Search

Poxvirus molecular structure has been a long-standing problem in structural virology. Poxviruses are large, enveloped, double stranded DNA viruses that complete their entire replication cycle in the host cytoplasm. Despite >60 years of investigation into the ultra- and molecular structure of vaccinia virus, the prototypical poxvirus, various factors have rendered its molecular structure refractory to traditional structural and molecular biology approaches. This dissertation describes new insights into the vaccinia virion molecular structure, achieved by protein crosslinking mass spectrometry (XLMS) combined with protein structure prediction by deep learning. To examine the vaccinia molecular structure in situ, we developed an XLMS approach implementing a “strategy of variation” by which we were able identify protein-protein interaction interfaces for almost all of the ~75 packaged virion proteins. As a part of this process, we developed an updated virus purification protocol for high yield-high vaccinia virus with proteomic purity, along with new methods for full vaccinia protein solubilization for proteomics, that was able to significantly improve the completeness of digestion of virion proteins to peptides for mass spectrometry. We also implemented bioinformatic and other strategies to maximize the identification of crosslinked peptides, finally allowing us to achieve what we consider likely to be a saturating XLMS dataset. Alongside this work, we applied a structural homology prediction approach (HHsuite) to identify homologs of vaccinia proteins and to hopefully generate “placeholder” models that we could integrate with XLMS to generate higher order three dimensional models. We applied structural homology prediction to other viruses from the nucleocytoplasmic large DNA virus (NCLDV) phylum to the identify orthologous genes between the virus families that were undetectable by sequence homology alone. This contributed substantially to expanding annotations of previously uncharacterized proteins from NCLDV proteomes. Confident structural homologs for core structural proteins of the vaccinia virion, however, were not identified. Instead, we eventually pursued protein structure prediction by AlphaFold2 to generate high confidence models of vaccinia virion proteins to combine with XLMS data.Through the resulting combination of XLMS based structure validation of AlphaFold2 models and crosslink-guided protein docking, we describe previously unidentified higher order structural assemblies of vaccinia virion transmembrane and surface-associated proteins. The density of crosslinks within and between molecules of Vaccinia virion core structural P4a (the major component of the palisade layer of the core wall) allowed us to address in greater depth its structure, maturation, and possible assembly pathway. This work was able to identify a likely order of events in which P4a precursor is first proteolytically processed at a downstream site to release its P4a-3 fragment. Removal of P4a-3 releases a steric block to a major rearrangement between the two domains of P4a-1, resulting in a final conformation that is stabilized by disulfide-locking. Removal of a second proteolytic fragment, P4a-2 and trimerization of P4a-1 then allows the assembly of P4a-1 trimers into a higher order hexagonal lattice as the palisade layer of the Vaccinia virus core wall. Overall, these findings contributed to our understanding of the Vaccinia mature virion molecular architecture and open the door to future studies of virion dynamics and morphogenesis. This approach was then extended to the 23 transmembrane and virion surface proteins associated with the virion envelope.

BackgroundAutologous fat grafting is commonly used for soft-tissue repair (approximately 90,000 cases per year in the United States), but outcomes are limited by volume loss (20% to 80%) over time. Human allograft adipose matrix (AAM) stimulates de novo adipogenesis in vivo, but retention requires optimization. The extracellular matrix derived from superficial fascia, interstitial within the adipose layer, is typically removed during AAM processing. Thus, fascia, which contains numerous important proteins, might cooperate with AAM to stimulate de novo adipogenesis, improving long-term retention compared to AAM alone.MethodsHuman AAM and fascia matrix proteins (back and upper leg regions) were identified by mass spectrometry and annotated by gene ontology. A three-dimensional in vitro angiogenesis assay was performed. Finally, AAM and/or fascia (1 mL) was implanted into 6- to 8-week-old male Fischer rats. After 8 weeks, the authors assessed graft retention by gas pycnometry and angiogenesis (CD31) and adipocyte counts (hematoxylin and eosin) histologically.ResultsGene ontology annotation revealed an angiogenic enrichment pattern unique to the fascia, including lactadherin, collagen alpha-3(V) chain, and tenascin-C. In vitro, AAM stimulated 1.0 ± 0.17 angiogenic sprouts per bead. The addition of fascia matrix increased sprouting by 88% (2.0 ± 0.12; P < 0.001). A similar angiogenic response (CD31) was observed in vivo. Graft retention volume was 25% (0.25 ± 0.13) for AAM, significantly increasing to 60% (0.60 ± 0.14) for AAM/fascia ( P < 0.05). De novo adipogenesis was 12% (12.4 ± 7.4) for AAM, significantly increasing to 51% (51.2 ± 8.0) for AAM/fascia ( P < 0.001) by means of adipocyte quantification.ConclusionsCombining fascia matrix with AAM improves angiogenesis and adipogenesis compared to AAM alone in rats. These preliminary in vitro and pilot animal studies should be further validated before definitive clinical adoption.Clinical relevance statementWhen producing an off-the-shelf adipose inducing product by adding a connective tissue fascial component (that is normally discarded) to the mix of adipose matrix, vasculogenesis is increased and, thus, adipogenesis and graft survival is improved. This is a significant advance in this line of product.

