Single-cell analysis uncovers high-proliferative tumour cell subtypes and their interactions in the microenvironment of gastric cancer.

Gastric cancer (GC) remains a prominent malignancy that poses a significant threat to human well-being worldwide. Despite advancements in chemotherapy and immunotherapy, which have effectively augmented patient survival rates, the mortality rate associated with GC remains distressingly high. This can be attributed to the elevated proliferation and invasive nature exhibited by GC. Our current understanding of the drivers behind GC cell proliferation remains limited. Hence, in order to reveal the molecular biological mechanism behind the swift advancement of GC, we employed single-cell RNA-sequencing (scRNA-seq) to characterize the tumour microenvironment in this study. The scRNA-seq data of 27 patients were acquired from the Gene Expression Omnibus database. Differential gene analysis, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and Gene Set Enrichment Analysis were employed to investigate 38 samples. The copy number variation level exhibited by GC cells was determined using InferCNV. The CytoTRACE, Monocle and Slingshot analysis were used to discern the cellular stemness and developmental trajectory of GC cells. The CellChat package was utilized for the analysis of intercellular communication crosstalk. Moreover, the findings of the data analysis were validated through cellular functional tests conducted on the AGS cell line and SGC-7901 cell line. Finally, this study constructed a risk scoring model to evaluate the differences of different risk scores in clinical characteristics, immune infiltration, immune checkpoints, functional enrichment, tumour mutation burden and drug sensitivity. Within the microenvironment of GC, we identified the presence of 8 cell subsets, encompassing NK_T cells, B_Plasma cells, epithelial cells, myeloid cells, endothelial cells, mast cells, fibroblasts, pericytes. By delving deeper into the characterization of GC cells, we identified 6 specific tumour cell subtypes: C0 PSCA+ tumour cells, C1 CLDN7+ tumour cells, C2 UBE2C+ tumour cells, C3 MUC6+ tumour cells, C4 CHGA+ tumour cells and C5 MUC2+ tumour cells. Notably, the C2 UBE2C+ tumour cells demonstrated a close association with cell mitosis and the cell cycle, exhibiting robust proliferative capabilities. Our findings were fortified through enrichment analysis, pseudotime analysis and cell communication analysis. Meanwhile, knockdown of the transcription factor CREB3, which is highly active in UBE2C+ tumour cells, significantly impedes the proliferation, migration and invasion of GC cells. And the prognostic score model constructed with CREB3-related genes showcased commendable clinical predictive capacity, thus providing valuable guidance for patients' prognosis and clinical treatment decisions. We have identified a highly proliferative cellular subgroup C2 UBE2C+ tumour cells in GC for the first time. The employment of a risk score model, which is based on genes associated with UBE2C expression, exhibits remarkable proficiency in predicting the prognosis of GC patients. In our investigation, we observed that the knockdown of the transcription factor CREB3 led to a marked reduction in cellular proliferation, migration and invasion in GC cell line models. Implementing a stratified treatment approach guided by this model represents a judicious and promising methodology.

Germination responses of Lens Culiunaris L. seeds to osmotic potentials at cardinal temperatures using hydrothermal time model.

BACKGROUND: Lentil is a significant legume that are consumed as a staple food and have a significant economic impact around the world. The purpose of the present research on lentil was to assess the hydrothermal time model's capacity to explain the dynamics of Lens culinaris L. var. Markaz-09 seed germination, as well as to ascertain the germination responses at various sub-optimal temperatures (T) and water potentials (Ψ). In order to study lentil seed germination (SG) behavior at variable water potentials (Ψs) and temperatures (Ts). A lab experiment employing the hydrothermal time model was created. Seeds were germinated at six distinct temperatures: 15 0С, 20 0С, 25 0С, 30 0С, 35 0С, and 40 0С, with five Ψs of 0, -0.3, -0.6, -0.9, and - 1.2 MPa in a PEG-6000 (Polyethylene glycol 6000) solution. RESULTS: The results indicated that the agronomic parameters like Germination index (GI), Germination energy (GE), Timson germination index (TGI), were maximum in 25 0C at (-0.9 MPa) and lowest at 40 0C in 0 MPa. On other hand, mean germination time (MGT) value was highest at 15 0C in -1.2 MPa and minimum at 40 0C in (-0.6 MPa) while Mean germination rate (MGR) was maximum at 40 0C in (0 MPa) and minimum at 15 0C in (-0.6 MPa). CONCLUSIONS: The HTT model eventually defined the germination response of Lens culinaris L. var. Markaz-09 (Lentil) for all Ts and Ψs, allowing it to be employed as a predictive tool in Lens culinaris L. var. Markaz-09 (Lentil) seed germination simulation models.

