Cumulative effect of unemployment on suicide mortality in South Korean workers (2018-2019).

BACKGROUND: While unemployment is known to increase the risk of suicide, its cumulative effect remains underexplored. This study investigates how unemployment affects suicide mortality and whether the effect varies based on the number of unemployment spells using two years of nationwide data. METHODS: Using the data from the National Statistical Office and Employment Insurance Database for 2018 and 2019, we identified an average of 2365 cases of suicide over two years among 7.76 million workers aged 25-64 years who had been employed within one year before their suicide. The number of unemployment spells was counted using the employment history of the past five years. We calculated crude suicide mortality rates per 100 000 population, age- and sex- standardized mortality rates (SMRs), and proportionate mortality rates (PMRs) for suicide. RESULTS: Over the two years, the crude suicide rate was 30.0 per 100 000 among the general population and 30.5 among workers. Workers with no unemployment spells in the past five years had a significantly lower SMR (0.44; 0.42-0.46), while those with four or more unemployment spells had a significantly higher SMR (3.13; 2.92-3.35) than the general population. These findings were consistent across all sex and age groups. Additionally, workers with four or more unemployment spells had a significantly higher PMR than the general population. CONCLUSION: The impact of unemployment on suicide mortality intensifies as the number of unemployment spells increases. These results underscore the necessity for additional social and psychological support along with economic assistance for individuals facing recurrent unemployment.

Formation of nuclear CPSF6/CPSF5 biomolecular condensates upon HIV-1 entry into the nucleus is important for productive infection.

The early events of HIV-1 infection involve the transport of the viral core into the nucleus. This event triggers the translocation of CPSF6 from paraspeckles into nuclear speckles forming puncta-like structures. Our investigations revealed that neither HIV-1 integration nor reverse transcription is required for the formation of puncta-like structures. Moreover, HIV-1 viruses without viral genome are competent for the induction of CPSF6 puncta-like structures. In agreement with the notion that HIV-1 induced CPSF6 puncta-like structures are biomolecular condensates, we showed that osmotic stress and 1,6-hexanediol induced the disassembly of CPSF6 condensates. Interestingly, replacing the osmotic stress by isotonic media re-assemble CPSF6 condensates in the cytoplasm of the cell. To test whether CPSF6 condensates were important for infection we utilized hypertonic stress, which prevents formation of CPSF6 condensates, during infection. Remarkably, preventing the formation of CPSF6 condensates inhibits the infection of wild type HIV-1 but not of HIV-1 viruses bearing the capsid changes N74D and A77V, which do not form CPSF6 condensates during infection1,2. We also investigated whether the functional partners of CPSF6 are recruited to the condensates upon infection. Our experiments revealed that CPSF5, but not CPSF7, co-localized with CPSF6 upon HIV-1 infection. We found condensates containing CPSF6/CPSF5 in human T cells and human primary macrophages upon HIV-1 infection. Additionally, we observed that the integration cofactor LEDGF/p75 changes distribution upon HIV-1 infection and surrounds the CPSF6/CPSF5 condensates. Overall, our work demonstrated that CPSF6 and CPSF5 are forming biomolecular condensates that are important for infection of wild type HIV-1 viruses.

Early impact of COVID-19 social distancing on social determinants of health and their effects on mental health and quality of life of Korean undergraduate students.