This article presents the ideological controversies that arose between the Armenian Apostolic church and the bolshevik regime following the invasion of soviet troops into Armenia. From its first days in power bolshevik authorities implemented radical steps against its ideological rival – the armenian church. Following the harsh anti-church political line of Russian bolsheviks, the soviet authorities in Armenia started a massive appropriation of Armenian church properties. The article mentions also the first attempts of the soviet administration to organize state institutions for the preservation and study of national cultural heritage.

The research aims to reveal the typical expressions of cognitive dissonance and consonance among the primary-school children by their argumentations upon their deed. The relevance of the research stems from the applicability of the study of the phenomena of dissonance and consonance, which inherently emerge in the sphere of argumentation among the children who study how to play chess. Cognitive dissonance and consonance in the sphere of argumentation in the course of the game of chess are gradually transforming both into the respective reference points – relevant to the situation given, and into the emotional and behavioural manifestations – either adequately or inadequately expressed and, by the logical and inner conflict, are observed as the result of juxtaposition of the realms of emotions and logic. The present research evidences that, in the case of similar chess skills, the manifestation of cognitive dissonance, and the own argumentation as well are detectable within the domain of extroversion – emotional instability. Such children, even more than the introverts, need the school psychologist’s support. The cognitive consonance is typical of those of the children involved who are ready to gain new experience and appear to be more positively charged towards their rivals or opponents.

The auditory brainstem relies on precise circuitry to facilitate sound source localization. In the chick, the development of this specialized circuitry requires non-apoptotic activity of caspase-3, for which we previously identified several hundred proteolytic substrates. Here we tested whether the sequence of the caspase cleavage site differentially encodes proteolytic preference in apoptotic and non-apoptotic contexts. We constructed a consensus sequence for caspase activity in the non-apoptotic chick auditory brainstem comprising the four residues N-terminal to the cleavage site: IX(G/R)D↓ where X represents no significant enrichment and ↓ represents the cleavage site. We identified GO terms significantly enriched among caspase substrates containing motifs found in the above consensus sequence. (G/R)D↓ was associated with the term “Structural Constituent of Cytoskeleton” (SCoC), suggesting that SCoC proteins may be specifically targeted by caspase activity during non-apoptotic developmental processes. To ascertain whether this consensus sequence was specific to the non-apoptotic auditory brainstem at embryonic day (E) 10, we used protein mass spectrometry of brainstems harvested at a time when auditory brainstem neurons undergo apoptotic cell death (E13). The apoptotic motif VD was significantly enriched among E13 cleavage sites, indicating that motif preference at the P2 subsite had shifted toward the canonical caspase consensus sequence. Additionally, Monte Carlo simulations revealed that only the GD motif was associated with SCoC substrates in the apoptotic auditory brainstem, indicating that GD encodes specificity for SCoC proteins in both non-apoptotic and apoptotic contexts, despite not being preferred in the latter. Finally, to identify candidate human non-apoptotic consensus sequences, we used Monte Carlo analyses to determine motifs and motif pairs associated with SCoC caspase substrates in the Degrabase, a database of cleavage sites in human apoptotic cell lines. We found 11 motifs significantly associated with SCoC proteolysis, including IXXD and GD. We employed a stepwise method to select motif pairs that optimized SCoC specificity for a given coverage of SCoC cleavage events, yielding 11 motif pairs likely to be preferred in SCoC-directed human non-apoptotic caspase consensus sequences. GD + IXXD was among these motif pairs, suggesting a conservation of non-apoptotic consensus sites among vertebrates.

