Biophysical Electrical and Mechanical Stimulations for Promoting Chondrogenesis of Stem Cells on PEDOT:PSS Conductive Polymer Scaffolds.

The investigation of the effects of electrical and mechanical stimulations on chondrogenesis in tissue engineering scaffolds is essential for realizing successful cartilage repair and regeneration. The aim of articular cartilage tissue engineering is to enhance the function of damaged or diseased articular cartilage, which has limited regenerative capacity. Studies have shown that electrical stimulation (ES) promotes mesenchymal stem cell (MSC) chondrogenesis, while mechanical stimulation (MS) enhances the chondrogenic differentiation capacity of MSCs. Therefore, understanding the impact of these stimuli on chondrogenesis is crucial for researchers to develop more effective tissue engineering strategies for cartilage repair and regeneration. This study focuses on the preparation of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) conductive polymer (CP) scaffolds using the freeze-drying method. The scaffolds were fabricated with varying concentrations (0, 1, 3, and 10 wt %) of (3-glycidyloxypropyl) trimethoxysilane (GOPS) as a crosslinker and an additive to tailor the scaffold properties. To gain a comprehensive understanding of the material characteristics and the phase aggregation phenomenon of PEDOT:PSS scaffolds, the researchers performed theoretical calculations of solubility parameters and surface energies of PSS, PSS-GOPS, and PEDOT polymers, as well as conducted material analyses. Additionally, the study investigated the potential of promoting chondrogenic differentiation of human adipose stem cells by applying external ES or MS on a PEDOT:PSS CP scaffold. Compared to the group without stimulation, the group that underwent stimulation exhibited significantly up-regulated expression levels of chondrogenic characteristic genes, such as SOX9 and COL2A1. Moreover, the immunofluorescence staining images exhibited a more vigorous fluorescence intensity of SOX9 and COL II proteins that was consistent with the trend of the gene expression results. In the MS experiment, the strain excitation exerted on the scaffold was simulated and transformed into stress. The simulated stress response showed that the peak gradually decreased with time and approached a constant value, with the negative value of stress representing the generation of tensile stress. This stress response quantification could aid researchers in determining specific MS conditions for various materials in tissue engineering, and the applied stress conditions could be further optimized. Overall, these findings are significant contributions to future research on cartilage repair and biophysical ES/MS in tissue engineering.

Applications of Hyaluronic Acid in Ophthalmology and Contact Lenses.

Hyaluronic acid (HA) is a glycosaminoglycan that was first isolated and identified from the vitreous body of a bull's eye. HA is ubiquitous in the soft connective tissues of animals and therefore has high tissue compatibility for use in medication. Because of HA's biological safety and water retention properties, it has many ophthalmology-related applications, such as in intravitreal injection, dry eye treatment, and contact lenses. Due to its broad range of applications, the identification and quantification of HA is a critical topic. This review article discusses current methods for analyzing HA. Contact lenses have become a widely used medical device, with HA commonly used as an additive to their production material, surface coating, and multipurpose solution. HA molecules on contact lenses retain moisture and increase the wearer's comfort. HA absorbed by contact lenses can also gradually release to the anterior segment of the eyes to treat dry eye. This review discusses applications of HA in ophthalmology.

Extension of C. elegans lifespan using the ·NO-delivery dinitrosyl iron complexes.

The ubiquitous and emerging physiology function of endogenous nitric oxide in vascular, myocardial, immune, and neuronal systems prompts chemists to develop a prodrug for the controlled delivery of ·NO in vivo and for the translational biomedical application. Inspired by the discovery of natural [Fe(NO)2] motif, herein, we develop the synthetic dinitrosyl iron complexes (DNICs) [Fe2(µ-SR)2(NO)4] (1) as a universal platform for the O2-triggered release of ·NO, for the regulation of ·NO-release kinetics (half-life = 0.6-27.4 h), and for the activation of physiological function of ·NO. Using C. elegans as a model organism, the ·NO-delivery DNIC 1 regulates IIS signaling pathway, AMPK signaling pathway, and mitochondrial function pathway to extend the lifespan and to delay the aging process based on the lifespan analysis, SA-ßgal activity assay, and next-generation RNA sequencing analysis. This study unveils the anti-aging effect of ·NO and develops DNICs as a chemical biology probe for the continued discovery of unprecedented NO physiology.

