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Despite prolonged surveillance and interventions, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses continue to pose a severe global health burden. Thus, we developed a chimpanzee adenovirus-based combination vaccine, AdC68-HATRBD, with dual specificity against SARS-CoV-2 and influenza virus. When used as a standalone vaccine, intranasal immunization with AdC68-HATRBD induced comprehensive and potent immune responses consisting of immunoglobin (Ig) G, mucosal IgA, neutralizing antibodies, and memory T cells, which protected the mice from BA.5.2 and pandemic H1N1 infections. When used as a heterologous booster, AdC68-HATRBD markedly improved the protective immune response of the licensed SARS-CoV-2 or influenza vaccine. Therefore, whether administered intranasally as a standalone or booster vaccine, this combination vaccine is a valuable strategy to enhance the overall vaccine efficacy by inducing robust systemic and mucosal immune responses, thereby conferring dual lines of immunological defenses for these two viruses. [ABSTRACT FROM AUTHOR]

The safe and efficient development of natural gas hydrate requires a deep understanding of the deformation behaviors of reservoirs. In this study, a series of triaxial shearing tests are carried out to investigate the deformation properties of hydrate-bearing sediments. Variations of volumetric and lateral strains versus hydrate saturation are analyzed comprehensively. Results indicate that the sediments with high hydrate saturation show dilative behaviors, which lead to strain-softening characteristics during shearing. The volumetric strain curves have a tendency to transform gradually from dilatation to compression with the increase in effective confining pressure. An easy prediction model is proposed to describe the relationship between volumetric and axial strains. The model coefficient β is the key dominating factor for the shape of volumetric strain curves and can be determined by the hydrate saturation and stress state. Moreover, a modified model is established for the calculation of lateral strain. The corresponding determination method is provided for the easy estimation of model coefficients for medium sand sediments containing hydrate. This study provides a theoretical and experimental reference for deformation estimation in natural gas hydrate development. [ABSTRACT FROM AUTHOR]

It has been evidenced that shallow gas hydrate resources are abundant in deep oceans worldwide. Their geological background, occurrence, and other characteristics differ significantly from deep-seated hydrates. Because of the high risk of well construction and low production efficiency, they are difficult to be recovered by using conventional oil production methods. As a result, this paper proposes an alternative design based on a combination of radial drilling, heat injection, and backfilling methods. Multi-branch holes are used to penetrate shallow gas hydrate reservoirs to expand the depressurization area, and heat injection is utilized as a supplement to improve gas production. Geotechnical information collected from an investigation site close to the offshore production well in the South China Sea is used to assess the essential components of this plan, including well construction stability and gas production behavior. It demonstrates that the hydraulic fracturing of the 60 mbsf overburden layer can be prevented by regulating the drilling fluid densities. However, the traditional well structure is unstable, and the suction anchor is advised for better mechanical performance. The gas production rate can be significantly increased by combining hot water injection and depressurization methods. Additionally, the suitable production equipment already in use is discussed. [ABSTRACT FROM AUTHOR]

Marine natural gas hydrate (NGH) is a promising substitutive low-carbon energy resource, whereas NGH-production induced geoengineering concerns remain challenging. Advanced forecast of possible geoengineering risks is the fundamental for eco-friendly NGH exploitation. Reservoir creep deformation is an early symptom of the geoengineering risks. However, whether the creep deformation behaviors of the NGH-bearing strata is predictable remains controversial. In this study, a series of multi-step loading creep test are conducted for sandy gas hydrate bearing sediment (GHBS) samples, during which the ultrasonic responses are recorded simultaneously. The acoustic velocity, compression-to-shear velocity ratio, Poission's ratio, main frequency, and main frequency amplitude are used to characterize creep failures of the GHBS for the first time. Combining analyses of the creep behaviors and acoustic responses yield the following conclusions. Firstly, the long-term strength derived from creeping test is 0.45–0.60 times of the shear strength derived from triaxial shearing. Ignoring the creep effect might underestimate the scale and intensity of possible geoengineering risks during long-term NGH exploitation. Secondly, the acoustic velocity increases gently and then decreases continuously during creeping. Once the accelerated creep appears, the acoustic velocity plummets significantly, together with a sudden decrease in the compression-to-shear velocity ratio, and fluctuations in the main frequency and its amplitude. Furthermore, the main frequency and its amplitude shall fluctuate abruptly prior to the emergence of the accelerated creep. Therefore, we anticipate that the combination of abnormal fluctuations of main frequency and its amplitude can be used as early-warning indicators for possible creep failure of the GHBS. The results might have great significance for in-situ detection and prediction of possible reservoir failure during long-term NGH exploitation. [ABSTRACT FROM AUTHOR]