Salicylic acid and Tocopherol improve wheat (Triticum aestivum L.) Physio-biochemical and agronomic features grown in deep sowing stress: a way forward towards sustainable production.

BACKGROUND: The rate of germination and other physiological characteristics of seeds that are germinating are impacted by deep sowing. Based on the results of earlier studies, conclusions were drawn that deep sowing altered the physio-biochemical and agronomic characteristics of wheat (Triticum aestivum L.). RESULTS: In this study, seeds of wheat were sown at 2 (control) and 6 cm depth and the impact of exogenously applied salicylic acid and tocopherol (Vitamin-E) on its physio-biochemical and agronomic features was assessed. As a result, seeds grown at 2 cm depth witnessed an increase in mean germination time, germination percentage, germination rate index, germination energy, and seed vigor index. In contrast, 6 cm deep sowing resulted in negatively affecting all the aforementioned agronomic characteristics. In addition, deep planting led to a rise in MDA, glutathione reductase, and antioxidants enzymes including APX, POD, and SOD concentration. Moreover, the concentration of chlorophyll a, b, carotenoids, proline, protein, sugar, hydrogen peroxide, and agronomic attributes was boosted significantly with exogenously applied salicylic acid and tocopherol under deep sowing stress. CONCLUSIONS: The results of the study showed that the depth of seed sowing has an impact on agronomic and physio-biochemical characteristics and that the negative effects of deep sowing stress can be reduced by applying salicylic acid and tocopherol to the leaves.

Efficient method for modeling large-scale arrays of optical nanoresonators based on the coupling theory of quasinormal mode.

We propose an efficient method for calculating the electromagnetic field of a large-scale array of optical nanoresonators based on the coupling theory of quasinormal mode (QNM). In this method, two approaches of the scattered-field reconstruction and stationary-phase-principle calculated plane-wave expansion are developed to obtain the regularized QNM (RQNM) in different regions. This accurate and efficient calculation of RQNM resolves the far-field divergence issue of QNMs in the QNM-coupling theory, thus enabling a rapid computation of the electromagnetic field of a large-scale array of optical nanoresonators, which is a challenging task for full-wave numerical methods. Using this method, we consider the numerical example of the radiation problem of a single point source in a large-scale periodic array of optical nanoantennas. In comparison to full-wave numerical methods, this method significantly reduces the computation time by 1∼2 orders of magnitude while maintaining accuracy. The high computational efficiency and physical intuitiveness of the method enables to clarify the impact of array size (exceeding 50 × 50 wavelengths), period and field-coupling range (far beyond the tight-binding approximation) on the optical response. The proposed method and results can provide an efficient tool and guidance for the design of large-scale arrays of optical nanoresonators.

Exploring the relationship between Treg-mediated risk in COPD and lung cancer through Mendelian randomization analysis and scRNA-seq data integration.

BACKGROUND: Evidence from observational studies suggests an association between chronic obstructive pulmonary disease (COPD) and lung cancer. The potential interactions between the immune system and the lungs may play a causative role in COPD and lung cancer and offer therapeutic prospects. However, the causal association and the immune-mediated mechanisms between COPD and lung cancer remain to be determined. METHODS: We employed a two-sample Mendelian randomization (MR) approach to investigate the causal association between COPD and lung cancer. Additionally, we examined whether immune cell signals were causally related to lung cancer, as well as whether COPD was causally associated with immune cell signals. Furthermore, through two-step Mendelian randomization, we investigated the mediating effects of immune cell signals in the causal association between COPD and lung cancer. Leveraging publicly available genetic data, our analysis included 468,475 individuals of European ancestry with COPD, 492,803 individuals of European ancestry with lung cancer, and 731 immune cell signatures of European ancestry. Additionally, we conducted single-cell transcriptome sequencing analysis on COPD, lung cancer, and control samples to validate our findings. FINDINGS: We found a causal association between COPD and lung cancer (odds ratio [OR] = 1.63, 95% confidence interval [CI] = 1.31-2.02, P-value < 0.001). We also observed a causal association between COPD and regulatory T cells (odds ratio [OR] = 1.19, 95% confidence interval [CI] = 1.01-1.40, P-value < 0.05), as well as a causal association between regulatory T cells and lung cancer (odds ratio [OR] = 1.02, 95% confidence interval [CI] = 1.002-1.045, P-value < 0.05). Furthermore, our two-step Mendelian randomization analysis demonstrated that COPD is associated with lung cancer through the mediation of regulatory T cells. These findings were further validated through single-cell sequencing analysis, confirming the mediating role of regulatory T cells in the association between COPD and lung cancer. INTERPRETATION: As far as we are aware, we are the first to combine single-celled immune cell data with two-sample Mendelian randomization. Our analysis indicates a causal association between COPD and lung cancer, with regulatory T cells playing an intermediary role.