Specific Interaction between eEF1A and HIV RT Is Critical for HIV-1 Reverse Transcription and a Potential Anti-HIV Target.

Reverse transcription is the central defining feature of HIV-1 replication. We previously reported that the cellular eukaryotic elongation factor 1 (eEF1) complex associates with the HIV-1 reverse transcription complex (RTC) and the association is important for late steps of reverse transcription. Here we show that association between the eEF1 and RTC complexes occurs by a strong and direct interaction between the subunit eEF1A and reverse transcriptase (RT). Using biolayer interferometry and co-immunoprecipitation (co-IP) assays, we show that association between the eEF1 and RTC complexes occurs by a strong (KD ~3-4 nM) and direct interaction between eEF1A and reverse transcriptase (RT). Biolayer interferometry analysis of cell lysates with titrated levels of eEF1A indicates it is a predominant cellular RT binding protein. Both the RT thumb and connection domains are required for interaction with eEF1A. A single amino acid mutation, W252A, within the thumb domain impaired co-IP between eEF1A and RT, and also significantly reduced the efficiency of late reverse transcription and virus replication when incorporated into infectious HIV-1. Molecular modeling analysis indicated that interaction between W252 and L303 are important for RT structure, and their mutation to alanine did not impair heterodimerisation, but negatively impacted interaction with eEF1A. Didemnin B, which specifically binds eEF1A, potently inhibited HIV-1 reverse transcription by greater than 2 logs at subnanomolar concentrations, especially affecting reverse transcription late DNA synthesis. Analysis showed reduced levels of RTCs from HIV-1-infected HEK293T treated with didemnin B compared to untreated cells. Interestingly, HIV-1 with a W252A RT mutation was resistant to didemnin B negative effects showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. The combined evidence indicates a direct interaction between eEF1A and RT is crucial for HIV reverse transcription and replication, and the RT-eEF1A interaction is a potential drug target.

A mutant Tat protein inhibits infection of human cells by strains from diverse HIV-1 subtypes.

BACKGROUND: Nullbasic is a mutant HIV-1 Tat protein that inhibits HIV-1 replication via three independent mechanisms that disrupts 1) reverse transcription of the viral RNA genome into a DNA copy, 2) HIV-1 Rev protein function required for viral mRNA transport from the nucleus to the cytoplasm and 3) HIV-1 mRNA transcription by RNA Polymerase II. The Nullbasic protein is derived from the subtype B strain HIV-1BH10 and has only been tested against other HIV-1 subtype B strains. However, subtype B strains only account for ~10% of HIV-1 infections globally and HIV-1 Tat sequences vary between subtypes especially for subtype C, which is responsible for ~50% HIV-1 infection worldwide. These differences could influence the ability of Tat to interact with RNA and cellular proteins and thus could affect the antiviral activity of Nullbasic. Therefore, Nullbasic was tested against representative HIV-1 strains from subtypes C, D and A/D recombinant to determine if it can inhibit their replication. METHODS: Nullbasic was delivered to human cells using a self-inactivating (SIN) γ-retroviral system. We evaluated Nullbasic-mCherry (NB-mCh) fusion protein activity against the HIV-1 strains in TZM-bl cell lines for inhibition of transactivation and virus replication. We also examined antiviral activity of Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein against the same strains in primary CD4+ T cells. The Nullbasic expression was monitored by western blot and flow cytometry. The effects of Nullbasic on primary CD4+ T cells cytotoxicity, proliferation and apoptosis were also examined. RESULTS: The results show that Nullbasic inhibits Tat-mediated transactivation and virus replication of all the HIV-1 strains tested in TZM-bl cells. Importantly, Nullbasic inhibits replication of the HIV-1 strains in primary CD4+ T cells without affecting cell proliferation, cytotoxicity or level of apoptotic cells. CONCLUSION: A SIN-based γ-retroviral vector used to express Nullbasic fusion proteins improved protein expression particularly in primary CD4+ T cells. Nullbasic has antiviral activity against all strains from the subtypes tested although small differences in viral inhibition were observed. Further improvement of in γ-retroviral vector stable expression of Nullbasic expression may have utility in a future gene therapy approach applicable to genetically diverse HIV-1 strains.