Introduction: This study aimed to investigate the association between social determinants of health and perception of COVID-19 social distancing/mental health/quality of life during COVID-19 social distancing in Korean undergraduate students using online survey data augmented with natural language processing. Methods: An online cross-sectional survey including sociodemographic characteristics, students' perceptions of COVID-19 social distancing, and social determinants of health was conducted between July and November in 2020. We conducted logistic regression analysis to investigate the relationship between social determinants of health (independent variables) and perceptions of COVID-19 social distancing, mental health, and quality of life (dependent variables). This association was augmented using sentiment analysis and word clouds by visualizing open-ended comments on COVID-19 social-distancing policies. Results: Data were collected from 1,276 undergraduate students. Participants who experienced negative impacts on their social-networking activities due to COVID-19 social distancing were at significantly higher odds to perceive COVID-19 social distancing as not being beneficial [odds ratio (OR) = 1.948, 95% confidence interval (CI) 1.254-3.027], to have increased stress levels (OR = 1.619, 95% CI 1.051-2.496), and to experience decreased quality of life over 5 weeks (OR = 2.230, 95% CI 1.448-3.434) against those who answered neutrally. In contrast, Participants who reported positive perceptions of social-networking activities during the COVID-19 pandemic had lower odds of feeling depressed or anxious (OR = 0.498, 95% CI 0.278-0.894) and reporting a low quality of life over 5 weeks (OR = 0.461, 95% CI 0.252-0.842) compared to those who reported neutral perceptions. Furthermore, the results of the word cloud and sentiment analyses showed that most students perceived social distancing negatively. Conclusions: The government's social-distancing policy to prevent the spread of COVID-19 may have had a negative impact, particularly on undergraduate students' social-networking activities. This highlights the need for greater social support for this population, including access to psychotherapeutic resources, and improvements in policies to prevent infectious diseases while still maintaining social connections.

The HIV-1 capsid core is an opportunistic nuclear import receptor.

The movement of viruses and other large macromolecular cargo through nuclear pore complexes (NPCs) is poorly understood. The human immunodeficiency virus type 1 (HIV-1) provides an attractive model to interrogate this process. HIV-1 capsid (CA), the chief structural component of the viral core, is a critical determinant in nuclear transport of the virus. HIV-1 interactions with NPCs are dependent on CA, which makes direct contact with nucleoporins (Nups). Here we identify Nup35, Nup153, and POM121 to coordinately support HIV-1 nuclear entry. For Nup35 and POM121, this dependence was dependent cyclophilin A (CypA) interaction with CA. Mutation of CA or removal of soluble host factors changed the interaction with the NPC. Nup35 and POM121 make direct interactions with HIV-1 CA via regions containing phenylalanine glycine motifs (FG-motifs). Collectively, these findings provide additional evidence that the HIV-1 CA core functions as a macromolecular nuclear transport receptor (NTR) that exploits soluble host factors to modulate NPC requirements during nuclear invasion.

Tetrazolium-Based Visually Indicating Bacteria Sensor for Colorimetric Detection of Point of Contamination.

Protective equipment for detecting bacterial contamination has been in high demand with increasing interest in public health and hygiene. Herein, a fiber-based visually indicating bacteria sensor (VIBS) embedded with iodonitrotetrazolium chloride is developed for the general purpose of detecting live bacteria, and its chromogenic effectiveness is investigated for Gram-negative Escherichia coli and Gram-positive Micrococcus luteus. The developed color intensity is measured by the light absorption coefficient to the scattering coefficient (K/S) based on the Kubelka-Munk equation, and the colorimetric sensitivities of different membranes are examined by calculating the limit of detection (LOD) and the limit of quantification (LOQ). The results demonstrate that the interactions between VIBS and bacteria depend on the wetting properties of membranes. A hydrophobic membrane shows excessive interactions at high concentrations of Gram-negative E. coli bacteria, whose cell membrane is lipophilic. The membrane blended with hydrophobic and hydrophilic polymers displays linear colorimetric responses for both Gram-negative and Gram-positive bacteria strains, demonstrating a reliable sensing capability in the range of the tested bacteria concentration. This study is significant in that explorative experimentations are performed to conceive a proof of concept of a fiber-based bacteria sensor, which is readily applicable in various fields where bacteria pose a threat.

Validation of the Patient Health Questionnaire-9 for Screening Depressive Disorders among Korean Employees: A Longitudinal Study of the National Health Examination Data.