Understanding the Intrinsic Water Wettability of Hexagonal Boron Nitride.

The water wettability of hexagonal boron nitride (hBN) has attracted a lot of research interest in the past 15 years. Experimentally, the static water contact angle (WCA) has been widely utilized to characterize the intrinsic water wettability of hBN. In the current study, we have investigated the effect of airborne hydrocarbons and defects on both static and dynamic WCAs of hBN. Our results showed that the static WCA is impacted by defects, which suggests that previously reported static WCAs do not characterize the intrinsic water wettability of hBN since the state-of-the-art hBN samples always have relatively high defect density. Instead, we found that the advancing WCA of freshly exfoliated hBN is not affected by the defects and airborne hydrocarbons. As a result, the advancing WCA on freshly exfoliated hBN, determined to be 79 ± 3°, best represents the intrinsic water wettability of hBN. A qualitative model has been proposed to describe the effect of airborne hydrocarbons and defects on the static and dynamic WCA of hBN, which is well supported by the experimental results. The finding here has important implications for the water wettability of 2D materials.

Glycine-Ti3C2Tx Hybrid Material Improves the Electrochemical Corrosion Resistance of a Water-Borne Epoxy Coating.

Improving the dispersibility and compatibility of nanomaterials in water-borne epoxy resins is an important means to improve the protection ability and corrosion resistance of coatings. In this study, glycine-functionalized Ti3C2Tx (GT) was used to prepare an epoxy composite coating. The results of Fourier transform infrared spectroscopy and X-ray diffraction showed that glycine was successfully modified. The scanning electron microscopy and transmission electron microscopy results showed that the aggregation of Ti3C2Tx was alleviated. Electrochemical impedance spectroscopy test results show that, after 60 days of immersion, GT coating still shows the best protection performance, and the composite coating |Z|f = 0.01 Hz is 3 orders of magnitude higher than that of the pure epoxy coating. This is mainly because, after adding glycine, the -COOH group on the surface of glycine binds to the -OH group on the surface of Ti3C2Tx, improving the aggregation of Ti3C2Tx itself. At the same time, the -NH group of glycine can also participate in the curing reaction of epoxy resin to strengthen the bonding strength between the coating and the metal. The good dispersion of GT in epoxy resin makes it fill the pores and holes left by epoxy resin curing and strengthen the corrosion resistance. The easy availability and green properties of glycine provide a simple and environmentally friendly method for the modification of Ti3C2Tx.

Wide Bandgap Polymer Donors Based on Succinimide-Substituted Thiophene for Nonfullerene Organic Solar Cells.

The advent of nonfullerene acceptors (NFAs) has greatly improved the photovoltaic performance of organic solar cells (OSCs). However, to compete with other solar cell technologies, there is a pressing need for accelerated research and development of improved NFAs as well as their compatible wide bandgap polymer donors. In this study, a novel electron-withdrawing building block, succinimide-substituted thiophene (TS), is utilized for the first time to synthesize three wide bandgap polymer donors: PBDT-TS-C5, PBDT-TSBT-C12, and PBDTF-TSBT-C16. These polymers exhibit complementary bandgaps for efficient sunlight harvesting and suitable frontier energy levels for exciton dissociation when paired with the extensively studied NFA, Y6. Among these donors, PBDTF-TSBT-C16 demonstrates the highest hole mobility and a relatively low highest occupied molecular orbital (HOMO) energy level, attributed to the incorporation of thiophene spacers and electron-withdrawing fluorine substituents. OSC devices based on the blend of PBDTF-TSBT-C16:Y6 achieve the highest power conversion efficiency of 13.21%, with a short circuit current density (Jsc) of 26.83 mA cm-2, an open circuit voltage (Voc) of 0.80 V, and a fill factor of 0.62. Notably, the Voc × Jsc product reaches 21.46 mW cm-2, demonstrating the potential of TS as an electron acceptor building block for the development of high-performance wide bandgap polymer donors in OSCs.

