Probing Oxidation Mechanisms in Plasmonic Catalysis: Unraveling the Role of Reactive Oxygen Species.

Plasmon-induced oxidation has conventionally been attributed to the transfer of plasmonic hot holes. However, this theoretical framework encounters challenges in elucidating the latest experimental findings, such as enhanced catalytic efficiency under uncoupled irradiation conditions and superior oxidizability of silver nanoparticles. Herein, we employ liquid surface-enhanced Raman spectroscopy (SERS) as a real-time and in situ tool to explore the oxidation mechanisms in plasmonic catalysis, taking the decarboxylation of p-mercaptobenzoic acid (PMBA) as a case study. Our findings suggest that the plasmon-induced oxidation is driven by reactive oxygen species (ROS) rather than hot holes, holding true for both the Au and Ag nanoparticles. Subsequent investigations suggest that plasmon-induced ROS may arise from hot carriers or energy transfer mechanisms, exhibiting selectivity under different experimental conditions. The observations were substantiated by investigating the cleavage of the carbon-boron bonds. Furthermore, the underlying mechanisms were clarified by energy level theories, advancing our understanding of plasmonic catalysis.

Overexpression of MiR-181c-5p Attenuates Human Umbilical Vascular Endothelial Cell Injury in Deep Vein Thrombosis by Targeting FOS.

Deep venous thrombosis (DVT) is the third most common cardiovascular disease. Its clinical therapeutic effect is unsatisfactory due to the high rate of postthrombotic syndrome. Several studies have demonstrated the involvement of miRNAs in DVT. Therefore, we identified differentially expressed miRNAs in patients with DVT and explored their effects and underlying mechanism on endothelial cell (EC) injury.Differentially expressed miRNAs were identified via microRNA sequencing and verified using real-time quantitative PCR. The biological function of miR-181c-5p in human umbilical vein endothelial cell (HUVEC) injury stimulated by oxidized low-density lipoprotein (ox-LDL) was investigated. The target gene of miR-181c-5p was analyzed using bioinformatics and verified via dual-luciferase reporter assay.miRNA sequencing showed that miR-181c-5p was downregulated in the peripheral blood of patients with DVT. Furthermore, miR-181c-5p had a high clinical diagnostic value for DVT by receiver operating characteristic curve analysis. An in vitro cell model of EC injury, miR-181c-5p, was repressed in ox-LDL-treated HUVECs. Enhancing miR-181c-5p expression could alleviate the inhibition cell viability, cell apoptosis, raising ROS and MDA production, the reducing SOD level, and the elevated levels of thrombosis-related factor, ET-1 and vWF induced by ox-LDL. Further analysis revealed that FBJ osteosarcoma oncogene (FOS) is a target of miR-181c-5p and could antagonize the protective role of miR-181c-5p in ox-LDL-induced HUVEC injury.Our research demonstrated that miR-181c-5p could attenuate ox-LDL-induced EC injury and thrombosis-related factor expression by negatively regulating FOS. These findings suggest that the miR-181c-5p/FOS axis is a promising therapeutic target for DVT.

Long-term phosphorus addition alleviates CO2 and N2O emissions via altering soil microbial functions in secondary rather primary tropical forests.

Tropical forests, where the soils are nitrogen (N) rich but phosphorus (P) poor, have a disproportionate influence on global carbon (C) and N cycling. While N deposition substantially alters soil C and N retention in tropical forests, whether P input can alleviate these N-induced effects by regulating soil microbial functions remains unclear. We investigated soil microbial taxonomy and functional traits in response to 10-year independent and interactive effects of N and P additions in a primary and a secondary tropical forest in Hainan Island. In the primary forest, N addition boosted oligotrophic bacteria and phosphatase and enriched genes responsible for C-, P-mineralization, nitrification and denitrification, suggesting aggravated P limitation while N excess. This might stimulate P excavation via organic matter mineralization, and enhance N losses, thereby increasing soil CO2 and N2O emissions by 86% and 110%, respectively. Phosphorus and NP additions elevated C-mining enzymes activity mainly due to intensified C limitation, causing 82% increase in CO2 emission. In secondary forest, P and NP additions reduced phosphatase activity, enriched fungal copiotrophs and increased microbial biomass, suggesting removal of nutrient deficiencies and stimulation of fungal growth. Meanwhile, soil CO2 emission decreased by 25% and N2O emission declined by 52-82% due to alleviated P acquisition from organic matter decomposition and increased microbial C and N immobilization. Overall, N addition accelerates most microbial processes for C and N release in tropical forests. Long-term P addition increases C and N retention via reducing soil CO2 and N2O emissions in the secondary but not primary forest because of strong C limitation to microbial N immobilization. Further, the seasonal and annual variations in CO2 and N2O emissions should be considered in future studies to test the generalization of these findings and predict and model dynamics in greenhouse gas emissions and C and N cycling.

Canopy nitrogen addition enhance the photosynthetic rate of canopy species by improving leaf hydraulic conductivity in a subtropical forest.

Elucidating the effects of atmospheric nitrogen (N) deposition on the photosynthetic capacity of plants is critical to understand forest growth and conservation under global change. However, studies on this topic generally consider only understory N addition, which ignores the effect of canopy interception. In this study, we conducted a field experiment in a subtropical forest to compare the effects of canopy vs. understory N addition on the photosynthetic rate of canopy and understory species. We found that canopy N addition enhanced the photosynthetic rate of canopy species by increasing leaf hydraulic conductivity and shortening the distance of CO2 transportation. In contrast, understory N addition had non-significant effects on the photosynthetic rate of canopy species. Moreover, the photosynthetic rate of understory species was not affected by canopy or understory N addition. Interestingly, changes in hydraulic conductivity contributed more to accelerating the photosynthetic rate than changes in CO2 transport distance. Our results provide important insights into the dissimilar effects of canopy and understory N addition on the photosynthetic rates of species in subtropical forests. Based on our findings, we highlighted the urgent need to consider canopy processes in future studies on N deposition.

Retention of deposited ammonium and nitrate and its impact on the global forest carbon sink.

The impacts of enhanced nitrogen (N) deposition on the global forest carbon (C) sink and other ecosystem services may depend on whether N is deposited in reduced (mainly as ammonium) or oxidized forms (mainly as nitrate) and the subsequent fate of each. However, the fates of the two key reactive N forms and their contributions to forest C sinks are unclear. Here, we analyze results from 13 ecosystem-scale paired 15N-labelling experiments in temperate, subtropical, and tropical forests. Results show that total ecosystem N retention is similar for ammonium and nitrate, but plants take up more labelled nitrate ([Formula: see text]%) ([Formula: see text]) than ammonium ([Formula: see text]%) while soils retain more ammonium ([Formula: see text]%) than nitrate ([Formula: see text]%). We estimate that the N deposition-induced C sink in forests in the 2010s  is [Formula: see text] Pg C yr-1, higher than previous estimates because of a larger role for oxidized N and greater rates of global N deposition.

Gross ecosystem product accounting of national park: Taking Hainan Tropical Rainforest National Park as an example.