This nationwide longitudinal study examined the screening utility of the Patient Health Questionnaire-9 (PHQ-9) for Korean workers (aged 20, 30, 40, 50, 60, and 70 years) who completed the questionnaire in 2018. Data on disease names and health-related behaviors were collected from the National Health Insurance Service (NHIS). Follow-up began on 1 January 2018, and the primary endpoint was the hospitalization date for depression, self-harm, or suicide or 31 December 2019. Of the 766,351 participants, 741,423 received depression screening. Those screened were classified into normal (n = 716,760) and high-risk groups (n = 24,663) based on PHQ-9 scores. The incidence of hospital admissions for depression, self-harm, or suicide in the non-screened, normal, and high-risk groups was analyzed, and the PHQ-9's validity was examined. There were more females in the high-risk group than in the normal group, and the income distribution differed. The two-year cumulative incidence was highest for the high-risk group (4.21%), followed by the normal (0.89%) and non-screened groups (0.80%). The PHQ-9's sensitivity was low (males: 14.2%; females: 13.8%). Its specificity for males and females was 97.1% and 96.3%, respectively. Our findings may help develop a system to prevent suicides and hospitalizations attributed to workplace depression.

GS-CA1 and lenacapavir stabilize the HIV-1 core and modulate the core interaction with cellular factors.

The HIV-1 capsid is the target for the antiviral drugs GS-CA1 and Lenacapavir (GS-6207). We investigated the mechanism by which GS-CA1 and GS-6207 inhibit HIV-1 infection. HIV-1 inhibition by GS-CA1 did not require CPSF6 in CD4+ T cells. Contrary to PF74 that accelerates uncoating of HIV-1, GS-CA1 and GS-6207 stabilized the core. GS-CA1, unlike PF74, allowed the core to enter the nucleus, which agrees with the fact that GS-CA1 inhibits infection after reverse transcription. Unlike PF74, GS-CA1 did not disaggregate preformed CPSF6 complexes in nuclear speckles, suggesting that PF74 and GS-CA1 have different mechanisms of action. GS-CA1 stabilized the HIV-1 core, possibly by inducing a conformational shift in the core; in agreement, HIV-1 cores bearing N74D regained their ability to bind CPSF6 in the presence of GS-CA1. We showed that GS-CA1 binds to the HIV-1 core, changes its conformation, stabilizes the core, and thereby prevents viral uncoating and infection.

Sequential Multiscale Simulation of a Filtering Facepiece for Prediction of Filtration Efficiency and Resistance in Varied Particulate Scenarios.

This study explores a novel approach of multiscale modeling and simulation to characterize the filtration behavior of a facepiece in varied particulate conditions. Sequential multiscale modeling was performed for filter media, filtering facepiece, and testing setup. The developed virtual models were validated for their morphological characteristics and filtration performance by comparing with the data from the physical experiments. Then, a virtual test was conducted in consideration of a time scale, simulating diverse particulate environments with different levels of particle size distribution, particle concentration, and face velocity. An environment with small particles and high mass concentration resulted in a rapid buildup of resistance, reducing the service life. Large particles were accumulated mostly at the entrance of the filter layer, resulting in a lower penetration and slower buildup of resistance. This study is significant in that the adopted virtual approach enables the prediction of filtration behavior and service life, applying diverse environmental conditions without involving the costs of extra setups for the physical experiments. This study demonstrates a novel and economic research method that can be effectively applied to the research and development of filters.

Effects of the filter microstructure and ambient air condition on the aerodynamic dispersion of sneezing droplets: A multiscale and multiphysics simulation study.

Concerns have been ramping up with regard to the propagation of infectious droplets due to the recent COVID-19 pandemic. The effects of filter microstructures and ambient air flows on droplet dispersion by sneezing are investigated by a fully coupled Eulerian-Lagrangian computational modeling with a micro-to-macroscale bridging approach. Materials that are commonly applied to face masks are modeled to generate two different virtual masks with various levels of filtration efficiency, and the leakage percentages through the unsealed nose and cheek areas were set to 11% and 25%, respectively. The droplet propagation distance was simulated with and without mask wearing in still and windy conditions involving head wind, tail wind, and side wind. The results demonstrate that wearing a face mask reduces the transmittance distance of droplets by about 90%-95% depending on the mask type; nonetheless, the droplets can be transmitted to distances of 20-25 cm in the forward direction even with mask-wearing. Thus, a social distance of at least 20 cm between people would help to prevent them from becoming exposed to ejected droplets. This study is significant in that important aspects of mask materials, in this case the porous microstructure-dependent filtration efficiency and permeability under varied ambient flow conditions, were considered for the first time in an evaluation of the barrier performance against droplet transmittance through a multiphase computational fluid dynamics simulation of air-droplet interaction and turbulence flow dynamics.