Stability and chemical bonding in a series of inverse sandwich actinide boride clusters (An2B8) with δ bonding.

An inverse sandwich structure has been computationally predicted for uranium boride and extended to the series of actinide elements (An) from Th to Cm. The electronic structure and chemical bonding of these novel compounds have been analyzed using density functional theory and multireference wave-function based methods. We report the trends in electronic structure and bonding for An2B8, and found that (d-π)π and (d-p)δ are the most important factors in the stability of An2B8. The (f-p)δ bond provides extra stabilization for Pa2B8 and U2B8, owing to the extensive interactions of An-B8-An, resulting in a short distance for the Pa-Pa and U-U bonds.

Distinguishing the Geometric and Electronic Structures of Actinide Carbides AnxC8 (An = Th, U; x = 2, 3) through Exchange Interactions.

Global-minimum optimizations combined with relativistic quantum chemistry calculations have been performed to characterize the ground-state stable structures of four titled compounds and to analyze the bonding properties. Th2C8 was identified as being a ThC4-Th(C2)2 structure, U2C8 has been found to favor the U-U(C8) structure, and both Th3C8 and U3C8 adopt the (AnC3)2-(AnC2) structure. Then, the wave function analyses reveal that the interactions between the Th 7s-based orbital and the σg molecular orbital of the C2 unit compensate for the excitation energy of 7s16d1 → 6d2 and lead to the stabilization of two Th(IV)s in the ThC4-Th(C2)2 structure. It also reveals that the U species exhibit magnetic exchange coupling behavior in UxC8, for instance, as seen in the direct interaction of U2C8 and the superexchange pathway of U3C8, which effectively stabilizes their low-spin states. This interpretation indicates that the geometric and electronic structures of AnxC8 species are largely influenced by the local magnetic moment and spin correlation.

A way to identify whether a DFT gap is from right reasons or error cancellations: The case of copper chalcogenides.

Gap opening remains elusive in copper chalcogenides (Cu2X, X = S, Se, and Te), not least because Hubbard + U, hybrid functional, and GW methods have also failed. In this work, we elucidate that their failure originates from a severe underestimation of the 4s-3d orbital splitting of the Cu atom, which leads to a band-order inversion in the presence of an anionic crystal field. As a result, the Fermi energy is pinned due to symmetry, yielding an invariant zero gap. Utilizing the hybrid pseudopotentials to correct the underestimation on the atomic side opens up gaps of experimental magnitude in Cu2X, suggesting their predominantly electronic nature. Our work not only clarifies the debate about the Cu2X gap but also provides a way to identify which of the different methods really captures the physical essence and which is the result of error cancellation.

Characterization of environmental airborne hydrocarbon contaminants by surface-enhanced Raman scattering.

This paper explores the unintentional contamination of Surface-Enhanced Raman Scattering (SERS) substrates by ambient hydrocarbon contaminants and their contribution to SERS spectra. Previous studies have identified amorphous carbon as a potential complicating factor in data analysis in SERS experiments, although its origin has been elusive. Our work showed that ambient hydrocarbon contamination and its decomposition products can be detected by SERS on a gold substrate. We propose that ambient air itself is a source of amorphous carbon contamination on SERS substrates. This understanding is crucial for the correct interpretation of SERS data and highlights the need for careful consideration of potential environmental contaminants in SERS analysis.

Combination of Polygonatum Rhizoma and Scutellaria baicalensis triggers apoptosis through downregulation of PON3-induced mitochondrial damage and endoplasmic reticulum stress in A549 cells.