Interaction between cytochrome P450 2A6 and Catechol-O-Methyltransferase genes and their association with smoking risk in young men.

BACKGROUND: Although some effects of gene-gene interactions on nicotine-dopamine metabolism for smoking behavior have been reported, polymorphisms of cytochrome P450 (CYP) 2A6 and catechol-O-methyltransferase (COMT) have not been studied together to determine their effects on smokers. The aim of this study was to investigate the effects of the interaction between the CYP 2A6 and COMT genes on smoking behavior in young Taiwanese men. RESULTS: A self-report questionnaire regarding smoking status was administered to 500 young men. Polymorphisms of the CYP 2A6 and COMT genes as well as urinary nicotine and urinary cotinine levels were determined. The odds ratio for starting smoking was significantly lower in subjects carrying a CYP2A6 low activity/variant COMT rs4680 genotype than in those possessing a CYP2A6 wild-type/variant COMT rs4680 genotype (0.44, 95% confidence interval = 0.19-0.98, P = 0.043). Comparisons of Fagerstrom Test for Nicotine Dependence (FTND), Physiological Cigarette Dependence Scale (PCDS), and Cigarette Withdrawal symptoms (CWS-21) among the smokers with different CYP2A6/COMT polymorphisms were not significantly different. The adjusted urinary nicotine concentrations were not significantly different between the two groups carrying different genotypes. The adjusted urinary cotinine level was significantly different between the COMT rs4680 wild-type group and COMT rs4680 variant group [92.46 ng/µL vs. 118.24 ng/µL (median value), P = 0.041] and between the COMT rs4680 wild-type/COMT rs165599 variant group and COMT rs4680 variant/COMT rs165599 variant group (97.10 ng/µL vs. 122.18 ng/µL, P = 0.022). CONCLUSIONS: These findings suggest that a single nucleotide polymorphism (rs4680) of the COMT gene and the interaction between the CYP 2A6 and COMT genes affect smoking status in young Taiwanese men.

A mutant tat protein inhibits HIV-1 reverse transcription by targeting the reverse transcription complex.

UNLABELLED: Previously, we reported that a mutant of Tat referred to as Nullbasic inhibits HIV-1 reverse transcription although the mechanism of action is unknown. Here we show that Nullbasic is a reverse transcriptase (RT) binding protein that targets the reverse transcription complex rather than directly inhibiting RT activity. An interaction between Nullbasic and RT was observed by using coimmunoprecipitation and pulldown assays, and a direct interaction was measured by using a biolayer interferometry assay. Mixtures of recombinant 6×His-RT and Nullbasic-FLAG-V5-6×His at molar ratios of up to 1:20,000 did not inhibit RT activity in standard homopolymer primer template assays. An analysis of virus made by cells that coexpressed Nullbasic showed that Nullbasic copurified with virus particles, indicating that it was a virion protein. In addition, analysis of reverse transcription complexes (RTCs) isolated from cells infected with wild type or Nullbasic-treated HIV-1 showed that Nullbasic reduced the levels of viral DNA in RTC fractions. In addition, a shift in the distribution of viral DNA and CAp24 to less-dense non-RTC fractions was observed, indicating that RTC activity from Nullbasic-treated virus was impaired. Further analysis showed that viral cores isolated from Nullbasic-treated HIV undergo increased disassembly in vitro compared to untreated HIV-1. To our knowledge, this is the first description of an antiviral protein that inhibits reverse transcription by targeting the RTC and affecting core stability. IMPORTANCE: HIV-1 infection is treated by using combinations of antiretroviral drugs that target independent steps of virus replication. A newly described antiviral protein called Nullbasic can also inhibit a combination of different steps in virus replication (transcription, reverse transcription, and Rev-mediated viral mRNA transport), although the precise mechanism of action is unknown. This study shows that Nullbasic can inhibit reverse transcription by binding to the viral enzyme called reverse transcriptase, which results in accelerated uncoating of the viral core and instability of the viral apparatus called the reverse transcription complex (RTC). This unique antiviral activity may inform development of other RTC inhibitors, as well as providing a unique investigative tool for dissecting the RTC cellular composition.