Fiber-Based Gas Filter Assembled via In Situ Synthesis of ZIF-8 Metal Organic Frameworks for an Optimal Adsorption of SO2: Experimental and Theoretical Approaches.

For environmental protection from exposure to airborne toxic gases, metal organic frameworks (MOFs) have drawn great attention as gas adsorbent options, with their advantages in chemical tailorability and large porosity. To develop a fiber-based gas filter that is effective against SO2 gas, zeolite imidazole framework-8 (ZIF-8) was applied to polypropylene nonwoven by various methods. Among the tested methods, the sol-gel impregnation method showed the highest ZIF-8 loading efficiency. There existed an optimal loading of ZIF-8 for the maximum adsorption efficiency, and it was associated with the accessibility of gas molecules to the ZIF-8 pores and active sites. Dominant adsorption processes and mechanisms were investigated by fitting the theoretical sorption models to experimental data. The results demonstrate that the increased ZIF-8 loading to fibers, beyond a certain level, may hinder the diffusivity and increase the barrier effect, eventually decreasing the adsorption efficiency. This study is novel and significant in that a multifaceted approach, including experimental analysis, theoretical investigation, and computational modeling, was made for scrutinizing the intricate phenomena occurring in the gas sorption process. The results of this study provide the fundamental yet practical information on the manufacturing considerations for the optimal design of MOF-loaded fibrous adsorbents.

Disinfection Treatments of Disposable Respirators Influencing the Bactericidal/Bacteria Removal Efficiency, Filtration Performance, and Structural Integrity.

In the outbreak of COVID-19, the extended wear of single-use, disposable respirators was inevitable due to limited supplies. As a respirator is front-line protection against particulate matter, including bioaerosol and droplets, a comprehensive understanding for the reuse strategy is needed. In this study, eight different disinfection methods commonly applied for the reuse of respirators were compared for their influence on the filtration and bactericidal/bacteria removal performance, with in-depth discussion on the cause of effects. Treatments including oven-dry, ultraviolet irradiation (UV), microwaving, laundering with and without detergent, and immersion in hypochlorite, isopropanol, and ethanol were performed to respirators. Immersion in ethanol or isopropanol was effective for inactivation and removal of bacteria, yet such a treatment significantly deteriorated the filtration efficiency in about 20-28%, dissipating the surface charges. Laundering, while effective in removing the attached bacteria, triggered physical damage, leading to a possible reduction of filtration performance. A short-term oven-dry, UV irradiation, and microwaving mostly preserved the filtration performance, yet the drawback lied in the incomplete bactericidal efficiency. This study would contribute to the public health and safety by providing scientific background on the effect of disinfection treatment methods for respirators.

CPSF6-Dependent Targeting of Speckle-Associated Domains Distinguishes Primate from Nonprimate Lentiviral Integration.