OBJECTIVE: Scutellaria baicalensis (SB) and Polygonatum Rhizoma (PR), two traditional Chinese medicines, are both known to suppress cancer. However, the mechanism and effect of combined treatment of them for lung cancer are rarely known. Investigating the combined effect of SB and PR (hereafter referred to as SP) in potential mechanism of lung cancer is required. This study was to evaluate the inhibitory effects of SP on A549 cell growth and to explore the underlying molecular mechanisms. METHODS: According to the theory of Chinese medicine and network pharmacology, in the in vivo experiment, a mouse model of carcinoma in situ was constructed, and lung carcinoma in situ tissues were collected for proteomics analysis, hematoxylin-eosin staining, and CK19 immunohistochemistry. In the in vitro experiment, lung cancer A549 cells at logarithmic growth stage were taken, and the inhibitory effect of SP on the proliferation of A549 cells was detected by CCK8 method. The expression of PON3 was detected by quantitative polymerase chain reaction and western blot. In addition, the effect of SP on the induction of apoptosis in A549 cells and the changes of membrane potential and reactive oxygen species (ROS) content were detected by flow cytometry. The changes of PON3 content in endoplasmic reticulum (ER) are observed by laser confocal microscopy, whereas the effects of SP on the expression of apoptosis-related proteins and ER stress-related proteins in A549 cells were examined by western blot. RESULT: By searching the Traditional Chinese Medicines of Systems Pharmacology (TCMSP) (https://www.tcmspe.com/index.php) database and SymMap database, the respective target genes of PR and SB were mapped into protein network interactions, and using Venn diagrams to show 38 genes in common between PR and SB and lung cancer, SP was found to play a role in the treatment of lung cancer. In vivo experiments showed that in a lung carcinoma in situ model, lung tumor tissue was significantly lower in the SP group compared with the control group, and PON3 was shown to be downregulated by lung tissue proteomics analysis. The combination of SP was able to inhibit the proliferation of A549 cells in a concentration-dependent manner (p < .0001). The expression levels of apoptosis-related proteins and ER stress proteins were significantly increased and the expression levels of PON3 and anti-apoptosis-related proteins were decreased in A549 cells. At the same time, knockdown of PON3 could inhibit tumor cell proliferation (p < .0001). The combination of different concentrations of SP significantly induced apoptosis in A549 cells (p < .05; p < .0001), increased ROS content (p < .01), and damaged mitochondrial membrane potential of A549 cells (p < .05; p < .0001), and significantly increased the expression levels of apoptosis-related proteins and ER stress proteins in lung cancer A549 cells. CONCLUSION: SP inhibits proliferation of lung cancer A549 cells by downregulating PON3-induced apoptosis in the mitochondrial and ER pathways.

An improved method of local coloring in vertical-slot fishways to enhance the fish migration effect of grass carp.

Vertical-slot fishway (VSF) has been used in many water conservancy projects to restore the river connectivity. A high-quality fishway project should facilitate fish to discovering the exit and passing through, avoiding to long stay in the fishway and delay the migration. Current research on fishway engineering has not yielded an expected passing ratio of fish migration, and it is therefore of great significance to further study the assisting effect of VSF in fish migration. To begin with, we preliminarily determined the attractive and repelling colors of grass carps based on their swimming behavior in a static water pool configured with local colors. Combined with the migration route of the grass carp in a VSF pool without local coloring, four local coloring cases were designed. Based on the camera results of the four experimental local coloring cases, a comparative analysis was conducted with the blank control group frame by frame. This was followed by the statistics of the number of successfully migrated grass carps and their total completion time. On that basis, the assisting effect of VSF in fish migration under the four cases was evaluated in terms of the reduction rate of migration route length, the reduction rate of completion time, and the improvement rate of passing ratio. The research outcomes indicated that green and blue act as attractive colors while yellow and red serve as repelling colors for grass carp. Adding colors to the training wall and dividing wall in the VSF pool, the migration route of grass carp was appropriately adjusted, alongside a shortened completion time and an improved passing ratio. Of the four local coloring cases, the recommended case showed a significant effect on migration route, with more concentrated moving trajectories and shortened route length. Typically, the migration route length decreased by 26%, and the frequency of fish long staying at the junction between the training wall and dividing wall was markedly reduced, as well as the frequency of fish swimming along the water flow from upstream to downstream. The completion time was shortened by 26%, and the passing ratio was enhanced by 44%. The approach of combining local coloring with fish behavior and fishway hydraulics in the pool surpassed the method that optimizes the fishway design only from the fishway hydraulics. The improved method greatly shortened the migration route length, reduced the completion time, and significantly improved the passing ratio of fish passage objects in the VSF. The present research mainly focuses on using model experiments to evaluate the local coloring cases. In the future studies, we will configure local colors to the sidewalls of on-site fishways using environmentally friendly paint or colored organic glass panels. With the monitoring results of the completion time and passing ratio of fish passage objects, the recommended case can be further verified and optimized, thereby providing a more reasonable and feasible local coloring case for assisting fish migration in the VSF project.