Motor neuron-derived Thsd7a is essential for zebrafish vascular development via the Notch-dll4 signaling pathway.

BACKGROUND: Development of neural and vascular systems displays astonishing similarities among vertebrates. This parallelism is under a precise control of complex guidance signals and neurovascular interactions. Previously, our group identified a highly conserved neural protein called thrombospondin type I domain containing 7A (THSD7A). Soluble THSD7A promoted and guided endothelial cell migration, tube formation and sprouting. In addition, we showed that thsd7a could be detected in the nervous system and was required for intersegmental vessels (ISV) patterning during zebrafish development. However, the exact origin of THSD7A and its effect on neurovascular interaction remains unclear. RESULTS: In this study, we discovered that zebrafish thsd7a was expressed in the primary motor neurons. Knockdown of Thsd7a disrupted normal primary motor neuron formation and ISV sprouting in the Tg(kdr:EGFP/mnx1:TagRFP) double transgenic zebrafish. Interestingly, we found that Thsd7a morphants displayed distinct phenotypes that are very similar to the loss of Notch-delta like 4 (dll4) signaling. Transcript profiling further revealed that expression levels of notch1b and its downstream targets, vegfr2/3 and nrarpb, were down-regulated in the Thsd7a morphants. These data supported that zebrafish Thsd7a could regulate angiogenic sprouting via Notch-dll4 signaling during development. CONCLUSIONS: Our results suggested that motor neuron-derived Thsd7a plays a significant role in neurovascular interactions. Thsd7a could regulate ISV angiogenesis via Notch-dll4 signaling. Thus, Thsd7a is a potent angioneurin involved in the development of both neural and vascular systems.

Direct-growth carbon nanotubes on 3D structural microelectrodes for electrophysiological recording.

A novel 3D carbon nanotube (CNT) microelectrode was developed through direct growth of CNTs on a gold pin-shaped 3D microelectrode at a low temperature (400 °C) for applications in neural and cardiac recording. With an electroplated Ni catalyst layer covering the entire surface of the pin-shaped structure, CNTs were synthesized on a 3D microelectrode by catalytic thermal chemical vapor deposition (CVD). According to the analyses by electrochemical impedance spectroscopy, the impedance of 3D microelectrodes after CNT growth and UV/O3 treatment decreased from 9.3 Ω mm(-2) to 1.2 Ω mm(-2) and the capacitance increased largely from 2.2 mF cm(-2) to 73.3 mF cm(-2). The existence of UVO3-treated CNT led to a large improvement of interfacial capacitance, contributing to the decrease of impedance. The electrophysiological detection capability of this 3D CNT microelectrode was demonstrated by the distinguished P waves, QRS complex and T waves in the electrocardiogram of the zebrafish heart and the action potential recorded from individual rat hippocampal neurons. The compatibility of integration with ICs, high resolution in space, electrophysiological signals, and non-invasive long-term recording suggest that the 3D CNT microelectrode exhibits promising potential for applications in electrophysiological research and clinical trials.

Binding of the eukaryotic translation elongation factor 1A with the 5'UTR of HIV-1 genomic RNA is important for reverse transcription.

BACKGROUND: The cellular protein eukaryotic translation elongation factor 1A (eEF1A) binds to aminoacylated transfer RNAs and delivers them to the ribosome during translation. eEF1A also binds to RNA secondary structures present in genomes of several RNA viruses and plays important roles in their replication. As a RNA binding protein, whether eEF1A can bind with HIV-1 genomic RNA has not been investigated and was the aim of the study. METHODS: RNA-protein interaction was determined by reversible crosslink co-immunoprecipitation (RC-Co-IP) and biolayer Interferometry assay (BLI). eEF1A binding region within RNA was mapped by deletion and mutation analysis. Virus with genomic RNA mutations were examined for eEF1A-RT interaction by proximity ligation assay, for reverse transcription by qPCR and for replication by CAp24 ELISA in cells. RESULTS: The interaction of eEF1A with 5'UTR of HIV-1 genomic RNA was detected in cells and in vitro. Truncation and substitution mutations in the 5'UTR RNA demonstrated that a stem-loop formed by nucleotides 142 to 170, which encompass a reported tRNA anticodon-like-element, binds to eEF1A. Mutations that altered the stem-loop structure by changing two highly conserved sequence clusters in the stem-loop region result in reduction of the interaction with eEF1A in vitro. HIV-1 virus harbouring the same 5'UTR mutations significantly reduced the interaction of eEF1A with HIV-1 reverse transcription complex (RTC), reverse transcription and replication. CONCLUSION: eEF1A interacts with 5'UTR of HIV-1 genomic RNA and the interaction is important for late DNA synthesis in reverse transcription.