Hainan Tropical Rainforest National Park has the most representative and largest contiguous tropical rainforest in China, which has advantages in exploring the realization mechanism of ecological product value in national parks. Based on the basic framework of "The Technical Guideline on Gross Ecosystem Product (GEP)", we constructed a GEP accounting system in line with the characteristics of tropical rain forest national park, and calculated the GEP of Hainan Tropical Rainforest National Park in 2019. The results showed that the GEP of Hainan Tropical Rainforest National Park in 2019 was 204.513 billion yuan, and the GEP per unit area was 0.046 billion yuan·km-2. Among all the service types, the value of material services was 4.850 billion yuan, accounting for 2.4% of the total GEP in the national park. The ecosystem regulation service value was 168.891 billion yuan, accounting for 82.6%. The value of cultural services was 30.772 billion yuan, accounting for 15.0%. Among different ecosystem types, the unit area value of the tropical rain forest ecosystem represented by mountain rain forest, lowland rain forest, deciduous monsoon forest, and tropical cloud forest was much higher than that of plantation or other ecosystems, indicating the dominant role of tropical rain forest ecosystem in providing ecosystem services. In addition, based on the GEP accounting results of the national park, we put forward relevant suggestions for further exploring the realization path and realization mechanism of ecological product value.

Contrasting Hydraulic Efficiency and Photosynthesis Strategy in Differential Successional Stages of a Subtropical Forest in a Karst Region.

Understanding the successional process from a disturbed forest to a mature forest is essential for species recovery and conservation initiatives. The resource acquisition and drought tolerance of plants can be instructive to predictions of species abundance and distribution for different forests. However, they have not been adequately tested at different successional stages in karst regions. Here, we selected seven dominant species in an early-succession forest and 17 species in a late-succession forest in a karst region of southwestern China. Resource acquisition-related traits such as hydraulic conductivity and photosynthetic rate, and drought tolerance-related traits, including turgor loss point and wood density, were measured. We found that species in the early-succession forest had a higher hydraulic conductance and photosynthetic rate than those in the late-succession forest, while leaf water potential at turgor loss point and wood density showed nonsignificant differences between the two forests. In addition, we observed a significant negative relationship between photosynthetic rate and drought tolerance in the early-succession forest, which was not identified in late-succession forests. Our study indicates that resource acquisition rather than drought tolerance was the key factor explaining plant distributions in forests at different successional stages in karst regions. We also suggest that the resource acquisition and drought tolerance trade-off hypothesis is not always supported for karst region species. Our study could inform about the design of species replacements in successional forests and provide forest management and restoration guidelines for karst regions.

Regioselective C-H Phosphorothiolation of (Hetero)arenes Enabled by the Synergy of Electrooxidation and Ultrasonic Irradiation.

An electrochemically regioselective C-H phosphorothiolation of (hetero)arenes with thiocyanate as the S source under ultrasonic irradiation has been developed. The synergistic cooperation of electrooxidation and ultrasonication markedly accelerated the C-H phosphorothiolation reaction. This mechanistically different method is distinguished by its wide substrate scope and transition-metal-free and external-oxidant-free conditions, thus complementing the existing metal-catalyzed or peroxide-mediated protocols for the green synthesis of S-(hetero)aryl phosphorothioates.

Quantification of trivalent non-replicating rotavirus vaccine antigens in the presence of aluminum adjuvant.

Parenterally administered rotavirus vaccines may overcome the low efficacy observed in resource-poor regions that use live oral formulations. We have reported work on a trivalent nonreplicating rotavirus vaccine (NRRV) for parenteral administration consisting of the recombinant tetanus toxoid P2 CD4 epitope fused to a truncated VP8* fragment (P2-VP8*) for the P[4], P[6], and P[8] serotypes of rotavirus adjuvanted with aluminum. An essential part of developing this vaccine candidate was devising quantification methods for each antigen in the trivalent NRRV in the presence of aluminum adjuvant. This report describes the development of quantitative inhibition enzyme-linked immunosorbent assays (ELISAs) for in vitro antigenicity determination of the adjuvanted trivalent NRRV using serotype-specific monoclonal antibodies (mAbs) against each of the P2-VP8* antigens. Adjuvanted trivalent vaccine samples are titrated and incubated with a constant concentration of specific mAbs against each NRRV P2-VP8* antigen variant. Unbound mAbs are measured by ELISA to indirectly quantify the amount of each antigen present in the trivalent vaccine. Sensitive, specific, and reproducible inhibition ELISAs were developed and qualified for each antigen and used for final product quantification and release testing without desorption of the vaccine antigen.