Laboratory visual detection on the hydrate accumulation process provides an effective and low-cost method to uncover hydrate accumulation mechanisms in nature. However, the spatial hydrate distribution and its dynamic evolutionary behaviors are still not fully understood due to the lack of methods and experimental systems. Toward this goal, we built a two-dimensional electrical resistivity tomography (ERT) apparatus capable of measuring spatial and temporal characteristics of hydrate-bearing porous media. Beach sand (0.05–0.85 mm) was used to form artificial methane hydrate-bearing sediment. The experiments were conducted at 1°C under excess water conditions and the ERT data were acquired and analyzed. This study demonstrates the utility of the ERT method for hydrate mapping in laboratory-scale. The results indicate that the average electrical conductivity decreases nonlinearly with the formation of the hydrate. At some special time-intervals, the average conductivity fluctuates within a certain scope. The plane conductivity fields evolve heterogeneously and the local preferential hydrate-forming positions alternate throughout the experimental duration. We speculate that the combination of hydrate formation itself and salt-removal effect plays a dominant role in the spatial and temporal hydrate distribution, as well as geophysical parameters changing behaviors during hydrate accumulation. [ABSTRACT FROM AUTHOR]

Massive production of efficacious SARS-CoV-2 vaccines is essential for controlling the ongoing COVID-19 pandemic. We report here the preclinical development of yeast-produced receptor-binding domain (RBD)-based recombinant protein SARS-CoV-2 vaccines. We found that monomeric RBD of SARS-CoV-2 could be efficiently produced as a secreted protein from transformed Pichia pastoris (P. pastoris) yeast. Yeast-derived RBD-monomer possessed functional conformation and was able to elicit protective level of neutralizing antibodies in mice. We further designed and expressed a genetically linked dimeric RBD protein in yeast. The engineered dimeric RBD was more potent than the monomeric RBD in inducing long-lasting neutralizing antibodies. Mice immunized with either monomeric RBD or dimeric RBD were effectively protected from live SARS-CoV-2 virus challenge even at 18 weeks after the last vaccine dose. Importantly, we found that the antisera raised against the RBD of a single SARS-CoV-2 prototype strain could effectively neutralize the two predominant circulating variants B.1.1.7 and B.1.351, implying broad-spectrum protective potential of the RBD-based vaccines. Our data demonstrate that yeast-derived RBD-based recombinant SARS-CoV-2 vaccines are feasible and efficacious, opening up a new avenue for rapid and cost-effective production of SARS-CoV-2 vaccines to achieve global immunization. [ABSTRACT FROM AUTHOR]

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses. The SARS-CoV-2 spike protein contains a multi-basic cleavage site. Here, the authors show how this multi-basic cleavage site affects entry of SARS-CoV-2 into cells and transmission in the hamster model and identify host factors affecting entry of SARS-CoV-2 in a genome-wide CRISPR screen. [ABSTRACT FROM AUTHOR]

