High-Resolution Structure of the N-Terminal Endonuclease Domain of the Lassa Virus L Polymerase in Complex with Magnesium Ions

Lassa virus (LASV) causes deadly hemorrhagic fever disease for which there are no vaccines and limited treatments. LASV-encoded L polymerase is required for viral RNA replication and transcription. The functional domains of L–a large protein of 2218 amino acid residues–are largely undefined, except for the centrally located RNA-dependent RNA polymerase (RdRP) motif. Recent structural and functional analyses of the N-terminal region of the L protein from lymphocytic choriomeningitis virus (LCMV), which is in the same Arenaviridae family as LASV, have identified an endonuclease domain that presumably cleaves the cap structures of host mRNAs in order to initiate viral transcription. Here we present a high-resolution crystal structure of the N-terminal 173-aa region of the LASV L protein (LASV L173) in complex with magnesium ions at 1.72 Å. The structure is highly homologous to other known viral endonucleases of arena- (LCMV NL1), orthomyxo- (influenza virus PA), and bunyaviruses (La Crosse virus NL1). Although the catalytic residues (D89, E102 and K122) are highly conserved among the known viral endonucleases, LASV L endonuclease structure shows some notable differences. Our data collected from in vitro endonuclease assays and a reporter-based LASV minigenome transcriptional assay in mammalian cells confirm structural prediction of LASV L173 as an active endonuclease. The high-resolution structure of the LASV L endonuclease domain in complex with magnesium ions should aid the development of antivirals against lethal Lassa hemorrhagic fever

Crystal structure of the outer membrane protein OmpU from Vibrio cholerae at 2.2 Å resolution

Vibrio cholerae causes a severe disease that kills thousands of people annually. The outer membrane protein OmpU is the most abundant outer membrane protein in V. cholerae, and has been identified as an important virulence factor that is involved in host-cell interaction and recognition, as well as being critical for the survival of the pathogenic V. cholerae in the host body and in harsh environments. The mechanism of these processes is not well understood owing to a lack of the structure of V. cholerae OmpU. Here, the crystal structure of the V. cholerae OmpU trimer is reported to a resolution of 2.2 Å. The protomer forms a 16-β-stranded barrel with a noncanonical N-terminal coil located in the lumen of the barrel that consists of residues Gly32–Ser42 and is observed to participate in forming the second gate in the pore. By mapping the published functional data onto the OmpU structure, the OmpU structure reinforces the notion that the long extracellular loop L4 with a β-hairpin-like motif may be critical for host-cell binding and invasion, while L3, L4 and L8 are crucially implicated in phage recognition by V. cholerae

Crystal structure of the lipopolysaccharide outer core galactosyltransferase WaaB involved in pathogenic bacterial invasion of host cells

Lipopolysaccharide (LPS) is essential for most gram-negative bacteria and plays an important role in serum resistance, pathogenesis, drug resistance, and protection from harsh environments. The outer core oligosaccharide of LPS is involved in bacterial recognition and invasion of host cells. The D-galactosyltransferase WaaB is responsible for the addition of D-galactose to the outer core oligosaccharide of LPS, which is essential for Salmonella typhimurium invasion. Here we report the first crystal structures of WaaB and WaaB in complex with UDP to resolutions of 1.8 and 1.9 Å, respectively. Mutagenesis and enzyme activity assays confirmed that residues V186, K195, I216, W243, E276, and E269 of WaaB are essential for the binding and hydrolysis of UDP-galactose. The elucidation of the catalytic mechanism of WaaB is of great importance and could potentially be used for the design of novel therapeutic reagents

THE PROBLEM OF THE RELATIONSHIP OF SCIENCE AND RELIGION IN THE SYSTEM OF SECULAR GENERAL EDUCATION

The relationship between science and religion throughout history ranged from opposition to their unity. The long-standing prevalence of theology over science has caused a counter-atheistic reaction, which has grown into the absolutization of science in all spheres of life. The introduction to the secular general education of the course “Fundamentals of Religious Cultures and Secular Ethics” was ambiguously accepted by society. The author substantiates the position that science and religion are not mutually exclusive, but rather can complement each other, forming a unified picture of the world among the younger generation.Взаимоотношения науки и религии на протяжении всей истории выстраивались от противопоставления до их единства. Многолетнее преобладание богословия над наукой вызвало встречную атеистическую реакцию, переросшую в абсолютизацию науки во всех сферах бытия. Введение в светское общее образование курса «Основы религиозных культур и светской этики» было неоднозначно воспринято обществом. Автор обосновывает позицию, что наука и религия не взаимно исключают друг друга, а напротив, могут дополнять друг друга, формируя у подрастающего поколения единую картину мира

Blocking interaction between SHP2 and PD‐1 denotes a novel opportunity for developing PD‐1 inhibitors

