3D Structure of the Tail Complex of E. coli Bacteriophage DT57C

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Abstract P-46: Structure of A. Baumannii Phage Tapaz, Revealed with Cryo-Electron Microscopy

Background: Acinetobacter baumannii is an opportunistic pathogen and one of the six most important multidrug resistant microorganisms in hospitals worldwide. Some of its strains are resistant to most of the antibiotics, A. baumannii is included into the Priority 1 part of Global Priority List of Antibiotic-resistant Bacteria. Phage therapy is considered to be an alternative strategy to antibiotic treatments. Methods: A. baumannii strain NIPH601 cells were grown till OD6000.4 and infected with the phage at MOI 10:1. After complete lysis took place cell debris was spined down and phage particles were precipitated with the PEG6000 (final concentration 10% PEG 6000, 0.5 NaCl). Virus particles were collected by centrifugation, resuspended at SM buffer and applied on CsCl step gradient. Gradient was spinned down for 2 hours at 40000g and the fraction containing phage particles was collected and dialyzed against SM buffer. Purified phage particles were applied to Quantifoil 1.2/1.3 grids and plunge-froze in Vitrobot Mark IV (TFS) Micrographs were collected in HKU, Shenzhen campus with Titan Krios cryoelectron microscope (TFS), equipped with Gatan K3 direct electron detector. The micrographs were acquired with 1.06 Å pixel size and 1.5 um average defocus value in counting mode with 50 frames and 1.2 e/Å2/frame dose rate. All image processing was performed with Relion3.0 software, except for the particle picking step performed with cryolo. Results: Lytic A. baumannii phage TaPaz belongs to the family Myoviridae. BLAST search over NCBI “nr” (non-redundant) database revealed close homology with previously published sequences of Acinetobacter phage vB_AbaM_B9 and Acinetobacter phage BS46. However, no structural information about any homologous proteins was found among the PDB structures. The cryo-EM map was reconstructed with single particle analysis independently for the capsid, tail and baseplate regions. The capsid was reconstructed at 3.9 Å resolution with I3 symmetry applied (Fig. 1A). The baseplate region of the phage was reconstructed at 3.5 Å resolution with C3 symmetry (Fig. 1B). The tail region was reconstructed at 2.6 Å resolution with helical symmetry (Rise 36.4 Å, Twist 25.7 deg). Initial atomic model for the tail region was built from sequence with Deeptracer and was further refined in coot (Fig. 1C). Conclusion: We successfully obtained the near-atomic resolution structural map of phage TaPaz. The data obtained contribute to enhancing knowledge of structural diversity of bacterial viruses infecting A. baumannii

Evolutionary Relationships Between the Laccase Genes of Polyporales: Orthology-Based Classification of Laccase Isozymes and Functional Insight From Trametes hirsuta

Laccase is one of the oldest known and intensively studied fungal enzymes capable of oxidizing recalcitrant lignin-resembling phenolic compounds. It is currently well established that fungal genomes almost always contain several non-allelic copies of laccase genes (laccase multigene families); nevertheless, many aspects of laccase multigenicity, for example, their precise biological functions or evolutionary relationships, are mostly unknown. Here, we present a detailed evolutionary analysis of the sensu stricto laccase genes (CAZy – AA1_1) from fungi of the Polyporales order. The conducted analysis provides a better understanding of the Polyporales laccase multigenicity and allows for the systemization of the individual features of different laccase isozymes. In addition, we provide a comparison of the biochemical and catalytic properties of the four laccase isozymes from Trametes hirsuta and suggest their functional diversification within the multigene family

Intraductal photodynamic therapy and its combination with intra-arterial chemoinfusion in the treatment of inoperable patients with Klatskin tumor