Gasification biochar from horticultural waste: An exemplar of the circular economy in Singapore.

Organic waste, the predominant component of global solid waste, has never been higher, resulting in increased landfilling, incineration, and open dumping that releases greenhouse gases and toxins that contribute to global warming and environmental pollution. The need to create and adopt sustainable closed-loop systems for waste reduction and valorization is critical. Using organic waste as a feedstock, gasification and pyrolysis systems can produce biooil, syngas, and thermal energy, while reducing waste mass by as much as 85-95% through conversion into biochar, a valuable byproduct with myriad uses from soil conditioning to bioremediation and carbon sequestration. Here, we present a novel case study detailing the circular economy of gasification biochar in Singapore's Gardens by the Bay. Biochar produced from horticultural waste within the Gardens was tested as a partial peat moss substitute in growing lettuce, pak choi, and pansy, and found to be a viable substitute for peat moss. At low percentages of 20-30% gasification biochar, fresh weight yields for lettuce and pak choi were comparable to or exceeded those of plants grown in pure peat moss. The biochar was also analyzed as a potential additive to concrete, with a 2% biochar mortar compound found to be of suitable strength for non-structural functions, such as sidewalks, ditches, and other civil applications. These results demonstrate the global potential of circular economies based on local biochar creation and on-site use through the valorization of horticultural waste via gasification, generating clean, renewable heat or electricity, and producing a carbon-neutral to -negative byproduct in the form of biochar. They also indicate the potential of scaled-up pyrolysis or gasification systems for a circular economy in waste management.

Dynamics and multi-annual fate of atmospherically deposited nitrogen in montane tropical forests.

The effects of nitrogen (N) deposition on forests largely depend on its fate after entering the ecosystem. While several studies have addressed the forest fate of N deposition using 15 N tracers, the long-term fate and redistribution of deposited N in tropical forests remains unknown. Here, we applied 15 N tracers to examine the fates of deposited ammonium ( NH 4 + ) and nitrate ( NO 3 - ) separately over 3 years in a primary and a secondary tropical montane forest in southern China. Three months after 15 N tracer addition, over 60% of 15 N was retained in the forests studied. Total ecosystem retention did not change over the study period, but between 3 months and 3 years following deposition 15 N recovery in plants increased from 10% to 19% and 13% to 22% in the primary and secondary forests, respectively, while 15 N recovery in the organic soil declined from 16% to 2% and 9% to 2%. Mineral soil retained 50% and 35% of 15 N in the primary and secondary forests, with retention being stable over time. The total ecosystem retention of the two N forms did not differ significantly, but plants retained more 15 NO 3 - than 15 NH 4 + and the organic soil more 15 NH 4 + than NO 3 - . Mineral soil did not differ in 15 NH 4 + and 15 NO 3 - retention. Compared to temperate forests, proportionally more 15 N was distributed to mineral soil and plants in these tropical forests. Overall, our results suggest that atmospherically deposited NH 4 + and NO 3 - is rapidly lost in the short term (months) but thereafter securely retained within the ecosystem, with retained N becoming redistributed to plants and mineral soil from the organic soil. This long-term N retention may benefit tropical montane forest growth and enhance ecosystem carbon sequestration.

Biochar industry to circular economy.

Biochar, produced as a by-product of pyrolysis/gasification of waste biomass, shows great potential to reduce the environment impact, address the climate change issue, and establish a circular economy model. Despite the promising outlook, the research on the benefits of biochar remains highly debated. This has been attributed to the heterogeneity of biochar itself, with its inherent physical, chemical and biological properties highly influenced by production variables such as feedstock types and treating conditions. Hence, to enable meaningful comparison of results, establishment of an agreed international standard to govern the production of biochar for specific uses is necessary. In this study, we analyzed four key uses of biochar: 1) in agriculture and horticulture, 2) as construction material, 3) as activated carbon, and 4) in anaerobic digestion. Then the guidelines for the properties of biochar, especially for the concentrations of toxic heavy metals, for its environmental friendly application were proposed in the context of Singapore. The international status of the biochar industry code of practice, feedback from Singapore local industry and government agencies, as well as future perspectives for the biochar industry were explained.

