Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high resolution crystal structures of wild-type B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 \uc5) and the variants D143A (1.3 \uc5), R232A (1.9 \uc5), and D143A/R232A (1.1 \uc5). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp 143 and Arg 232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin (LS) cyanide complex as well as on (iv) the stability and reactivity of an oxoiron(IV)porphyrin \u3c0-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527 and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs

This data upload contains variant call format files of a genetic screen in mouse embryonic stem cells identifying LDLR as a host factor for CCHFV.

This is an upload of vcf files identifying mutations present in colonies resistant to CCHFV infection</p

Anti-Bacterial Activity of Indoor Light Activated Photocatalysts

Nanocrystalline photocatalysts, prepared under ambient conditions using a microwave assisted syn-thesis, show indoor light photocatalytic activity for the degradation of Staphylococcus aureus and Escherichia coli. The zinc sulphide (ZnS) nanomaterials, prepared by a microwave assisted synthesis, are shown to be cubic blende structure with an average crystallite size of 4–6 nm. The anti-bacterial activ-ity of these nanomaterials is investigated under irradiation from a 60 W light bulb and photocatalytic activity is revealed to be due to the defects present in the crystal structure. The ZnS shows anti-bacterial action as both a bacteriostatic and bacteriocidal (88% reduction in the amount of bacteria in 5 h) material and the methods of bacterial degradation on the ZnS is discussed. The anti-bacterial actions of these materials were also compared with commercial ZnS and Evonik-Degussa P-25. A detailed mechanism for the light absorption in the visible light region of the microwave prepared ZnS is proposed based on the luminescence spectroscopy

A Highly Efficient TiO_2–<i>x</i>C_<i>x</i> Nano-heterojunction Photocatalyst for Visible Light Induced Antibacterial Applications

Visible-light-induced antibacterial activity of carbon-doped anatase-brookite titania nano-heterojunction photocatalysts are reported for the first time. These heterostructures were prepared using a novel low temperature (100 °C) nonhydrothermal low power microwave (300 W) assisted method. Formation of interband C 2p states was found to be responsible for the band gap narrowing of the carbon doped heterojunctions. The most active photocatalyst obtained after 60 min of microwave irradiation exhibits a 2-fold higher visible-light induced photocatalytic activity in contrast to the standard commercial photocatalyst Evonik-Degussa P-25. Staphylococcus aureus inactivation rate constant for carbon-doped nano-heterojunctions and the standard photocatalyst was 0.0023 and −0.0081 min^–1, respectively. It is proposed that the photoexcited electrons (from the C 2p level) are effectively transferred from the conduction band of brookite to that of anatase causing efficient electron–hole separation, which is found to be responsible for the superior visible-light induced photocatalytic and antibacterial activities of carbon-doped anatase-brookite nano-heterojunctions

Identification of CCZ1 as an essential lysosomal trafficking regulator in Marburg and Ebola virus infections

: Marburg and Ebola filoviruses are two of the deadliest infectious agents and several outbreaks have occurred in the last decades. Although several receptors and co-receptors have been reported for Ebola virus, key host factors remain to be elucidated. In this study, using a haploid cell screening platform, we identify the guanine nucleotide exchange factor CCZ1 as a key host factor in the early stage of filovirus replication. The critical role of CCZ1 for filovirus infections is validated in 3D primary human hepatocyte cultures and human blood-vessel organoids, both critical target sites for Ebola and Marburg virus tropism. Mechanistically, CCZ1 controls early to late endosomal trafficking of these viruses. In addition, we report that CCZ1 has a role in the endosomal trafficking of endocytosis-dependent SARS-CoV-2 infections, but not in infections by Lassa virus, which enters endo-lysosomal trafficking at the late endosome stage. Thus, we have identified an essential host pathway for filovirus infections in cell lines and engineered human target tissues. Inhibition of CCZ1 nearly completely abolishes Marburg and Ebola infections. Thus, targeting CCZ1 could potentially serve as a promising drug target for controlling infections caused by various viruses, such as SARS-CoV-2, Marburg, and Ebola

IDENTIFICATION OF AN IMPORTANT FACTOR INVOLVED IN CCHFV INFECTION

Despite intensive research, much of the molecular pathogenesis of CCHFV is still unknown. Genome-wide screening methods (particularly CRISPR/Cas9-based screens and insertional mutagenesis in haploid cell systems) have facilitated and accelerated the identification and characterization of host genes involved in infectious diseases. Combining haploid cells with genome saturating chemical mutagenesis using N-Ethyl-N-nitrosourea, we have developed an unbiased screening system that interrogates single nucleotide variants for their relevance in viral infections. To identify host factors involved in CCHFV infections, we performed resistant screens with a viral RNA replication competent vesicular stomatitis virus, pseudotyped with the glycoproteins of the CCHFV (VSV-CCHF_G). Resistant clones were individually selected, expanded and rescreened using the infectious CCHFV IbAr10200 laboratory strain. Subsequently, whole exome sequencing was conducted on the resistant clones. Three clones showing nearly 100% resistance to CCHFV displayed mutations in the gene encoding for protein we named X. Through the use of knocked out haploid and diploid cells as well as soluble form of this protein on VSV-CCHF_G, CCHFV IbAr 10200 and CCHFV isolate, we showed that this protein is important for CCHFV infection. These data were then confirmed in vivo, in a mice model. By using an unbiaised screening system, our study identified an important factor involved for CCHFV cell entry and infection

Crimean-Congo haemorrhagic fever virus uses LDLR to bind and enter host cells

Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean-Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV. Laboratory and clinical strains of Crimean-Congo haemorrhagic fever virus use LDLR to bind and enter host cells in blood vessel organoids and mice. Infection can also occur through ApoE, possibly present on virus particles

A Highly Efficient TiO2-XCx Nano-Heterojunction Photocatalyst for Visible-Light Induced Antibacterial Applications

Visible-light-induced antibacterial activity of carbon-doped anatase-brookite titania nano-heterojunction photocatalysts are reported for the first time. These heterostructures were prepared using a novel low temperature (100 °C) non-hydrothermal low power microwave (300 W) assisted method. Formation of interband C 2p states was found to be responsible for the band gap narrowing of the carbon doped heterojunctions. The most active photocatalyst obtained after 60 minutes of microwave irradiation exhibits a 2-fold higher visible-light induced photocatalytic activity in contrast to the standard commercial photocatalyst Evonik-Degussa P-25. Staphylococcus aureus inactivation rate constant for carbon-doped nano-heterojunctions and the standard photocatalyst was 0.0023 and -0.0081 min-1 respectively. It is proposed that the photo-excited electrons (from the C 2p level) are effectively transferred from the conduction band of brookite to that of anatase causing efficient electron-hole separation, which is found to be responsible for the superior visible-light induced photocatalytic and antibacterial activities of carbon-doped anatase-brookite nano-heterojunctions. KEYWORDS: Titanium dioxide; Microwave synthesis; Carbon-doping; Visible-light induced photocatalysis; Antibacterial activity; Heterojunction

CRISPR-UMI : single-cell lineage tracing of pooled CRISPR-Cas9 screens

Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens