RNA Helicase DDX6 Regulates A-to-I Editing and Neuronal Differentiation in Human Cells.

The DEAD-box proteins, one family of RNA-binding proteins (RBPs), participate in post-transcriptional regulation of gene expression with multiple aspects. Among them, DDX6 is an essential component of the cytoplasmic RNA processing body (P-body) and is involved in translational repression, miRNA-meditated gene silencing, and RNA decay. In addition to the cytoplasmic function, DDX6 is also present in the nucleus, but the nuclear function remains unknown. To decipher the potential role of DDX6 in the nucleus, we performed mass spectrometry analysis of immunoprecipitated DDX6 from a HeLa nuclear extract. We found that adenosine deaminases that act on RNA 1 (ADAR1) interact with DDX6 in the nucleus. Utilizing our newly developed dual-fluorescence reporter assay, we elucidated the DDX6 function as negative regulators in cellular ADAR1p110 and ADAR2. In addition, depletion of DDX6 and ADARs results in the opposite effect on facilitation of RA-induced differentiation of neuronal lineage cells. Our data suggest the impact of DDX6 in regulation of the cellular RNA editing level, thus contributing to differentiation in the neuronal cell model.

Evolving trends in the prevalence of hepatitis C virus antibody positivity among HIV-infected men in a community-based primary care setting.

Hepatitis C virus (HCV) infections in the United States occurred mostly among those born between 1945 and 1965. However, new infections continue to increase in recent years. To understand the changes in the prevalence and risk factors of HCV infection in different age and risk groups among men living with HIV, we performed a retrospective cross-sectional analyses of 1948 HIV-infected men at a multisite community health centre in urban/suburban and rural Maryland from 2003 through 2014. We used multivariate logistic regression to determine factors associated with HCV antibody (anti-HCV) positivity and restricted cubic spline method to model trends in anti-HCV prevalence over time. The overall anti-HCV prevalence was 24.2%. The annual prevalence declined in the full cohort, from 38% in 2003 to 24% in 2014, and among those ≥ 40 years old. However, the annual prevalence increased initially and then stabilized in the groups of men who were younger (<40 years old) or had injection-drug use and/or sex with men. Among the younger injection-drug users, the prevalence rose from 33% in 2003 to 79% in 2009 and then stabilized. The independent predictors for anti-HCV positivity differed between the men with and without injection-drug use and between those < 40 and ≥ 40 years old. Notably, a high prevalence of anti-HCV was observed among the younger, white injection-drug users residing in rural areas. Thus, the HCV epidemic continued unabated among high-risk individuals in this diverse population of HIV-infected men. The ongoing HCV transmission among young HIV-infected men poses a challenge en route to HCV eradication.

Dendritic cells and hepatocytes use distinct pathways to process protective antigen from plasmodium in vivo.

Malaria-protective CD8+ T cells specific for the circumsporozoite (CS) protein are primed by dendritic cells (DCs) after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2K(b) epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d) and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS) motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase.

The interplay among HIV, monocytes/macrophages, and extracellular vesicles: a systematic review.

Despite effective antiretroviral therapies, chronic inflammation and spontaneous viral "blips" occur in HIV-infected patients. Given the roles for monocytes/macrophages in HIV pathogenesis and extracellular vesicles in intercellular communication, we performed this systematic review to delineate the triad of HIV, monocytes/macrophages, and extracellular vesicles in the modulation of immune activation and HIV activities. We searched PubMed, Web of Science, and EBSCO databases for published articles, up to 18 August 2022, relevant to this triad. The search identified 11,836 publications, and 36 studies were deemed eligible and included in this systematic review. Data were extracted for the characteristics of HIV, monocytes/macrophages, and extracellular vesicles used for experiments and the immunologic and virologic outcomes in extracellular vesicle recipient cells. Evidence for the effects on outcomes was synthesized by stratifying the characteristics by outcomes. In this triad, monocytes/macrophages were potential producers and recipients of extracellular vesicles, whose cargo repertoires and functionalities were regulated by HIV infection and cellular stimulation. Extracellular vesicles derived from HIV-infected monocytes/macrophages or the biofluid of HIV-infected patients enhanced innate immune activation and HIV dissemination, cellular entry, replication, and latency reactivation in bystander or infected target cells. These extracellular vesicles could be synthesized in the presence of antiretroviral agents and elicit pathogenic effects in a wide range of nontarget cells. At least eight functional types of extracellular vesicles could be classified based on the diverse extracellular vesicle effects, which were linked to specific virus- and/or host-derived cargos. Thus, the monocyte/macrophage-centered multidirectional crosstalk through extracellular vesicles may help sustain persistent immune activation and residual viral activities during suppressed HIV infection.

Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.

The association between neutrophil extracellular traps (NETs) and response to inhaled corticosteroids (ICS) in asthma is unclear. To better understand this relationship, we analyzed the blood transcriptomes from children with controlled and uncontrolled asthma in the Taiwanese Consortium of Childhood Asthma Study using weighted gene coexpression network analysis and pathway enrichment methods. We identified 298 uncontrolled asthma-specific differentially expressed genes and one gene module associated with neutrophil-mediated immunity, highlighting a potential role for neutrophils in uncontrolled asthma. We also found that NET abundance was associated with nonresponse to ICS in patients. In a neutrophilic airway inflammation murine model, steroid treatment could not suppress neutrophilic inflammation and airway hyperreactivity. However, NET disruption with deoxyribonuclease I (DNase I) efficiently inhibited airway hyperreactivity and inflammation. Using neutrophil-specific transcriptomic profiles, we found that CCL4L2 was associated with ICS nonresponse in asthma, which was validated in human and murine lung tissue. CCL4L2 expression was also negatively correlated with pulmonary function change after ICS treatment. In summary, steroids fail to suppress neutrophilic airway inflammation, highlighting the potential need to use alternative therapies such as leukotriene receptor antagonists or DNase I that target the neutrophil-associated phenotype. Furthermore, these results highlight CCL4L2 as a potential therapeutic target for individuals with asthma refractory to ICS.

Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites.

Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization--a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

A protein concentration measurement system using a flexural plate-wave frequency-shift readout technique.

A protein concentration measurement system with two-port flexural plate-wave (FPW) biosensors using a frequency-shift readout technique is presented in this paper. The proposed frequency-shift readout method employs a peak detecting scheme to measure the amount of resonant frequency shift. The proposed system is composed of a linear frequency generator, a pair of peak detectors, two registers, and a subtractor. The frequency sweep range of the linear frequency generator is limited to 2 MHz to 10 MHz according to the characteristics of the FPW biosensors. The proposed frequency-shift readout circuit is carried out on silicon using a standard 0.18 µm CMOS technology. The sensitivity of the peak detectors is measured to be 10 mV. The power consumption of the proposed protein concentration measurement system is 48 mW given a 0.1 MHz system clock.

Protective CD8 T cells against Plasmodium liver stages: immunobiology of an 'unnatural' immune response.

SUMMARY: Immunization with high doses of irradiated sporozoites delivered by the bites of infected mosquitoes has been shown to induce protective responses against malaria, mediated in part by CD8(+) T cells. In contrast, natural transmission involving low exposure to live sporozoite antigen fails to elicit strong immunity. In this review, we examine how irradiated sporozoite immunization breaks the natural host-parasite interaction and induces protective CD8(+) T cells. Upon biting, the malaria-infected mosquitoes deposit parasites in the skin, many of which eventually exit to the bloodstream and infect hepatocytes. However, certain antigens, including the circumsporozoite (CS) protein, remain in the skin and are presented in the draining lymph node. These antigens prime specific CD8(+) T cells, which migrate to the liver where they eliminate parasitized hepatocytes. We discuss the relevance of the different tissue compartments involved in the induction and effector phases of this response, as well as the cellular requirements for priming and memory development of CD8(+) T cells, which include a complete dependence on dendritic cells and a near absolute need for CD4(+) T-cell help. Finally, we discuss the impact of the immunodominant CS protein on this protection and the implications of these findings for vaccine design.

The Role of Zn Substitution in Improving the Electrical Properties of CuI Thin Films and Optoelectronic Performance of CuI MSM Photodetectors.

