrode array to deliver ES at the basal cochlea and detect intracochlear electrocochleography (iECochG) generated from apical AS. Within iECochG, 3 or 6 dB (double or quadruple intensity of ECAP threshold) electric stimulation, 1 ms-forward ES significantly increased CAP amplitudes of 4 kHz/20 dB AS compared to 0 dB ES. Notably, 1 ms-forward 3 dB ES significantly increased CAP amplitudes of 4 kHz/20 dB AS, while 3 or 5 ms-forward ES did not change the CAP amplitudes. The elevation in CAP amplitude of 40 dB/4 kHz AS induced by 1 ms-forward 3 dB ES was significantly lower than that in 20 dB/4 kHz AS. With 1 ms-forward 3 dB ES, AS frequency and stimulating electrode location have no significant impact on relative CAP amplitudes of 20 dB AS. These results suggest that the basal forward ES and the following apical AS could produce a cumulative effect on the auditory nerve response.

Using Extracochlear Multichannel Electrical Stimulation to Relieve Tinnitus and Reverse Tinnitus-Related Auditory-Somatosensory Plasticity in the Cochlear Nucleus.

OBJECTIVES: Tinnitus has no reliable cure but may be significantly relieved by the usage of cochlear implants. However, not all tinnitus patients necessitate cochlear implantation that can impair hearing. This study was to investigate whether a novel extracochlear electrical stimulation (EES) strategy could relieve tinnitus of guinea pigs without hearing impairment, and the roles of auditory-somatosensory plasticity in the cochlear nucleus in the tinnitus relief. MATERIALS AND METHODS: We used a novel four-electrode extracochlear implant to electrically stimulate the cochlea of tinnitus guinea pigs. Tinnitus was assessed by the gap-prepulse inhibition of the acoustic startle reflex (GPIAS) ratios and the tinnitus index. The plasticity of auditory and somatosensory innervation in the different subdivisions of cochlear nucleus was evaluated by immunostaining of vesicular glutamate transporter 1 (VGLUT1) and VGLUT2, respectively. RESULTS: The EES induced significant decreases of GPIAS ratios and the tinnitus index of tinnitus guinea pigs, indicating reductions of tinnitus behavioral manifestations. Meanwhile, the EES reversed the abnormal auditory-somatosensory innervation in the cochlear nucleus of tinnitus animals but did not change the hearing and the numbers of inner hair cell synapses. CONCLUSIONS: This study demonstrated that the novel EES strategy could effectively relieve tinnitus without impairment to hearing and cochlear structure of tinnitus animals. The reversal of tinnitus-related auditory-somatosensory plasticity in the cochlear nucleus was correlated with the tinnitus relief induced by the EES.

Therapeutic potential of paeoniflorin in atherosclerosis: A cellular action and mechanism-based perspective.

Epidemiological studies have shown that the incidence, prevalence and mortality of atherosclerotic cardiovascular disease (ASCVD) are increasing globally. Atherosclerosis is characterized as a chronic inflammatory disease which involves inflammation and immune dysfunction. P. lactiflora Pall. is a plant origin traditional medicine that has been widely used for the treatment of various diseases for more than a millennium in China, Japan and Korean. Paeoniflorin is a bioactive monomer extracted from P. lactiflora Pall. with anti-atherosclerosis effects. In this article, we comprehensively reviewed the potential therapeutic effects and molecular mechanism whereby paeoniflorin protects against atherosclerosis from the unique angle of inflammation and immune-related pathway dysfunction in vascular endothelial cells, smooth muscle cells, monocytes, macrophages, platelets and mast cells. Paeoniflorin, with multiple protective effects in atherosclerosis, has the potential to be used as a promising therapeutic agent for the treatment of atherosclerosis and its complications. We conclude with a detailed discussion of the challenges and future perspective of paeoniflorin in translational cardiovascular medicine.

The Biogenesis, Biological Functions, and Applications of Macrophage-Derived Exosomes.

Macrophage-derived exosomes have been implicated on the modulation of inflammatory processes. Recent studies have shown that macrophage-derived exosomes contribute to the progression of many diseases such as cancer, atherosclerosis, diabetes and heart failure. This review describes the biogenesis of macrophage-derived exosomes and their biological functions in different diseases. In addition, the challenges facing the use of macrophage-derived exosomes as delivery tools for drugs, genes, and proteins in clinical applications are described. The application of macrophage-derived exosomes in the diagnosis and treatment of diseases is also discussed.

Doxorubicin-Induced Cancer Cell Senescence Shows a Time Delay Effect and Is Inhibited by Epithelial-Mesenchymal Transition (EMT).

BACKGROUND Senescence is a natural barrier for the body to resist the malignant transformation of its own cells. This work investigated the senescence characteristics of cancer cells in vitro. MATERIAL AND METHODS Human cervical cancer HeLa cells were treated with different concentrations of doxorubicin for 3 days, with or without subsequent extended culture in drug-free medium for 6 days. Senescent cell ratios between these 2 culture schemes were calculated. Expression of 2 senescence-associated secretory factors, IL-6 and IL-8, were detected by RT-PCR and ELISA. Doxorubicin treatment induced epithelial-mesenchymal transition in cancer cells. The proportions of senescent cells in epithelial-like and mesenchymal-like sub-groups were calculated. Doxorubicin-treated HeLa cells were stained with Vimentin antibody and sorted by flow cytometry. Senescent cell marker p16ᴵᴺᴷ4ᵃ and IL-8 expression in Vimentin-high and Vimentin-low cells were detected by Western blot. RESULTS We found that less than 1% of HeLa cells showed senescence phenotype after treatment with doxorubicin for 3 days. However, the proportion of senescent cells was significantly increased when the doxorubicin-treated cells were subsequently cultured in drug-free medium for another 6d. RT-PCR and ELISA results showed that this prolonged culture method could further improve the expression of IL-6 and IL-8. We also found that the senescent cells were mainly epithelial-like type and few presented mesenchymal-like shape. p16ᴵᴺᴷ4ᵃ and IL-8 expression were decreased in cell fraction with higher Vimentin expression. CONCLUSIONS Our results suggested the existence of time delay effect in doxorubicin-induced senescence of HeLa cells, and epithelial- mesenchymal transition may resist doxorubicin-induced cell senescence.

Recent research on bioactive xanthones from natural medicine: Garcinia hanburyi.

Garcinia hanburyi, a tropical plant found in south Asia, has a special long history in the development of both medicine and art. This review mainly focuses on the pharmacy research of the bioactive compounds from the plant in recent years. Preparative and analysis separation methods were introduced. Moreover, the chemical structure of the isolated compounds was included. The studies of biological activities of the caged xanthones from the plant, including antitumor, anti-HIV-1, antibacterial, and neurotrophic activities, were reviewed in detail. Furthermore, the mechanisms of its antitumor activity were also reviewed. As mentioned above, some of the xanthones from G. hanburyi can be promising drug candidates, which is worth studying. However, we still need much evidence to prove their efficacy and safety. So, further research is critical for the future application of xanthones from G. hanburyi.