Lentiviral DNA integration favors transcriptionally active chromatin. We previously showed that the interaction of human immunodeficiency virus type 1 (HIV-1) capsid with cleavage and polyadenylation specificity factor 6 (CPSF6) localizes viral preintegration complexes (PICs) to nuclear speckles for integration into transcriptionally active speckle-associated domains (SPADs). In the absence of the capsid-CPSF6 interaction, PICs uncharacteristically accumulate at the nuclear periphery and target heterochromatic lamina-associated domains (LADs) for integration. The integrase-binding protein lens epithelium-derived growth factor (LEDGF)/p75 in contrast to CPSF6 predominantly functions to direct HIV-1 integration to interior regions of transcription units. Though CPSF6 and LEDGF/p75 can reportedly interact with the capsid and integrase proteins of both primate and nonprimate lentiviruses, the extents to which these different viruses target SPADs versus LADs, as well as their dependencies on CPSF6 and LEDGF/p75 for integration targeting, are largely unknown. Here, we mapped 5,489,157 primate and nonprimate lentiviral integration sites in HEK293T and Jurkat T cells as well as derivative cells that were knocked out or knocked down for host factor expression. Despite marked preferences of all lentiviruses to target genes for integration, nonprimate lentiviruses only marginally favored SPADs, with corresponding upticks in LAD-proximal integration. While LEDGF/p75 knockout disrupted the intragenic integration profiles of all lentiviruses similarly, CPSF6 depletion specifically counteracted SPAD integration targeting by primate lentiviruses. CPSF6 correspondingly failed to appreciably interact with nonprimate lentiviral capsids. We conclude that primate lentiviral capsid proteins evolved to interact with CPSF6 to optimize PIC localization for integration into transcriptionally active SPADs.IMPORTANCE Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on the factors that guide provirus formation and associated chromatin environments that regulate transcriptional reactivation, and studies of orthologous infectious agents such as nonprimate lentiviruses can inform basic principles of HIV biology. HIV-1 utilizes the integrase-binding protein LEDGF/p75 and the capsid interactor CPSF6 to target speckle-associated domains (SPADs) for integration. However, the extent to which these two host proteins regulate integration of other lentiviruses is largely unknown. Here, we mapped millions of retroviral integration sites in cell lines that were depleted for LEDGF/p75 and/or CPSF6. Our results reveal that primate lentiviruses uniquely target SPADs for integration in a CPSF6-dependent manner.

Experimental and Computational Investigation of Intra- and Interlayer Space for Enhanced Depth Filtration and Reduced Pressure Drop.

The buildup of pressure drop with mass loading of particles aggravates the breathing resistance and energy consumption of filters. This study investigated the role of intra- and interlayer space of filter media on the pressure drop development with continued particle loading. Five basic morphologies, including microfibers, nanofibers, microbeads-on-strings, and a mix of those morphologies were fabricated via electrospinning. Then the variations of layered constructions were made, to include a total 14 different filter structures. For a single layer filter media, the pore size rather than the percent porosity had a major impact on the pressure drop. For dual layers, the space between the layers and the placement order of webs were important factors affecting the pressure drop and depth loading of particles. Computational modeling was used to interpret the role of the interlayer space on the pressure drop, by monitoring the air flow and particle movement within the filter constructions, where the computational prediction corresponded to the tendency of the experimental findings. The novelty of this study lies in the combined approach of the experimental and computational work to understand the particle capture phenomenon during the mass loading.

Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating.

HIV-1 reverse transcription (RT) occurs before or during uncoating, but the cellular compartment where RT and uncoating occurs is unknown. Using imaging and biochemical assays to track HIV-1 capsids in the nucleus during infection, we demonstrated that higher-order capsid complexes and/or complete cores containing the viral genome are imported into the nucleus. Inhibition of RT does not prevent capsid nuclear import; thus, RT may occur in nuclear compartments. Cytosolic and nuclear fractions of infected cells reveal that most RT intermediates are enriched in nuclear fractions, suggesting that HIV-1 RT occurs in the nucleus alongside uncoating. In agreement, we find that capsid in the nucleus induces recruitment of cleavage and polyadenylation specific factor 6 (CPSF6) to SC35 nuclear speckles, which are highly active transcription sites, suggesting that CPSF6 through capsid is recruiting viral complexes to SC35 speckles for the occurrence of RT. Thus, nuclear import precedes RT and uncoating, which fundamentally changes our understanding of HIV-1 infection.

Interferon-Inducible MicroRNA miR-128 Modulates HIV-1 Replication by Targeting TNPO3 mRNA.

The HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR), miR-128, represses retrotransposon long interspaced element 1 (L1) by a dual mechanism, namely, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three TNPO proteins (transportins TNPO1, TNPO2, and TNPO3). Here, we establish that miR-128 also influences HIV-1 replication by repressing TNPO3, a factor that regulates HIV-1 nuclear import and viral; replication of TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that type I interferon (IFN)-inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly downregulating TNPO3 mRNA and protein expression levels. Challenging miR-modulated Jurkat cells or primary CD4+ T-cells with wild-type (WT), replication-competent HIV-1 demonstrated that miR-128 reduces viral replication and delays spreading of infection. Manipulation of miR-128 levels in HIV-1 target cell lines and in primary CD4+ T-cells by overexpression or knockdown showed that reduction of TNPO3 levels by miR-128 significantly affects HIV-1 replication but not murine leukemia virus (MLV) infection and that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus, suggesting that miR-128-indued TNPO3 repression contributes to the inhibition of HIV-1 replication. Finally, we determine that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Thus, we have established a novel role of miR-128 in antiviral defense in human cells, namely inhibiting HIV-1 replication by altering the cellular milieu through targeting factors that include TNPO3.IMPORTANCE HIV-1 is the causative agent of AIDS. During HIV-1 infection, type I interferons (IFNs) are induced, and their effectors limit HIV-1 replication at multiple steps in its life cycle. However, the cellular targets of INFs are still largely unknown. In this study, we identified the interferon-inducible microRNA (miR) miR-128, a novel antiviral mediator that suppresses the expression of the host gene TNPO3, which is known to modulate HIV-1 replication. Notably, we observe that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Elucidation of the mechanisms through which miR-128 impairs HIV-1 replication may provide novel candidates for the development of therapeutic interventions.

l-Citrulline restores nitric oxide level and cellular uptake at the brain capillary endothelial cell line (TR-BBB cells) with glutamate cytotoxicity.

OBJECTIVE: Glutamate excitotoxicity provokes neuronal cell damage and death, leading to collapse of the blood-brain barrier (BBB). Recently, it has been reported that l-citrulline, a neutral amino acid and a major precursor of l-arginine in the nitric oxide (NO) cycle, can prevent both neuronal cell death and cerebrovascular cell loss in brain ischemia. Therefore, the objective of this study was to investigate the effect of l-citrulline on glutamate cytotoxicity in the BBB using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells) as an in vitro model of the BBB. METHODS: Cell viability was determined using MTT assay. Cellular uptake of [14C] l-citrulline and expression levels of rat large neutral amino acid transporter 1 (rLAT1), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) at mRNA level were performed using quantitative real-time polymerase chain reaction (PCR) analysis. NO production from TR-BBB cells was measured using Griess reagents. All experiments were performed after treatment of TR-BBB cells with glutamate alone or co-treatment with l-citrulline, l-arginine, and/or taurine for 24 h. RESULTS: l-Citrulline treatment increased cell viability, [14C] l-citrulline uptake, and the mRNA levels of LAT1 and eNOS in TR-BBB cells treated with glutamate. However, iNOS mRNA expression was inhibited by l-citrulline. NO production and transcript level of iNOS were markedly increased by glutamate treatment alone. However, co-treatment with l-citrulline, taurine, or both l-citrulline and taurine decreased NO levels and mRNA levels of iNOS in TR-BBB cells treated with glutamate. In co-treatment of TR-BBB cells with l-arginine, a NO donor, and glutamate, NO levels were increased and expression levels of iNOS mRNA were similar compared to those in cells treated with glutamate alone. CONCLUSION: l-Citrulline can restore NO level and its cellular uptake in TR-BBB cells with glutamate cytotoxicity. Supplying l-citrulline at the BBB may provide neuroprotective effect to improve cerebrovascular dysfunction such as a brain ischemia.

Characteristics of L-citrulline transport through blood-brain barrier in the brain capillary endothelial cell line (TR-BBB cells).