A Study on the Growth and Physiological Toxicity Effects of the Combined Exposure of Microplastics and Cadmium on the Vicia faba L. Seedlings.

To investigate the toxicological effects of polystyrene microplastics (PS-MPs), cadmium (Cd), and their combined contamination on the growth and physiological responses of V. faba seedlings, this experiment employed a hydroponic method. The Hoagland nutrient solution served as the control, changes in root growth, physiological and biochemical indicators of V. faba seedlings under different concentrations of PS-MPs (10, 100 mg/L) alone and combined with 0.5 mg/L Cd. The results demonstrated that the root biomass, root vitality, generation rate of superoxide radicals (O2·-), malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity increased with increasing concentration under the influence of PS-MPs alone, while the soluble sugar content and peroxidase (POD) activity decreased. In the combined treatment with Cd, the trends of these indicators are generally similar to the PS-MPs alone treatment group. However, root vitality and SOD activity showed an inverse relationship with the concentration of PS-MPs. Furthermore, laser confocal and electron microscopy scanning revealed that the green fluorescent polystyrene microspheres entered the root tips of the V. faba and underwent agglomeration in the treatment group with a low concentration of PS-MPs alone and a high concentration of composite PS-MPs with Cd.

Comparative transcriptome analysis reveals candidate genes related to the sex differentiation of Schisandra chinensis.

Schisandra chinensis is a monoecious plant with unisex flowers. The fruit of S. chinensis is of high medical with economic value. The yield of S. chinensis fruit is related to the ratio of its female and male flowers. However, there is little research on its floral development and sex differentiation. To elucidate the possible mechanism for the sex differentiation of S. chinensis, we collected 18 samples of female and male flowers from three developmental stages and performed a comparative RNA-seq analysis aimed at identifying differentially expressed genes (DEGs) that may be related to sex differentiation. The results showed 936, 7179, and 6890 differentially expressed genes between female and male flowers at three developmental stages, respectively, and 466 candidate genes may play roles in sex differentiation. KEGG analysis showed genes involved in the flavonoid biosynthesis pathway and DNA replication pathway were essential for the development of female flowers. 51 MADS-box genes and 10 YABBY genes were identified in S. chinensis. The DEGs analysis indicated that MADS-box and YABBY genes were strongly related to the sex determination of S. chinensis. RT-qPCR confirmed the RNA-seq results of 20 differentially expressed genes, including three male-biased genes and 17 female-biased genes. A possible regulatory model of sex differentiation in S. chinensis was proposed according to our results. This study helps reveal the sex-differentiation mechanism of S. chinensis and lays the foundation for regulating the male-female ratio of S. chinensis in the future.

Regulation of Hepatitis B Virus Virion Release and Envelopment Timing by Nucleocapsid and Envelope Interactions.

Interactions between the N-terminal (assembly) domain (NTD), the linker region of the hepatitis B virus (HBV) capsid protein, and the large (L) envelope protein are required for virion formation, which occurs via budding of cytoplasmic mature nucleocapsids (NCs) containing the relaxed circular (RC) DNA genome into an intracellular membrane compartment containing viral envelope proteins. L-capsid interactions also negatively regulate covalently closed circular (CCC) DNA formation, which occurs after RC DNA release from mature NCs and nuclear import. We have now found that L could increase RC DNA in cytoplasmic mature NCs that are destabilized due to mutations in the NTD or the linker, even in those that apparently fail to support secretion of complete virions extracellularly. Other mutations in the capsid linker could block the effects of L on both cytoplasmic NC DNA and nuclear CCC DNA. Furthermore, the maturity of RC DNA in cytoplasmic NCs that was enhanced by L or found in secreted virions was modulated by the capsid linker sequence. The level and maturity of the cytoplasmic RC DNA were further influenced by the efficiency of extracellular virion secretion dependent on viral genotype-specific envelope proteins. These results suggest that interactions between the capsid and envelope proteins regulate one or more steps during virion secretion beyond initial capsid envelopment and highlight the critical role of the capsid linker in regulating capsid-envelope interaction, including the timing of envelopment during NC maturation. IMPORTANCE Hepatitis B virus (HBV) is a major human pathogen causing serious liver diseases, including cancer. Interactions between the HBV capsid and the large (L) envelope protein are required for formation of infectious viral particles and also negatively regulate formation of an HBV DNA episome in the host cell nucleus, which serves as the sole transcriptional template capable of supporting all viral gene expression to sustain HBV replication and, therefore, is the molecular basis of HBV persistence. Here, we report evidence indicating that L-capsid interactions modulate the timing of formation of infectious HBV particles during replication and facilitate extracellular release following their formation. Furthermore, a short linker sequence in the capsid protein plays a critical role in these processes as well as controls the amplification of the nuclear episome. These findings inform fundamental mechanisms of HBV replication as well as antiviral development targeting the HBV capsid and DNA episome.

