Structural and functional basis of low-affinity SAM/SAH-binding in the conserved MTase of the multi-segmented Alongshan virus distantly related to canonical unsegmented flaviviruses.

Alongshan virus (ALSV), a newly discovered member of unclassified Flaviviridae family, is able to infect humans. ALSV has a multi-segmented genome organization and is evolutionarily distant from canonical mono-segmented flaviviruses. The virus-encoded methyltransferase (MTase) plays an important role in viral replication. Here we show that ALSV MTase readily binds S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) but exhibits significantly lower affinities than canonical flaviviral MTases. Structures of ALSV MTase in the free and SAM/SAH-bound forms reveal that the viral enzyme possesses a unique loop-element lining side-wall of the SAM/SAH-binding pocket. While the equivalent loop in flaviviral MTases half-covers SAM/SAH, contributing multiple hydrogen-bond interactions; the pocket-lining loop of ALSV MTase is of short-length and high-flexibility, devoid of any physical contacts with SAM/SAH. Subsequent mutagenesis data further corroborate such structural difference affecting SAM/SAH-binding. Finally, we also report the structure of ALSV MTase bound with sinefungin, an SAM-analogue MTase inhibitor. These data have delineated the basis for the low-affinity interaction between ALSV MTase and SAM/SAH and should inform on antiviral drug design.

Development of a bispecific nanobody conjugate broadly neutralizes diverse SARS-CoV-2 variants and structural basis for its broad neutralization.

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and profound immune-escape capacity makes it an urgent need to develop broad-spectrum therapeutics. Nanobodies have recently attracted extensive attentions due to their excellent biochemical and binding properties. Here, we report two high-affinity nanobodies (Nb-015 and Nb-021) that target non-overlapping epitopes in SARS-CoV-2 S-RBD. Both nanobodies could efficiently neutralize diverse viruses of SARS-CoV-2. The neutralizing mechanisms for the two nanobodies are further delineated by high-resolution nanobody/S-RBD complex structures. In addition, an Fc-based tetravalent nanobody format is constructed by combining Nb-015 and Nb-021. The resultant nanobody conjugate, designated as Nb-X2-Fc, exhibits significantly enhanced breadth and potency against all-tested SARS-CoV-2 variants, including Omicron sub-lineages. These data demonstrate that Nb-X2-Fc could serve as an effective drug candidate for the treatment of SARS-CoV-2 infection, deserving further in-vivo evaluations in the future.

Factors influencing the farmer's chemical fertilizer reduction behavior from the perspective of farmer differentiation.

Farmers are the primary decision-makers in chemical fertilizer application. Identifying their chemical fertilizer reduction behavior and its influencing factors is critical to controlling the surface source pollution caused by excessive fertilizer. This paper incorporates farmer characteristic, technology cognition and social capital into the analytical framework of farmers' fertilizer reduction behavior. Based on 889 farmers' questionnaire data, this paper constructs a Structural Equation Model to analyze farmers' fertilizer reduction behavior and its influencing factors from the perspective of farmers' differentiation. The findings are as follows. (1) Farmer characteristics, technology cognition and social capital all positively influences farmers' chemical fertilizer reduction behavior. Among them, social capital has the highest degree of influence on farmers' chemical fertilizer reduction behavior, followed by farmer characteristics, while technology cognition shows the lowest influence. (2) The effect of technology cognition on small-scale pure farmers' chemical fertilizer reduction behavior is insignificant. However, the effect on part-time farmers and large professional farmers is significant, and the effect is higher for large professional farmers. (3) The effect of farmer characteristics on part-time farmers' chemical fertilizer reduction behavior is insignificant. However, the effect on small-scale pure farmers and large professional farmers is significant, and the effect is higher for large professional farmers. (4) The effect of social capital on chemical fertilizer reduction behavior of small-scale pure farmers, part-time farmers, and large professional farmers is significant. This effect is the highest for part-time farmers, followed by large professional farmers, and the lowest for small-scale pure farmers. Accordingly, to continuously promote chemical fertilizer reduction, the government should focus on strengthening policy support, technical support, education guidance and classification.

An engineered 5-helix bundle derived from SARS-CoV-2 S2 pre-binds sarbecoviral spike at both serological- and endosomal-pH to inhibit virus entry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related sarbecoviruses enter host cells by receptor-recognition and membrane-fusion. An indispensable step in fusion is the formation of 6-helix bundle by viral spike heptad repeats 1 and 2 (HR1 and HR2). Here, we report the construction of 5-helix bundle (5HB) proteins for virus infection inhibition. The optimal construct inhibits SARS-CoV-2 pseudovirus entry with sub-micromolar IC50. Unlike HR2-based peptides that cannot bind spike in the pre-fusion conformation, 5HB features with the capability of binding to pre-fusion spike. Furthermore, 5HB binds viral HR2 at both serological- and endosomal-pH, highlighting its entry-inhibition capacity when SARS-CoV-2 enters via either cell membrane fusion or endosomal route. Finally, we show that 5HB could neutralize S-mediated entry of the predominant SARS-CoV-2 variants and a wide spectrum of sarbecoviruses. These data provide proof-of-concept evidence that 5HB might be developed for the prevention and treatment of SARS-CoV-2 and other emerging sarbecovirus infections.

Thermal degradation of hexachlorobenzene in the presence of calcium oxide at 340-400 °C.

Hexachlorobenzene (HCB) in the milligram range was co-heated with calcium oxide (CaO) powder in sealed glass ampoules at 340-400 °C. The heated samples were characterized and analyzed by Raman spectroscopy, elemental analysis, gas chromatography/mass spectrometry, ion chromatography, and thermal/optical carbon analysis. The degradation products of HCB were studied at different temperatures and heated times. The amorphous carbon was firstly quantitatively evaluated and was thought to be important fate of the C element of HCB. The yield of amorphous carbon in products increased with heating time, for samples treated for 8h at 340, 380 °C and 400 °C, the value were 17.5%, 34.8% and 50.2%, respectively. After identification of the dechlorination products, the HCB degradation on CaO at 340-400 °C was supposed to through dechlorination/polymerization pathway, which is induced by electron transfer, generate chloride ions and form high-molecular weight intermediates with significant levels of both hydrogen and chlorine, and finally form amorphous carbon. Higher temperature was beneficial for the dechlorination/polymerization efficiency. The results are helpful for clarifying the reaction mechanism for thermal degradation of chlorinated aromatics in alkaline matrices.

[Characteristics of municipal sludge and vacuum filtration thickening process].

It was found that sludge total solid (TS) concentration was equal to chemical oxygen demand (COD), while volatile solid (VS) was 1.5 times of COD concentration. R2 of linear regression of TS and VS with COD was 0.9314 and 0.9228 respectively. Total COD in sludge was approximately 60% of that removed in water treatment process. Sludge contained high level protein and low fat. The TS of present gravity thickening sludge was universally lower than 3.3%. Efficiency of vacuum filtration process was determined by sludge type, sludge solid concentration, PAM molecular weight and PAM addition dose. Under - 34.7 kPa pressure, sludge dry solid filtration thickening rate of primary sludge was up to 31 kg/(m2 x h). While, for wasted actived sludge the rate was lower than 15 kg/(m2 x h). Rate of gravity thickening sludge was up to 43 kg/(m2 x h). TS of vacuum filtrate were lower than 1.5 g/L.