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors.

Cellular proteins have been implicated as important for HIV-1 reverse transcription, but whether any are reverse transcription complex (RTC) cofactors or affect reverse transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous reverse transcription assay to identify cellular proteins that stimulated late steps of reverse transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as reverse transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late reverse transcription in vitro. We observed that the p51 subunit of reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with reverse transcriptase following infection of cells. Reverse transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of reverse transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of reverse transcription and trafficking of the RTC to the nucleus.

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.

BACKGROUND: Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. RESULTS: To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. CONCLUSIONS: Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev's activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function.

Overexpression of PRMT6 does not suppress HIV-1 Tat transactivation in cells naturally lacking PRMT6.

BACKGROUND: Protein arginine methyltransferase 6 (PRMT6) can methylate the HIV-1 Tat, Rev and nucleocapsid proteins in a manner that diminishes each of their functions in in vitro assays, and increases the stability of Tat in human cells. In this study, we explored the relationship between PRMT6 and HIV-1 Tat by determining the domains in each protein required for interaction. METHODS: Through domain mapping and immunoprecipitation experiments, we determined that both the amino and carboxyl termini of PRMT6, and the activation domain within Tat are essential for interaction. Mutation of the basic domain of Tat did not affect the ability of PRMT6 to interact with Tat. RESULTS: We next used the A549 human alveolar adenocarcinoma cell line, which naturally expresses undetectable levels of PRMT6, as a model for testing the effects of PRMT6 on Tat stability, transactivation, and HIV-1 replication. As previously observed, steady state levels and the protein half-life of Tat were increased by the ectopic expression of PRMT6. However, no down regulation of Tat transactivation function was observed, even with over 300-fold molar excess of PRMT6 plasmid. We also observed no negative effect on HIV-1 infectivity when A549 producer cells overexpressed PRMT6. CONCLUSIONS: We show that PRMT6 requires the activation domain, but surprisingly not the basic domain, of Tat for protein interaction. This interaction between Tat and PRMT6 may impact upon pathogenic effects attributed to Tat during HIV-1 infection other than its function during transactivation.

Defective Interfering Particles with Broad-Acting Antiviral Activity for Dengue, Zika, Yellow Fever, Respiratory Syncytial and SARS-CoV-2 Virus Infection.

More than 100 arboviruses, almost all of which have an RNA genome, cause disease in humans. RNA viruses are causing unprecedented health system challenges worldwide, many with little or no specific therapies or vaccines available. Certain species of mosquito can carry dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV), where co-infection of these viruses has occurred. Here, we found that purified synthetic defective interfering particles (DIPs) derived from DENV type 2 (DENV-2) strongly suppressed replication of the aforementioned viruses, respiratory syncytial virus (RSV) and also the novel emerging virus SARS-CoV-2 in human cells. DENV DIPs produced in bioreactors, purified by column chromatography, and concentrated are virus-like particles that are about half the diameter of a typical DENV particle, but with similar ratios of the viral structural proteins envelope and capsid. Overall, DIP-treated cells inhibited DENV, ZIKV, YFV, RSV, and SARS-CoV-2 by at least 98% by mechanisms which included interferon (IFN)-dependent cellular antiviral responses. IMPORTANCE DIPs are spontaneously derived virus mutants with deletions in genes that block viral replication. DIPs play important roles in modulation of viral disease, innate immune responses, virus persistence and virus evolution. Here, we investigated the antiviral activity of highly purified synthetic DIPs derived from DENV, which were produced in bioreactors. DENV DIPs purified by column chromatography strongly inhibited five different RNA viruses, including DENV, ZIKV, YFV, RSV, and SARS-CoV-2 in human cells. DENV DIPs inhibited virus replication via delivery of a small, noninfectious viral RNA that activated cellular innate immunity, resulting in robust type 1 interferon responses. The work here presents a pathway for DIP production which is adaptable to Good Manufacturing Practice, so that their preclinical testing should be suitable for evaluation in subjects.