Gas leakage is an important consideration in natural systems that experience gas hydrate accumulation. A number of velocity models have been created to study hydrate-bearing sediments, including the BGTL theory, the weighted equation, the Wood equation, the K-T equation, and the effective medium theory. In previous work, we regarded water as the pore fluid, which meant its density and bulk modulus values were those of water. This approach ignores the presence of gas, which results in a biased calculation of the pore fluid's bulk modulus and density. To take into account the effect of gas on the elastic wave velocity, it is necessary to recalculate the bulk modulus and density of an equivalent medium. Thus, a high-pressure reactor device for simulating leakage systems was developed to establish the relationship between wave velocity and hydrate saturation in methane-flux mode. A comparison of the values calculated by the velocity model with the experimental data obtained in this study indicates that the effective medium theory (EMT, which considers gas effects) is more applicable than other models. For hydrate saturations of 10%–30%, the result ranges between EMT-B (homogenous gas distribution) and EMT-B (patchy gas distribution). For hydrate saturations of 30%–60%, the results are similar to those of the EMT-B (homogenous gas distribution) mode, whereas hydrate saturations of 60%–70% yield results similar to those of the EMT-A mode. For hydrate saturations greater than 80%, the experimental results are similar to those of the EMT-B mode. These results have significance for hydrate exploitation in the South China Sea. [ABSTRACT FROM AUTHOR]

Cell membranes are a great obstacle for entrance of gene therapeutic agents. Cell-penetrating peptides (CPPs) have been proven as a promising gene delivery tool. However, the early TAT peptide derived from the HIV transcription activator protein has been proven that the sequence contains Furin protease cleaved motifs which limited the TAT application in delivery of exogenous active molecules. In the present study, through the bioinformatics and experimental approach, we have identified a novel CPP derived from the N terminus of VP1 protein of chicken anemia virus (CAV), designated as CVP1-N2, which is rich in arginine residues and contains α-helical structure. Then, the ability of CVP1-N2 cell penetrating was detected using confocal imaging and flow cytometry. FITC-labeled CVP1-N2 peptide could rapidly internalize into different types of live cells with dose dependence and without cytotoxic effects by MTT assay. Surprisingly, CVP1-N2 with a pattern of nuclear sub-location has shown the higher uptake efficiency than TAT. At 10, 1, and 0.1 μM, the mean relative internalization of CVP1-N2 was respectively 1.08-, 12-, and 75-fold higher than that of CVP1, as well as 1.6-, 56-, and 75-fold higher than that of TAT. Moreover, using endocytic inhibitors along with low-temperature stress validated that the CVP1-N2 internalization route is direct translocation pathway. Finally, the capacity of CVP1-N2 for delivery of gene into cells was determined, where it was able to carry red fluorescent protein (RFP) and apoptin genes into cells respectively and induce the apoptosis. All these data indicate that CVP1-N2 could be used as a novel gene delivery vehicle for gene therapy in the future. Key points: • 1CVP1-N2 was identified as a novel more efficient cell-penetrating peptide. • 2. CVP1-N2 localized to the nucleus through the direct transduction pathway. • 3. CVP1-N2 was able to deliver the apoptin gene into HCT116 cells and induce apoptosis. [ABSTRACT FROM AUTHOR]

A novel lactate dehydrogenase gene, named lrldh, was cloned from Lactobacillus rossiae and heterologously expressed in Escherichia coli. The lactate dehydrogenase LrLDH is NADH-dependent with a molecular weight of approximately 39 kDa. It is active at 40 °C and pH 6.5 and stable in a neutral to alkaline environment below 35 °C. The kinetic constants, including maximal reaction rate (Vmax), apparent Michaelis–Menten constant (Km), turnover number (Kcat) and catalytic efficiency (Kcat/Km) for phenylpyruvic acid were 1.95 U mg−1, 2.83 mM, 12.29 s−1, and 4.34 mM−1 s−1, respectively. Using whole cells of recombinant E. coli/pET28a-lrldh, without coexpression of a cofactor regeneration system, 20.5 g l−1d-phenyllactic acid with ee above 99% was produced from phenylpyruvic acid in a fed-batch biotransformation process, with a productivity of 49.2 g l−1 d−1. Moreover, LrLDH has broad substrate specificity to a range of ketones, keto acids and ketonic esters. Taken together, LrLDH is a promising biocatalyst for the efficient synthesis of d-phenyllactic acid and other fine chemicals. [ABSTRACT FROM AUTHOR]