Background: A palliative or symptomatic treatment is indicated for 7080% of patients with Klatskins tumor because of the advanced lesion volume and the patients grave condition. Hepatic arterial infusion chemotherapy, chemoembolization, radioembolization are successfully used in the treatment of hepatocellular carcinoma and liver metastases. Aim: to estimate the immediate and long-term results of photodynamic therapy (PDT) and its combination with hepatic arterial infusion in inoperable patients with Klatskin tumors. Methods: Between 2010 and 2021, 83 palliative PDT sessions (from 1 to 8, average 2.4) were performed in 82 patients as a single treatment or in combination with hepatic arterial infusion. In all cases, percutaneous transhepatic biliary drainage was previously performed; no chemotherapy was applied. Two groups of 48 patients were stratified according to the ECOG status (23) and the numbers of PDT sessions (no more than two). The treatment group of the combination therapy consisted of 24 patients (13 male, 11 female) aged 38 to 85 (mean 63) years with the ECOG status of 24 (mean 2.4). This group received PDT with hepatic arterial infusion using a GemCis regimen. On average, 1.4 PDT sessions were performed, the treatment started on the 89th (27225) day after the biliary drainage. The hepatic arterial infusion was performed on the 2d3th day after the PDT. The control group received only PDT and consisted of 24 patients (13 male, 11 female) aged 51 to 83 (66 on average) years, with the ECOG status of 23 (mean 2.6). On average, 1.4 PDT sessions were performed, starting on the 106th (32405) day after the biliary drainage. Results: There were no serious adverse events associated with PDT in both groups. Toxic complications of hepatic arterial infusion were observed in 13 of 24 patients (54%): III grade hematological (54%) and gastrointestinal (69%); all were eliminated with medical therapy. Complications of the percutaneous transhepatic biliary drainage in three patients (hemobilia, n=2, and sepsis, n=1) were estimated as grade III by the CIRSE classification (2017) and successfully treated without surgery. In the combination treatment group, the overall mean survival and median survival were higher than those in the control group: 327.939.8 days (10.9 mo) versus 246.931.2 days (8.2 mo) and 275 days versus 244 days. However, these differences did not reach the statistical significance (p=0.12). Conclusions: PDT is a safe method of a palliative treatment of critically ill patients with Klatskin tumor (ECOG 23). PDT alone has limited clinical efficacy. A combination of PDT and hepatic arterial infusion does not cause serious complications and may increase the survival rates

Abstract P-41: Contribution of Matrix-bound Vesicles Produced by Mesenchymal Stromal Cells in the Differentiation of Multipotent Stem Cells in vitro

Background: According to the current view on the extracellular matrix (ECM) composition and functions, it includes not only structural proteins and components of cell adhesion, but also various deposited components, including enzymes involved in ECM remodeling, growth factors, and matrix-bound vesicles (MBV). MBV can presumably participate in the formation of a specific microenvironment for stem cells and regulate their differentiation. However, the contribution of MBV to these processes remains poorly understood. In our work, we evaluated the effects of MBV within native ECM produced by mesenchymal stromal cells (MSCs) cultured in cell sheet on multipotent stem cell differentiation. Methods: We isolated MBV from decellularized MSC-produced ECM by treatment with the following enzymes: collagenase, hyaluronidase, or trypsin, and centrifugation on 1000 kDa filters. The nanostructure and relative size in each sample were observed using TEM. The particle size and concentration were also studied with NTA. In addition, the obtained MBV were examined for the presence of key exosome markers using Western blot. Then we investigated the effect of MBV on the formation of capillary-like structures by endothelial cells (in vitro model of angiogenesis) as well as on the differentiation of primary MSCs isolated from human adipose tissue in the adipogenic, osteogenic, and chondrogenic directions. Results: As a result of comparative analysis of isolation protocols, it was shown that all MBV samples had the characteristics of extracellular vesicles (EV), but differed in size and representation of exosomal markers. The MBV isolated from ECM did not stimulate the formation of capillary-like structures by endothelial cells, in contrast to EV secreted by MSCs to the conditioned medium, but maintained the viability of the endothelium. Isolated MBV stimulated osteogenic and adipogenic differentiation of MSCs similar to secreted EV. On the other hand, preincubation of MSCs with MBV leads to reorganization of cell monolayer to spheroid-like structures during chondrogenic differentiation. Conclusion: Here, we developed the protocol of isolation of MBV from ECM that have distinguished characteristics and functional activity

Structural and Immunoreactivity Properties of the SARS-CoV-2 Spike Protein upon the Development of an Inactivated Vaccine

Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by β-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein’s pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the β-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while β-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of β-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process

Structural and Immunoreactivity Properties of the SARS-CoV-2 Spike Protein upon the Development of an Inactivated Vaccine

Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by β-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein’s pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the β-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while β-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of β-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process

The Structure of the Potato Virus A Particles Elucidated by Small Angle X-Ray Scattering and Complementary Techniques

Potato virus A (PVA) protein coat contains on its surface partially unstructured N-terminal domain of the viral coat protein (CP), whose structural and functional characteristics are important for understanding the mechanism of plant infection with this virus. In this work, we investigated the properties and the structure of intact PVA and partially trypsinized PVAΔ32 virions using small-angle X-ray scattering (SAXS) and complimentary methods. It was shown that after the removal of 32 N-terminal amino acids of the CP, the virion did not disintegrate and remained compact, but the helical pitch of the CP packing changed. To determine the nature of these changes, we performed ab initio modeling, including the multiphase procedure, with the geometric bodies (helices) and restoration of the PVA structure in solution using available high-resolution structures of the homologous CP from the PVY potyvirus, based on the SAXS data. As a result, for the first time, a low-resolution structure of the filamentous PVA virus, both intact and partially degraded, was elucidated under conditions close to natural. The far-UV circular dichroism spectra of the PVA and PVAΔ32 samples differed significantly in the amplitude and position of the main negative maximum. The extent of thermal denaturation of these samples in the temperature range of 20-55°C was also different. The data of transmission electron microscopy showed that the PVAΔ32 virions were mostly rod-shaped, in contrast to the flexible filamentous particles typical of the intact virus, which correlated well with the SAXS results. In general, structural analysis indicates an importance of the CP N-terminal domain for the vital functions of PVA, which can be used to develop a strategy for combating this plant pathogen

Fine Structure of Plasmodesmata-Associated Membrane Bodies Formed by Viral Movement Protein

Cell-to-cell transport of plant viruses through plasmodesmata (PD) requires viral movement proteins (MPs) often associated with cell membranes. The genome of the Hibiscus green spot virus encodes two MPs, BMB1 and BMB2, which enable virus cell-to-cell transport. BMB2 is known to localize to PD-associated membrane bodies (PAMBs), which are derived from the endoplasmic reticulum (ER) structures, and to direct BMB1 to PAMBs. This paper reports the fine structure of PAMBs. Immunogold labeling confirms the previously observed localization of BMB1 and BMB2 to PAMBs. EM tomography data show that the ER-derived structures in PAMBs are mostly cisterns interconnected by numerous intermembrane contacts that likely stabilize PAMBs. These contacts predominantly involve the rims of the cisterns rather than their flat surfaces. Using FRET-FLIM (Förster resonance energy transfer between fluorophores detected by fluorescence-lifetime imaging microscopy) and chemical cross-linking, BMB2 is shown to self-interact and form high-molecular-weight complexes. As BMB2 has been shown to have an affinity for highly curved membranes at cisternal rims, the interaction of BMB2 molecules located at rims of adjacent cisterns is suggested to be involved in the formation of intermembrane contacts in PAMBs

Size Distribution of Inactivated Tick-Borne Encephalitis Virus Particles Revealed by a Comprehensive Physicochemical Approach

Tick-borne encephalitis virus (TBEV) is an enveloped RNA virus, a member of the genus Flavivirus (family Flaviviridae). Here, we provide a detailed analysis of the size and structure of the inactivated TBEV vaccine strain Sofjin-Chumakov. Four analytical methods were used to analyze individual TBEV particles—negative staining TEM, cryo-EM, atomic force microscopy (AFM), and nanoparticle tracking analysis (NTA). All methods confirmed that the particles were monodisperse and that their mean size was ~50 nm. Cryo-EM data allowed us to obtain a 3D electron density model of the virus with clearly distinguishable E protein molecules. STEM-EELS analysis detected phosphorus in the particles, which was interpreted as an indicator of RNA presence. Altogether, the described analytical procedures can be valuable for the characterization of inactivated vaccine virus samples