BACKGROUND: L-Citrulline is a neutral amino acid and a major precursor of L-arginine in the nitric oxide (NO) cycle. Recently it has been reported that L-citrulline prevents neuronal cell death and protects cerebrovascular injury, therefore, L-citrulline may have a neuroprotective effect to improve cerebrovascular dysfunction. Therefore, we aimed to clarify the brain transport mechanism of L-citrulline through blood-brain barrier (BBB) using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells), as an in vitro model of the BBB. METHODS: The uptake study of [14C] L-citrulline, quantitative real-time polymerase chain reaction (PCR) analysis, and rLAT1, system b0,+, and CAT1 small interfering RNA study were performed in TR-BBB cells. RESULTS: The uptake of [14C] L-citrulline was a time-dependent, but ion-independent manner in TR-BBB cells. The transport process involved two saturable components with a Michaelis-Menten constant of 30.9 ± 1.0 µM (Km1) and 1.69 ± 0.43 mM (Km2). The uptake of [14C] L-citrulline in TR-BBB cells was significantly inhibited by neutral and cationic amino acids, but not by anionic amino acids. In addition, [14C]L-citrulline uptake in the cells was markedly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), which is the inhibitor of the large neutral amino acid transporter 1 (LAT1), B0, B0,+ and harmaline, the inhibitor of system b0,+. Gabapentin and L-dopa as the substrates of LAT1 competitively inhibited the uptake of [14C] L-citrulline. IC50 values for L-dopa, gabapentin, L-phenylalanine and L-arginine were 501 µM, 223 µM, 68.9 µM and 33.4 mM, respectively. The expression of mRNA for LAT1 was predominantly increased 187-fold in comparison with that of system b0,+ in TR-BBB cells. In the studies of LAT1, system b0,+ and CAT1 knockdown via siRNA transfection into TR-BBB cells, the transcript level of LAT1 and [14C] L-citrulline uptake by LAT1 siRNA were significantly reduced compared with those by control siRNA in TR-BBB cells. CONCLUSIONS: Our results suggest that transport of L-citrulline is mainly mediated by LAT1 in TR-BBB cells. Delivery strategy for LAT1-mediated transport and supply of L-citrulline to the brain may serve as therapeutic approaches to improve its neuroprotective effect in patients with cerebrovascular disease.

Structural basis for nuclear import of splicing factors by human Transportin 3.

Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. Herein, we show that the majority of cellular Tnpo3 binding partners contain arginine-serine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran- and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible ß-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. Our results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3-CPSF6 nexus in HIV-1 biology.

Differential effects of human immunodeficiency virus type 1 capsid and cellular factors nucleoporin 153 and LEDGF/p75 on the efficiency and specificity of viral DNA integration.

Retroviruses integrate into cellular DNA nonrandomly. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) favor the bodies of active genes and gene-enriched transcriptionally active regions of chromosomes. The interaction between lentiviral integrase and the cellular protein lens epithelium-derived growth factor (LEDGF)/p75 underlies the targeting of gene bodies, whereas recent research has highlighted roles for the HIV-1 capsid (CA) protein and cellular factors implicated in viral nuclear import, including transportin 3 (TNPO3) and nucleoporin 358 (NUP358), in the targeting of gene-dense regions of chromosomes. Here, we show that CA mutations, which include the substitution of Asp for Asn74 (N74D), significantly reduce the dependency of HIV-1 on LEDGF/p75 during infection and that this difference correlates with the efficiency of viral DNA integration. The distribution of integration sites mapped by Illumina sequencing confirms that the N74D mutation reduces integration into gene-rich regions of chromosomes and gene bodies and reveals previously unrecognized roles for NUP153 (another HIV-1 cofactor implicated in viral nuclear import) and LEDGF/p75 in the targeting of the viral preintegration complex to gene-dense regions of chromatin. A role for the CA protein in determining the dependency of HIV-1 on LEDGF/p75 during infection highlights a connection between the viral capsid and chromosomal DNA integration.