GBP5 Repression Suppresses the Metastatic Potential and PD-L1 Expression in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype because of its high metastatic potential. Immune evasion due to aberrant expression of programmed cell death ligand 1 (PD-L1) has also been reported recently in metastatic TNBC. However, the mechanism underlying metastatic progression and PD-L1 upregulation in TNBC is still largely unknown. Here, we found that guanylate binding protein 5 (GBP5) is expressed in higher levels in TNBC tissues than in non-TNBC and normal mammary tissues and serves as a poorer prognostic marker in breast cancer patients. Transwell cultivation indicated that GBP5 expression is causally related to cellular migration ability in the detected TNBC cell lines. Moreover, the computational simulation of the gene set enrichment analysis (GSEA) program against the GBP5 signature generated from its coexpression with other somatic genes in TNBC revealed that GBP5 upregulation may be associated with the activation of interferon gamma (IFN-γ)-responsive and NF-κB-related signaling cascades. In addition, we found that the coexpression of GBP5 with PD-L1 was significantly positive correlation in TNBC tissues. Robustly, our data showed that GBP5 knockdown in TNBC cells harboring a higher GBP5 level dramatically suppresses the number of migrated cells, the activity of IFN-γ/STAT1 and TNF-α/NF-κB signaling axes, and the expression of PD-L1. Importantly, the signature combining a higher GBP5 and PD-L1 level predicted the shortest time interval of brain metastasis in breast cancer patients. These findings not only uncover the oncogenic function of GBP5 but also provide a new strategy to combat metastatic/immunosuppressive TNBC by targeting GBP5 activity.

Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity.

Dengue virus (DENV) is spread from human to human through the bite of the female Aedes aegypti mosquito and leads to about 100 million clinical infections yearly. Treatment options and vaccine availability for DENV are limited. Defective interfering particles (DIPs) are considered a promising antiviral approach but infectious virus contamination has limited their development. Here, a DENV-derived DIP production cell line was developed that continuously produced DENV-free DIPs. The DIPs contained and could deliver to cells a DENV serotype 2 subgenomic defective-interfering RNA, which was originally discovered in DENV infected patients. The DIPs released into cell culture supernatant were purified and could potently inhibit replication of all DENV serotypes in cells. Antiviral therapeutics are limited for many viral infection. The DIP system described could be re-purposed to make antiviral DIPs for many other RNA viruses such as SARS-CoV-2, yellow fever, West Nile and Zika viruses.

Arginine methylation increases the stability of human immunodeficiency virus type 1 Tat.

Arginine methylation of human immunodeficiency virus type 1 (HIV-1) Tat protein downregulates its key function in viral-gene transactivation. The fate of methylated Tat is unknown, so it is unclear whether methylated Tat is degraded or persists in the cell for additional functions. Here we show that the arginine methyltransferase PRMT6 increases Tat protein half-life by 4.7-fold. Tat stabilization depends on the catalytic activity of PRMT6 and requires arginine methylation within the Tat basic domain. In contrast, HIV-1 Rev, which is also methylated by PRMT6, is completely refractory to the stabilizing effect. Proteasome inhibition and silencing experiments demonstrated that Tat can be degraded by a REGgamma-independent proteasome, against which PRMT6 appears to act to increase Tat half-life. Our data reveal a proteasome-dependent Tat degradation pathway that is inhibited by arginine methylation. The stabilizing action of PRMT6 could allow Tat to persist within the cell and the extracellular environment and thereby enable functions implicated in AIDS-related cancer, neurodegeneration, and T-cell death.

Tat-Based Therapies as an Adjuvant for an HIV-1 Functional Cure.