Pure CuI and Zn-substituted CuI (CuI:Zn) semiconductor thin films, and metal-semiconductor-metal (MSM) photodetectors were fabricated on glass substrates by a low-temperature solution process. The influence of Zn substitution concentration (0-12 at%) on the microstructural, optical, and electrical characteristics of CuI thin films and its role in improving the optoelectronic performance of CuI MSM photodetectors were investigated in this study. Incorporation of Zn cation dopant into CuI thin films improved the crystallinity and increased the average crystalline size. XPS analysis revealed that the oxidation state of Cu ions in all the CuI-based thin films was +1, and the estimated values of [Cu]/[I] for the CuI:Zn thin films were lower than 0.9. It was found that the native p-type conductivity of polycrystalline CuI thin film was converted to n-type conductivity after the incorporation of Zn ions into CuI nanocrystals, and the electrical resistivity decreased with increases in Zn concentration. A time-resolved photocurrent study indicated that the improvements in the optoelectronic performance of CuI MSM photodetectors were obtained through the substitution of Zn ions, which provided operational stability to the two-terminal optoelectronic device. The 8 at% Zn-substituted CuI photodetectors exhibited the highest response current, responsivity, and EQE, as well as moderate specific detectivity.

CpG-enhanced CD8+ T-cell responses to peptide immunization are severely inhibited by B cells.

Synthetic peptides encoding protective pathogen-derived epitopes represent--in principle--an ideal approach to T-cell vaccination. Empirically, however, these strategies have not been successful. In the current study, we profiled the early activation of CD8+ T cells by MHC class I-restricted peptide immunization to better understand the biology of this response. We found that CD8+ T cells proliferated robustly in response to low doses of short synthetic peptides in PBS, but failed to acquire effector function or form memory populations in the absence of the TLR ligand CpG. CpG was unique among TLR ligands in its ability to enhance the response to peptide and its adjuvant effects had strict temporal requirements. Interestingly, CpG treatment modulated T-cell expression of the surface receptors PD-1 and CD25, providing insight into its possible adjuvant mechanism. The effects of CpG on peptide immunization were dramatically enhanced in the absence of B cells, demonstrating a unique system of regulation of T-cell responses by these lymphocytes. The results reported here provide insight into the complex response to a simple vaccination regimen, as well as a framework for a rational peptide-based vaccine design to both exploit and overcome targeted aspects of the immune response.

Trends in hepatitis C treatment initiation among HIV/hepatitis C virus-coinfected men engaged in primary care in a multisite community health centre in Maryland: a retrospective cohort study.

OBJECTIVES: Little is known about the cascade of hepatitis C care among HIV/hepatitis C virus (HCV)-coinfected patients in community-based clinics. Thus, we analysed our data from the interferon era to understand the barriers to HCV treatment, which may help improve getting patients into treatment in the direct-acting antivirals era. DESIGN: Retrospective cohort study. SETTING: Four HIV clinics of a multisite community health centre in the USA. PARTICIPANTS: 1935 HIV-infected men with >1 medical visit to the clinic between 2011 and 2013. Of them, 371 had chronic HCV and were included in the analysis for HCV care continuum during 2003-2014. OUTCOME MEASURES: HCV treatment initiation was designated as the primary outcome for analysis. Multivariate logistic regression was performed to identify factors associated with HCV treatment initiation. RESULTS: Among the 371 coinfected men, 57 (15%) initiated HCV treatment. Entering care before 2008 (adjusted OR [aOR, 3.89; 95% CI, 1.95 to 7.78), higher educational attainment (aOR, 3.20; 95% CI, 1.59 to 6.44), HCV genotype 1 versus non-1 (aOR, 0.21; 95% CI, 0.07 to 0.65) and HIV suppression (aOR, 2.13; 95% CI, 1.12 to 4.06) independently predicted treatment initiation. Stratification by entering care before or after 2008 demonstrated that higher educational attainment was the only factor independently associated with treatment uptake in both periods (aOR, 2.79; 95% CI, 1.13 to 6.88 and aOR, 4.10; 95% CI, 1.34 to 12.50, pre- and post-2008, respectively). Additional associated factors in those entering before 2008 included HCV genotype 1 versus non-1 (aOR, 0.09; 95% CI, 0.01 to 0.54) and HIV suppression (aOR, 2.35; 95% CI, 1.04 to 5.33). CONCLUSIONS: Some traditional barriers predicted HCV treatment initiation in those in care before 2008; however, the patients' level of educational attainment remained an important factor even towards the end of the interferon era. Further studies will need to determine whether educational attainment persists as an important determinant for initiating direct-acting antiviral therapies.