Region-Specific Hepatitis B Virus Genome Exposure from Nucleocapsid Modulated by Capsid Linker Sequence and Inhibitor: Implications for Uncoating.

Hepatitis B virus (HBV) contains a partially double-stranded, relaxed circular (RC) DNA genome synthesized within a nucleocapsid (NC) in the host cell cytoplasm. The release of RC DNA from the NC, in an ill-defined process called uncoating, to the nucleus is required for its conversion to the covalently closed circular (CCC) DNA, the viral episome serving as the transcriptional template for all viral RNAs necessary for replication and, thus, essential for establishing and sustaining viral infection. In efforts to better understand uncoating, we analyzed HBV core (HBc) mutants that show various levels of nuclear CCC DNA but little to no cytoplasmic RC DNA. We found that RC DNA could be synthesized by these mutants outside the cell, but in contrast to the wild type (wt), the mutant NCs were unable to protect RC DNA from digestion by the endogenous nuclease(s) in cellular lysates or exogenous DNase. Subcellular fractionation suggested that the major RC DNA-degrading activity was membrane associated. Digestion with sequence-specific and nonspecific DNases revealed the exposure of specific regions of RC DNA from the mutant NC. Similarly, treatment of wt NCs with a core inhibitor known to increase CCC DNA by affecting uncoating also led to region-specific exposure of RC DNA. Furthermore, a subpopulation of untreated wild type (wt) mature NCs showed site-specific exposure of RC DNA as well. Competition between RC DNA degradation and its conversion to CCC DNA during NC uncoating thus likely plays an important role in the establishment and persistence of HBV infection and has implications for the development of capsid-targeted antivirals. IMPORTANCE Disassembly of the hepatitis B virus (HBV) nucleocapsid (NC) to release its genomic DNA, in an ill-understood process called uncoating, is required to form the viral nuclear episome in the host cell nucleus, a viral DNA essential for establishing and sustaining HBV infection. The elimination of the HBV nuclear episome remains the holy grail for the development of an HBV cure. We report here that the HBV genomic DNA is exposed in a region-specific manner during uncoating, which is enhanced by mutations of the capsid protein and a capsid-targeted antiviral compound. The exposure of the viral genome can result in its rapid degradation or, alternatively, can enhance the formation of the nuclear episome, thus having a major impact on HBV infection and persistence. These results are thus important for understanding fundamental mechanisms of HBV replication and persistence and for the ongoing pursuit of an HBV cure.

Hydrocarbons in the Meniscus: Effects on Conductive Atomic Force Microscopy.

It is commonly accepted that during conductive atomic force microscopy (CAFM) measurement in ambient, a liquid meniscus can form between the tip and the sample. Such a liquid bridge, normally assumed to be composed of water, is a major factor in analyzing and understanding CAFM results. Here, we show that the adsorption of adventitious hydrocarbons from the air to a surface can greatly affect CAFM data both in imaging mode and in local spectroscopy (current-voltage or I-V curves). We propose a model to explain the phenomena whereby hydrocarbon contaminates contribute to the composition of the liquid bridge between the tip and the sample.

Influence of Atmospheric Contaminants on the Work Function of Graphite.

Airborne hydrocarbon contamination occurs rapidly on graphitic surfaces and negatively impact many of their material properties, yet much of the molecular details of the contamination remains unknown. We use Kelvin probe force microscopy (KPFM) to study the time evolution of the surface potential of graphite exposed to ambient. After exfoliation in air, the surface potential of graphite is not homogeneous and contains features that are absent in the topography image. In addition, the heterogeneity of the surface potential images increased in the first few days followed by a decrease at longer exposure times. These observations are strong support of slow conformation change, phase separation, and/or dynamic displacement of the adsorbed airborne contaminants.