Formulation and preclinical studies with a trivalent rotavirus P2-VP8 subunit vaccine.

More effective rotavirus vaccines are essential for preventing extensive diarrheal morbidity and mortality in children under five years of age in low-resource regions. Nonreplicating rotavirus vaccines (NRRV) administered parenterally provide an alternate vaccination method to the current licensed oral vaccine. Live attenuated vaccines and may generate increased efficacy in low-resource settings because the parenteral administration route bypasses some of the challenges associated with oral administration, including differences in intestinal environments. Work described here supports development of a trivalent NRRV vaccine for parenteral administration to avoid complications of the gastrointestinal route. Recombinant VP8* subunit proteins representing some of the most prevalent strains of rotavirus infecting humans - DS-1 (P[4]), 1076 (P[6]), and Wa (P[8]) - were combined with an aluminum adjuvant and the P2 epitope of tetanus toxoid to enhance the immune response to this NRRV antigen. Vaccine formulation development included selection of aluminum hydroxide (Alhydrogel®) as an appropriate adjuvant as well as an optimal buffer to maintain antigen stability and optimize antigen binding to the adjuvant. Characterization assays were used to select the lead vaccine formulation and monitor formulation stability. The NRRV liquid formulation was stable for one year at 2°C to 8°C and four weeks at 37°C. Immunogenicity of the NRRV formulation was evaluated using a guinea pig model, where we demonstrated that the adjuvant provided a 20-fold increase in neutralization titer against a homologous antigen and that the P2-fusion also enhanced the serum neutralizing antibody responses. This vaccine candidate is currently being evaluated in human clinical trials.

Small molecular modulators of JMJD1C preferentially inhibit growth of leukemia cells.

Histone demethylases are promising therapeutic targets as they play fundamental roles for survival of Mixed lineage leukemia rearranged acute leukemia (MLLr AL). Here we focused on the catalytic Jumonji domain of histone H3 lysine 9 (H3K9) demethylase JMJD1C to screen for potential small molecular modulators from 149,519 natural products and 33,765 Chinese medicine components via virtual screening. JMJD1C Jumonji domain inhibitor 4 (JDI-4) and JDI-12 that share a common structural backbone were detected within the top 15 compounds. Surface plasmon resonance analysis showed that JDI-4 and JDI-12 bind to JMJD1C and its family homolog KDM3B with modest affinity. In vitro demethylation assays showed that JDI-4 can reverse the H3K9 demethylation conferred by KDM3B. In vivo demethylation assays indicated that JDI-4 and JDI-12 could induce the global increase of H3K9 methylation. Cell proliferation and colony formation assays documented that JDI-4 and JDI-12 kill MLLr AL and other malignant hematopoietic cells, but not leukemia cells resistant to JMJD1C depletion or cord blood cells. Furthermore, JDI-16, among multiple compounds structurally akin to JDI-4/JDI-12, exhibits superior killing activities against malignant hematopoietic cells compared to JDI-4/JDI-12. Mechanistically, JDI-16 not only induces apoptosis but also differentiation of MLLr AL cells. RNA sequencing and quantitative PCR showed that JDI-16 induced gene expression associated with cell metabolism; targeted metabolomics revealed that JDI-16 downregulates lactic acids, NADP+ and other metabolites. Moreover, JDI-16 collaborates with all-trans retinoic acid to repress MLLr AML cells. In summary, we identified bona fide JMJD1C inhibitors that induce preferential death of MLLr AL cells.

Contrasting Soil Bacterial Community, Diversity, and Function in Two Forests in China.