Favorable Socioeconomic Status and Recreational Polydrug Use Are Linked With Sexual Hepatitis C Virus Transmission Among Human Immunodeficiency Virus-Infected Men Who Have Sex With Men.

Background. Sexual transmission of hepatitis C virus (HCV) among human immunodeficiency virus (HIV)-infected men who have sex with men (MSM) is an emerging issue. Studies addressing the temporal trends and risk factors associated with incident HCV in HIV-infected MSM in the community-based primary care settings in the United States are scarce. Methods. Using a retrospective cohort study design, HCV incidence, defined as HCV antibody seroconversion, was determined in 1147 HIV-infected men receiving care at Chase Brexton Health Care clinics in Baltimore, Maryland between 2004 and 2014. Multivariate regression analyses were used to identify factors associated with incident HCV. Results. There were 42 incident HCV infections during 5242 person-years (PY) of follow up (incidence rate [IR], 8.01/1000 PY). Thirty-seven (88%) of the incident infections were in MSM, of whom 31 (84%) reported no injection-drug use (IDU). The annual IRs for MSM were 13.1-15.8/1000 PY between 2004 and 2007, decreased to 2.7-6.2/1000 PY between 2008 and 2011, and increased to 10.4/1000 PY and 13.3/1000 PY in 2013 and 2014, respectively. Injection-drug use was strongly associated with incident HCV among all MSM (IR ratio [IRR], 14.15; P = .003); however, among MSM without IDU, entering care between 2010 and 2013 (IRR, 3.32; P = .01), being employed (IRR, 3.14; P = .03), and having a history of ulcerative sexually transmitted infections (IRR, 3.70; P = .009) or of polydrug use (IRR, 5.54; P = .01) independently predicted incident HCV. Conclusions. In this cohort of HIV-infected men, a re-emerging HCV epidemic was observed from 2011 to 2014 among MSM. In addition to IDU, high-risk sexual behaviors, favorable socioeconomic status, and polydrug use fueled this increase in HCV infections.

Clinicopathological Variation of Lauren Classification in Gastric Cancer.

The investigation of prognostic factor for gastric cancer is still desirable because of dismal prognosis in gastric cancer. Lauren's classification is currently a useful histological classification. There are few large series evaluating the prognostic significance of Lauren's classification in gastric cancer. From January 1987 to December 2013, a total of 3071 patients received gastrectomy for gastric cancer. According Lauren's classification, 1423(46.3%) patients were intestinal type, 1000 patients (32.6%) were diffuse type, and 648 patients (21.1%) were mixed type. The clinicopathological characteristics and prognosis in Lauren's classification were analyzed in these patients. Our results showed that patients with intestinal type gastric cancer (57.7%) had a better 5-year overall survival than diffuse type (45.6%) and mixed type (43.4%, P < 0.001). The clinicopathological characteristics showed that gastric cancer patients with intestinal type were older (P < 0.001), male predominant (P < 0.001), smaller tumor size (P < 0.001), distal stomach predominant (P < 0.001), relative well differentiated (P < 0.001), less advanced Borrmann type (P < 0.001), less scirrhous type stromal reaction(P < 0.001), less infiltrating type of Ming's histology type(P < 0.001), less tumor invasion depth and less lymphovascular invasion (P < 0.001). Multivariate analysis with overall survival as an endpoint showed that age (P = 0.005), Borrmann classification (P < 0.001), pathological T category (P = 0.023), pathological N category (P < 0.001) and Lauren's classification (P = 0.003) were significant correlated in gastric cancer. Lauren's classification is an independent prognostic factor in gastric cancer patient undergoing gastrectomy. Lauren's classification can serve as a prognostic marker for gastric cancer patient receiving gastrectomy. The clinicopathological appearance and prognosis of mixed type gastric cancer is similar to diffuse type gastric cancer.

Development and use of TCR transgenic mice for malaria immunology research.