The complex distribution of gas hydrate in sediments makes understanding the mechanical properties of hydrate-bearing sediments a challenging task. The mechanical behaviors of hydrate-bearing interlayered sediments are still poorly known. A series of triaxial shearing tests were conducted to investigate the strength parameters and deformation properties of methane hydrate-bearing interlayered sediments at the effective pressure of 1 MPa. The results indicate that the stress-strain curves of hydrate-bearing interlayered sediments are significantly different from that of hydrate-bearing sediments. The peak strength, Young's modulus, initial yielding modulus, and failure mode are deeply affected by the methane hydrate distribution. The failure behaviors and mechanism of strain softening and hardening patterns of the interlayered specimens are more complicated than those of the integrated specimens. This study compares the different mechanical behaviors between integrated and interlayered specimens containing gas hydrate, which can serve as a reference for the prediction and analysis of the deformation behaviors of natural gas hydrate reservoirs. [ABSTRACT FROM AUTHOR]

The undrained shear strength of shallow strata is a critical parameter for safety design in deep-water operations. In situ piezocone penetration tests (CPTU) and laboratory experiments are performed at Site W18-19 in the Shenhu area, northern South China Sea, where China's first marine hydrate exploitation operation is due to be located. The validation of the undrained shear strength prediction model based on CPTU parameters. Different laboratory tests, including pocket penetrometer, torvane, miniature vane and unconsolidated undrained triaxial tests, are employed to solve empirical cone coefficients by statistical and mathematical methods. Finally, an optimized model is proposed to describe the longitudinal distribution of undrained shear strength in calcareous clay strata in the Shenhu area. Research results reveal that average empirical cone coefficients based on total cone resistance, effective resistance, and excess-pore pressure are 13.8, 4.2 and 14.4, respectively. The undrained shear strength prediction model shows a good fit with the laboratory results only within specific intervals based on their compaction degree and gas-bearing conditions. The optimized prediction model in piecewise function format can be used to describe the longitudinal distribution of the undrained shear strength for calcareous clay within all depth intervals from the mud-line to the upper boundary of hydrate-bearing sediments (HBS). The optimized prediction result indicates that the effective cone resistance model is suitable for very soft to firm calcareous clays, the excess-pore pressure model can depict the undrained shear strength for firm to very stiff but gas-free clays, while the total cone resistance model is advantageous for evaluating the undrained shear strength for very stiff and gassy clays. The optimized model in piecewise function format can considerably improve the adaptability of empirical models for calcareous clay in the Shenhu area. These results are significant for safety evaluations of proposed hydrate exploitation projects. [ABSTRACT FROM AUTHOR]

Marine gas hydrates accumulate primarily in coarse-grained, high-permeability layers; however, highly saturated natural gas hydrates have been discovered in the fine-grained sediments of Shenhu area, South China Sea (SCS). This may be explained by key factors, such as the great abundance of foraminifera shells. In this paper, by analyzing the SCS foraminifera structure and performing hydrate formation experiments in the foraminifera shells, the contribution of foraminifera to hydrate accumulation in the SCS was investigated from a microscopic point of view. Simulations of hydrate formation were carried out in both pure SCS foraminifera shells and the host sediments. Pore structures in typical foraminifera were studied by use of micro-focus X-ray computed tomography (CT) and scanning electron microscopy (SEM). Hydrate growth and occurrence characteristics in the foraminifera shells were observed in-situ. The results showed that the presence of foraminifera significantly enhanced the effective porosity of the SCS sediments. Moreover, while the hydrates grew preferentially in the chambers of the coarse-grained foraminifera by adhering to the inner walls of the foraminifera shells, no apparent hydrate accumulation was observed in the fine-grained or argillaceous matrix. These findings provide a basis for further studies on the accumulation mechanism of hydrates and physical properties of hydrate reservoir in the South China Sea. [ABSTRACT FROM AUTHOR]