Small molecular PD‐1 inhibitors are lacking in current immuno‐oncology clinic. PD‐1/PD‐L1 antibody inhibitors currently approved for clinical usage block interaction between PD‐L1 and PD‐1 to enhance cytotoxicity of CD8+ cytotoxic T lymphocyte (CTL). Whether other steps along the PD‐1 signaling pathway can be targeted remains to be determined. Here, we report that methylene blue (MB), an FDA‐approved chemical for treating methemoglobinemia, potently inhibits PD‐1 signaling. MB enhances the cytotoxicity, activation, cell proliferation, and cytokine‐secreting activity of CTL inhibited by PD‐1. Mechanistically, MB blocks interaction between Y248‐phosphorylated immunoreceptor tyrosine‐based switch motif (ITSM) of human PD‐1 and SHP2. MB enables activated CTL to shrink PD‐L1 expressing tumor allografts and autochthonous lung cancers in a transgenic mouse model. MB also effectively counteracts the PD‐1 signaling on human T cells isolated from peripheral blood of healthy donors. Thus, we identify an FDA‐approved chemical capable of potently inhibiting the function of PD‐1. Equally important, our work sheds light on a novel strategy to develop inhibitors targeting PD‐1 signaling axis

Cryo-EM structures of lipopolysaccharide transporter LptB2FGC in lipopolysaccharide or AMP-PNP-bound states reveal its transport mechanism

Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through a bridge formed by seven membrane proteins (LptBFGCADE). The IM component LptB2FG powers the process through a yet unclarified mechanism. Here we report three high-resolution cryo-EM structures of LptB2FG alone and complexed with LptC (LptB2FGC), trapped in either the LPS- or AMP-PNP-bound state. The structures reveal conformational changes between these states and substrate binding with or without LptC. We identify two functional transmembrane arginine-containing loops interacting with the bound AMP-PNP and elucidate allosteric communications between the domains. AMP-PNP binding induces an inward rotation and shift of the transmembrane helices of LptFG and LptC to tighten the cavity, with the closure of two lateral gates, to eventually expel LPS into the bridge. Functional assays reveal the functionality of the LptF and LptG periplasmic domains. Our findings shed light on the LPS transport mechanism

Lipopolysaccharide is Inserted into the Outer Membrane through An Intramembrane Hole, A Lumen Gate, and the Lateral Opening of LptD

Lipopolysaccharide (LPS) is essential for the vitality of most Gram-negative bacteria and plays an important role in bacterial multidrug resistance. The LptD/E translocon inserts LPS into the outer leaflet, the mechanism of which is poorly understood. Here, we report mutagenesis, functional assays, and molecular dynamics simulations of the LptD/E complex, which suggest two distinct pathways for the insertion of LPS. The N-terminal domain of LptD comprises a hydrophobic slide that injects the acyl tails of LPS directly into the outer membrane through an intramembrane hole, while the core oligosaccharide and O-antigen pass a lumen gate that triggers the unzipping of the lateral opening between strands β1C and β26C of the barrel of LptD, to finalize LPS insertion. Mutation of the LPS transport related residues or block of the LPS transport pathways results in the deaths of Escherichia coli. These findings are important for the development of novel antibiotics

Neurodevelopmental toxicity assessments of alkyl phenanthrene and Dechlorane Plus co-exposure in zebrafish.

Alkyl phenanthrene (A-Phen) and Dechlorane Plus (DP) are ubiquitous environmental pollutants that widely co-exist in the environment. It has been established that both A-Phen and DP elicit neurotoxicity, but the potential interactive toxicity of these contaminants is not well-known. To determine whether a mixture of A-Phen and DP would exhibit interactive effects on neurodevelopment, we co-exposed 3-methylphenanthrene (3-MP), a representative of A-Phen, with DP. Our results illustrated that exposure to 5 or 20 μg/L 3-MP alone or in combination with 60 μg/L DP caused neurobehavioral anomalies in zebrafish. In accordance with the behavioral deficits, 3-MP alone or co-exposed with DP significantly decreased axonal growth of secondary motoneurons, altered intracellular Ca2+ homeostasis and induced cell apoptosis in the muscle of zebrafish. Additionally, 3-MP alone or co-exposed with DP significantly increased reactive oxygen species (ROS) and the mRNA levels of apoptosis-related genes. These findings indicate that 3-MP alone or co-exposed with DP induces neurobehavioral deficits through the combined effects on neuronal connectivity and muscle function. Chemical analysis revealed significant increases in 3-MP and DP bioaccumulation in zebrafish co-exposed with 3-MP and DP. Elevated bioaccumulation resulting from mixture exposure may represent a significant contribution of the synergistic effects observed in combined chemical exposure