T-cell receptor transgenic mice are powerful tools to study T cell responses to malaria parasites. They allow for a population of antigen specific T cells to be monitored during developing responses to immunization or parasite infection; this makes them particularly useful to study fundamental aspects of T cell activation, differentiation, and migration in different tissue compartments. Moreover, the use of these cells allows for a thorough analysis of the mechanisms of antiparasite activity by T cells.

Chaperon solvent plug design in size-exclusion chromatography protein refolding process.

Although the chaperon solvent plug was reported as a strategy to reduce aggregation before the column inlet in SEC (size-exclusion chromatography) protein refolding process, the appropriate position at which sample injected and the volume of the chaperon solvent plug have not been elucidated. Therefore, the detail of chaperon solvent plug design was investigated in this work. Our results indicated that, to ensure good performances in the SEC refolding process, the appropriate front and tail volumes of chaperon solvent plug should be slightly larger than the optimal values, which depend on the flow dispersion from the injector to the column inlet. However, with the front volume more than the optimum, it could have an adverse effect on activity recovery but not the mass recovery, while no effect at all if the tail volume exceeded the optimum. Furthermore, it might be economical to replace the eluent (refolding buffer) after the tail of chaperon solvent plug with a cheaper one.

CD4+ T cells modulate expansion and survival but not functional properties of effector and memory CD8+ T cells induced by malaria sporozoites.

CD4(+) helper T cells are critical orchestrators of immune responses to infection and vaccination. During primary responses, naïve CD8(+) T cells may need "CD4 help" for optimal development of memory populations. The immunological factors attributed to CD4 help depend on the context of immunization and vary depending on the priming system. In response to immunization with radiation-attenuated Plasmodium yoelii sporozoites, CD8(+) T cells in BALB/c mice fail to generate large numbers of effector cells without help from CD4(+) T cells--a defect not observed in most systems. Given this unique early dependence on CD4 help, we evaluated the effects of CD4(+) cells on the development of functional properties of CD8(+) T cells and on their ability to abolish infection. First, we determined that this effect was not mediated by CD4(+) non-T cells and did not involve CD1d-restricted NKT cells. We found that CD8(+) T cells induced by sporozoites without CD4 help formed memory populations severely reduced in magnitude that could not limit parasite development in the liver. The inability of these "helpless" memory T cells to protect is not a result of defects in effector function, as their capacity to produce cytokines and undergo cytotoxic degranulation was indistinguishable from control memory T cells. These data indicate that CD4(+) T help may not be necessary to develop the functional attributes of CD8(+) T cells; however they are crucial to ensure the survival of effector and memory cells induced in primary responses.

Activation of terminally differentiated human monocytes/macrophages by dengue virus: productive infection, hierarchical production of innate cytokines and chemokines, and the synergistic effect of lipopolysaccharide.

Dengue virus (DV) primarily infects blood monocytes (MO) and tissue macrophages (M phi). We have shown in the present study that DV can productively infect primary human MO/M phi regardless of the stage of cell differentiation. After DV infection, the in vitro-differentiated MO/M phi secreted multiple innate cytokines and chemokines, including tumor necrosis factor alpha, alpha interferon (IFN-alpha), interleukin-1 beta (IL-1 beta), IL-8, IL-12, MIP-1 alpha, and RANTES but not IL-6, IL-15, or nitric oxide. Secretion of these mediators was highlighted by distinct magnitude, onset, kinetics, duration, and induction potential. A chemokine-to-cytokine hierarchy was noted in the magnitude and induction potential of secretion, and a chemokine-to-cytokine-to-chemokine/Th1 cytokine cascade could be seen in the production kinetics. Furthermore, we found that terminally differentiated MO/M phi cultured for more than 45 days could support productive DV infection and produce innate cytokines and chemokines, indicating that these mature cells were functionally competent in the context of a viral infection. In addition, DV replication in primary differentiated human MO/M phi was enhanced and prolonged in the presence of lipopolysaccharide (LPS), and LPS-mediated synergistic production of IFN-alpha could be seen in DV-infected MO/M phi. The secretion of innate cytokines and chemokines by differentiated MO/M phi suggests that regional accumulation of these mediators may occur in various tissues to which DV has disseminated and may thus result in local inflammation. The LPS-mediated enhancement of virus replication and synergistic IFN-alpha production suggests that concurrent bacterial infection may modulate cytokine-mediated disease progression during DV infection.