The anaerobic oxidation of methane (AOM) has strongly developed at Core A, Site 79 of the middle Okinawa Trough, East China Sea, and a large amount of authigenic pyrite is preserved in the surface sediment. In this study, we analyze the characteristics of the authigenic pyrite and its sulfur isotopic values. The authigenic pyrite is stripy and tubular, and there were foraminifera compartments filled with pyrite. The pyrite is extracted using chromium reduction, and the values of δS are found to lie between -41.20‰ and 8.92‰ V-CDT. The bulk pyrite tends to be more enriched in 34S with increasing depth. Particularly, the δS value of the pyrite lies between -32.73‰ and -41.20‰ V-CDT above 278 cmbsf, but it quickly increases below this depth (-21.49‰-8.92‰ V-CDT). At the same time, the total sulfur content of the pyrite shows an abrupt increase above 100 cmbsf but is otherwise stable between 1.04% and 0.55% below 100 cmbsf. The stable and negative values of δS and the decreasing values of total sulfur above 278 cmbsf indicate reduced AOM activities in 17.18-5.3 ka. In addition, the increasing δS and pyrite content indicate strong AOM development and methane seep below 278 cmbsf in 18.8-17.18 ka. In particular, the highest positive value of δS occurring in 18.78 ka indicates the most intense AOM activity. The shallow sulfate-methane interface (SMI) and high methane flux below marine sediments also strongly support this activity. [ABSTRACT FROM AUTHOR]

Anaerobic oxidation of methane and sulfate reduction was studied in the pore waters of four cores at two stations of the middle Okinawa Trough. Pore water vertical distributions of sulfate, methane, sulfide, total alkalinity, ammonium, and phosphate were determined in this study. Our results show strong linear sulfate concentration gradients of 6.83 mmol m in Core A and 5.96 mmol m in Core C, which were collected from two stations. Concurrent variations of methane, total alkalinity and hydrogen sulfide all exhibit steep increases with depth at both cores, which indicate active methane seep activities around two stations. Pore water ammonium and phosphate concentrations reveal minor influences of organic matter degradation on sulfate reduction at two stations. Sulfate methane interface (SMI) was extrapolated from linear sulfate profiles in methane seep cores. Shallower SMI depths (A: 4.9 mbsf; C: 5.4 mbsf) indicate strong methane fluxes and active anaerobic oxidation of methane in the underlying sediments. [ABSTRACT FROM AUTHOR]

To revise P-wave velocity and thickness of the hydrate layer in the Shenhu area of the South China Sea, acoustic and resistivity logging curves are reanalyzed. The waterlogging phenomenon is found in the shallow sediments of five drilling wells, which causes P-wave velocity to approximate the propagation velocity of sea water (about 1500 m s). This also affects the identification of the hydrate layer and results in the underestimate of its thickness. In addition, because there could be about a 5 m thick velocity ramp above or below the hydrate layer as interpreted by acoustic and resistivity logging curves, the recalibrated thickness of this layer is less than the original estimated thickness. The recalibrated P-wave velocity of the hydrate layer is also higher than the original estimated velocity. For the drilling well with a relatively thin hydrate layer, the velocity ramp plays a more important role in identifying and determining the thickness of the layer. [ABSTRACT FROM AUTHOR]

The P-T stability conditions of gas hydrate in different systems (i.e., solution, silica sand, and marine sediment) were studied using multi-step decomposition method with our experimental equipment. The effects of different ions with various concentrations and sediment grains on the P-T stability conditions of gas hydrate were investigated. The results show that different ions have different influences on the phase equilibrium of gas hydrate. However, the influence of ions is in a similar trend: the larger the concentration, the bigger the P-T curve shifts to the left. For the silica sand, the influence of pore capillarity of coarse particles (> 460 μm) can be negligible. The P-T curve measured in coarse silica is in agreement with that in pure water. However, the influence of pore capillarity of fine particles (< 35 μm) is significant. The maximum reduction value of temperature is 1.5 K for methane hydrate under stable state. The sediment from the South China Sea significantly affects the P-T stability conditions of methane hydrate, with an average reduction value of 1.9 K within the experimental conditions. This is mainly the result of both the pore water salinity and the pore capillarity of sediment. Because the pore water salinity is keeping diluted by the fresh water released from hydrate dissociation, the measured P-T stability points fall on different P-T curves with the decreasing salinity. [ABSTRACT FROM AUTHOR]