The human immunodeficiency virus type 1 (HIV) establishes a chronic infection that can be well controlled, but not cured, by combined antiretroviral therapy (cART). Interventions have been explored to accomplish a functional cure, meaning that a patient remains infected but HIV is undetectable in the blood, with the aim of allowing patients to live without cART. Tat, the viral transactivator of transcription protein, plays a critical role in controlling HIV transcription, latency, and viral rebound following the interruption of cART treatment. Therefore, a logical approach for controlling HIV would be to block Tat. Tackling Tat with inhibitors has been a difficult task, but some recent discoveries hold promise. Two anti-HIV proteins, Nullbasic (a mutant of Tat) and HT1 (a fusion of HEXIM1 and Tat functional domains) inhibit viral transcription by interfering with the interaction of Tat and cellular factors. Two small molecules, didehydro-cortistatin A (dCA) and triptolide, inhibit Tat by different mechanisms: dCA through direct binding and triptolide through enhanced proteasomal degradation. Finally, two Tat-based vaccines under development elicit Tat-neutralizing antibodies. These vaccines have increased the levels of CD4+ cells and reduced viral loads in HIV-infected people, suggesting that the new vaccines are therapeutic. This review summarizes recent developments of anti-Tat agents and how they could contribute to a functional cure for HIV.

Expression of Rta in B Lymphocytes during Epstein-Barr Virus Latency.

Rta of Epstein-Barr virus (EBV) is thought to be expressed only during the lytic cycle to promote the transcription of lytic genes. However, we found that Rta is expressed in EBV-infected B cells during viral latency, at levels detectable by immunoblot analysis. Latent Rta expression cannot be attributed to spontaneous lytic activation, as we observed that more than 90% of Akata, P3HR1, and 721 cells latently infected by EBV express Rta. We further found that Rta is sequestered in the nucleolus during EBV latency through its interaction with MCRS2, a nucleolar protein. When Rta is sequestered in the nucleolus, it no longer activates RNA polymerase II-driven transcription, thus explaining why Rta expression during latency does not transactivate EBV lytic genes. Additional experiments showed that Rta can bind to 18S rRNA and become incorporated into ribosomes, and a transient transfection experiment showed that Rta promotes translation from an mRNA reporter. These findings reveal that Rta has novel functions beyond transcriptional activation during EBV latency and may have interesting implications for the concept of EBV latency.

DNA polymerase theta repression enhances the docetaxel responsiveness in metastatic castration-resistant prostate cancer.

OBJECTIVE: Docetaxel remains a main treatment for metastatic castration-resistant prostate cancer (mCRPC); however, the development of docetaxel resistance has been found in some mCRPC patients. The aim of this work is to identify an effective biomarker for predicting therapeutic effectiveness of docetaxel in mCRPC patients. METHODS: We examined DNA polymerase theta (POLQ) expression in The Cancer Genome Atlas (TCGA) database and Tissue microarray. Kaplan-Meier analyses were performed to estimate the prognostic significance of POLQ. A series of functional analyses were conducted in cell lines and xenograft models. Regulated pathways were predicted by Geneset Enrichment Analysis (GSEA) software and further investigated by luciferase reporter and RT-PCR assays. RESULTS: We found that POLQ mRNA levels in CRPC tissues was significantly higher than that of other DNA polymerases in non-CRPC prostate tissues. POLQ upregulation was extensively detected in mCRPC and strongly predicted a poor prognosis. POLQ knockdown enhanced docetaxel sensitivity in a cell-based cytotoxicity assay and promoted the therapeutic effect on the tumor growth of metastatic PC-3M cells in xenograft models. The computational simulation by GSEA software significantly predicted the association between POLQ upregulation and the activation of E2F/G2M checkpoint-related pathways. Moreover, luciferase reporter and RT-PCR assays demonstrated that POLQ knockdown downregulated the transcriptional regulatory activity of E2F and repressed E2F/G2M checkpoint-regulated CDK1 in mCRPC cells. CONCLUSION: Our results suggest that POLQ serves as a predictive factor for poor docetaxel response and provide a novel strategy to enhance the anticancer effects of docetaxel therapy by targeting POLQ in mCRPC patients.