Bacteria are the highest abundant microorganisms in the soil. To investigate bacteria community structures, diversity, and functions, contrasting them in four different seasons all the year round with/within two different forest type soils of China. We analyzed soil bacterial community based on 16S rRNA gene sequencing via Illumina HiSeq platform at a temperate deciduous broad-leaved forest (Baotianman, BTM) and a tropical rainforest (Jianfengling, JFL). We obtained 51,137 operational taxonomic units (OTUs) and classified them into 44 phyla and 556 known genera, 18.2% of which had a relative abundance >1%. The composition in each phylum was similar between the two forest sites. Proteobacteria and Acidobacteria were the most abundant phyla in the soil samples between the two forest sites. The Shannon index did not significantly differ among the four seasons at BTM or JFL and was higher at BTM than JFL in each season. The bacteria community at both BTM and JFL showed two significant (P < 0.05) predicted functions related to carbon cycle (anoxygenic photoautotrophy sulfur oxidizing and anoxygenic photoautotrophy) and three significant (P < 0.05) predicted functions related to nitrogen cycle (nitrous denitrificaton, nitrite denitrification, and nitrous oxide denitrification). We provide the basis on how changes in bacterial community composition and diversity leading to differences in carbon and nitrogen cycles at the two forests.

High nitrogen isotope fractionation of nitrate during denitrification in four forest soils and its implications for denitrification rate estimates.

Denitrification is a major process contributing to the removal of nitrogen (N) from ecosystems, but its rate is difficult to quantify. The natural abundance of isotopes can be used to identify the occurrence of denitrification and has recently been used to quantify denitrification rates at the ecosystem level. However, the technique requires an understanding of the isotopic enrichment factor associated with denitrification, which few studies have investigated in forest soils. Here, soils collected from two tropical and two temperate forests in China were incubated under anaerobic or aerobic laboratory conditions for two weeks to determine the N and oxygen (O) isotope enrichment factors during denitrification. We found that at room temperature (20°C), NO3- was reduced at a rate of 0.17 to 0.35µgNg-1h-1, accompanied by the isotope fractionation of N (15ε) and O (18ε) of 31‰ to 65‰ (48.3±2.0‰ on average) and 11‰ to 39‰ (18.9±1.7‰ on average), respectively. The N isotope effects were, unexpectedly, much higher than reported in the literature for heterotrophic denitrification (typically ranging from 5‰ to 30‰) and in other environmental settings (e.g., groundwater, marine sediments and agricultural soils). In addition, the ratios of Δδ18O:Δδ15N ranged from 0.28 to 0.60 (0.38±0.02 on average), which were lower than the canonical ratios of 0.5 to 1 for denitrification reported in other terrestrial and freshwater systems. We suggest that the isotope effects of denitrification for soils may vary greatly among regions and soil types and that gaseous N losses may have been overestimated for terrestrial ecosystems in previous studies in which lower fractionation factors were applied.

Assessing the potency and immunogenicity of inactivated poliovirus vaccine after exposure to freezing temperatures.

According to manufacturers, inactivated poliovirus vaccines (IPVs) are freeze sensitive and require storage between 2°C and 8°C, whereas oral poliovirus vaccine requires storage at -20 °C. Introducing IPV into ongoing immunization services might result in accidental exposure to freezing temperatures and potential loss of vaccine potency. To better understand the effect of freezing IPVs, samples of single-dose vaccine vials from Statens Serum Institut (VeroPol) and multi-dose vaccine vials from Sanofi Pasteur (IPOL) were exposed to freezing temperatures mimicking what a vaccine vial might encounter in the field. D-antigen content was measured to determine the in vitro potency by ELISA. Immunogenicity testing was conducted for a subset of exposed IPVs using the rat model. Freezing VeroPol had no detectable effect on in vitro potency (D-antigen content) in all exposures tested. Freezing of the IPOL vaccine for 7 days at -20 °C showed statistically significant decreases in D-antigen content by ELISA in poliovirus type 1 (p < 0.0001) and type 3 (p = 0.048). Reduction of poliovirus type 2 potency also approached significance (p = 0.062). The observed loss in D-antigen content did not affect immunogenicity in the rat model. Further work is required to determine the significance of the loss observed and the implications for vaccine handling policies and practices.

Preformulation studies with the Escherichia coli double mutant heat-labile toxin adjuvant for use in an oral vaccine.

Double mutant heat-labile toxin (dmLT) is a promising adjuvant for oral vaccine administration. The aims of our study were to develop sensitive methods to detect low concentrations of dmLT and to use the assays in preformulation studies to determine whether dmLT remains stable under conditions encountered by an oral vaccine. We developed a sandwich ELISA specific for intact dmLT and a sensitive SDS-PAGE densitometry method, and tested stability of dmLT in glass and plastic containers, in saliva, at the pH of stomach fluid, and in high-osmolarity buffers. The developed ELISA has a quantification range of 62.5 to 0.9ng/mL and lower limit of detection of 0.3ng/mL; the limit of quantification of the SDS-PAGE is 10µg/mL. This work demonstrates the application of dmLT assays in preformulation studies to development of an oral vaccine containing dmLT. Assays reported here will facilitate the understanding and use of dmLT as an adjuvant.

Development of a Freeze-Dried, Heat-Stable Influenza Subunit Vaccine Formulation.

An influenza pandemic remains a major public health concern. A key strategy to prevent a pandemic is to stockpile and pre-position stable influenza vaccine to allow rapid deployment in response to an outbreak. However, most influenza vaccines today are formulated as liquids that are stable only within a temperature range of 2°C to 8°C and require use of a cold chain, making vaccine transportation, distribution, and storage complicated and expensive, particularly for developing countries. To support the National Strategy for Pandemic Influenza preparedness in the United States and internationally, we developed two lead dry formulations of stable H1N1 influenza subunit vaccines using freeze-drying technology. The stable formulations contain an excipient combination of a disaccharide, such as sucrose or trehalose, and glycine, in addition to a surfactant and phosphate buffer. The freeze-dried vaccines were shown to be safe and remained immunogenic in an in vivo study in mice. Moreover, the lead formulations demonstrated no significant loss of activity after 40 months at storage temperatures of 25°C and 37°C. This stability can be particularly attractive as it could eliminate the need to use a cold chain for vaccine deployment and facilitate integration of vaccine distribution with general drug distribution where appropriate. These freeze-dried thermostable influenza subunit vaccines could also reduce the frequency of vaccine stockpile turnover, offering a cost-effective option for pandemic preparedness.

Development of a stable liquid formulation of live attenuated influenza vaccine.

Vaccination is the most effective means of preventing influenza. However, the cost of producing annual seasonal influenza vaccines puts them out of reach for most developing countries. While live attenuated influenza vaccines are among the most efficacious and can be manufactured at low cost, they may require lyophilization to be stable enough for developing-country use, which adds a significant cost burden. The development of a liquid live attenuated seasonal influenza vaccine that is stable for around a year-the duration of an annual influenza season-would significantly improve not only the production output but also the use and accessibility of influenza vaccines in low-resource settings. In this study, potential stabilizing excipients were screened and optimized using the least stable influenza vaccine strain presently known, H1N1 (A/California/07/2009), as a model. The stability-conferring properties of the lead formulations were also tested with a Type B strain of influenza virus (B/Brisbane/60/2008). Stability was also evaluated with higher titers of influenza virus and exposure to agitation and freeze-thaw stresses to further confirm the stability of the lead formulations. Through this process, we identified a liquid formulation consisting of sucrose phosphate glutamate buffer with 1% arginine and 0.5% recombinant human serum albumin that provided storage stability of one year at 2-8°C for the influenza A and B strains tested.