The mitochondrial and chloroplast mRNAs of the majority of land plants are modified through cytidine to uridine (C-to-U) RNA editing. Previously, forward and reverse genetic screens demonstrated a requirement for pentatricopeptide repeat (PPR) proteins for RNA editing. Moreover, chloroplast editing factors OZ1, RIP2, RIP9 and ORRM1 were identified in co-immunoprecipitation (co-IP) experiments, albeit the minimal complex sufficient for editing activity was never deduced. The current study focuses on isolated, intact complexes that are capable of editing distinct sites. Peak editing activity for four sites was discovered in size-exclusion chromatography (SEC) fractions ≥ 670 kDa, while fractions estimated to be approximately 413 kDa exhibited the greatest ability to convert a substrate containing the editing site rps14 C80. RNA content peaked in the ≥ 670 kDa fraction. Treatment of active chloroplast extracts with RNase A abolished the relationship of editing activity with high-MW fractions, suggesting a structural RNA component in native complexes. By immunoblotting, RIP9, OTP86, OZ1 and ORRM1 were shown to be present in active gel filtration fractions, though OZ1 and ORRM1 were mainly found in low-MW inactive fractions. Active editing factor complexes were affinity-purified using anti-RIP9 antibodies, and orthologs to putative Arabidopsis thaliana RNA editing factor PPR proteins, RIP2, RIP9, RIP1, OZ1, ORRM1 and ISE2 were identified via mass spectrometry. Western blots from co-IP studies revealed the mutual association of OTP86 and OZ1 with native RIP9 complexes. Thus, RIP9 complexes were discovered to be highly associated with C-to-U RNA editing activity and other editing factors indicative of their critical role in vascular plant editosomes.

We conducted an exhaustive search for three-dimensional structural homologs to the proteins of 20 key phylogenetically distinct nucleocytoplasmic DNA viruses (NCLDV). Structural matches covered 429 known protein domain superfamilies, with the most highly represented being ankyrin repeat, P-loop NTPase, F-box, protein kinase, and membrane occupation and recognition nexus (MORN) repeat. Domain superfamily diversity correlated with genome size, but a diversity of around 200 superfamilies appeared to correlate with an abrupt switch to paralogization. Extensive structural homology was found across the range of eukaryotic RNA polymerase II subunits and their associated basal transcription factors, with the coordinated gain and loss of clusters of subunits on a virus-by-virus basis. The total number of predicted endonucleases across the 20 NCLDV was nearly quadrupled from 36 to 132, covering much of the structural and functional diversity of endonucleases throughout the biosphere in DNA restriction, repair, and homing. Unexpected findings included capsid protein-transcription factor chimeras; endonuclease chimeras; enzymes for detoxification; antimicrobial peptides and toxin-antitoxin systems associated with symbiosis, immunity, and addiction; and novel proteins for membrane abscission and protein turnover. IMPORTANCE We extended the known annotation space for the NCLDV by 46%, revealing high-probability structural matches for fully 45% of the 9,671 query proteins and confirming up to 98% of existing annotations per virus. The most prevalent protein families included ankyrin repeat- and MORN repeat-containing proteins, many of which included an F-box, suggesting extensive host cell modulation among the NCLDV. Regression suggested a minimum requirement for around 36 protein structural superfamilies for a viable NCLDV, and beyond around 200 superfamilies, genome expansion by the acquisition of new functions was abruptly replaced by paralogization. We found homologs to herpesvirus surface glycoprotein gB in cytoplasmic viruses. This study provided the first prediction of an endonuclease in 10 of the 20 viruses examined; the first report in a virus of a phenolic acid decarboxylase, proteasomal subunit, or cysteine knot (defensin) protein; and the first report of a prokaryotic-type ribosomal protein in a eukaryotic virus.

The goal of this study was to determine if exosomes, isolated from Chlamydia muridarum infected HeLa cells (C. muridarum-exosomes), induce protective immune responses in mice following vaccination using CpG plus Montanide as adjuvants. Exosomes, collected from uninfected HeLa cells and PBS, formulated with the same adjuvants, were used as negative controls. Mass spectrometry analyses identified 113 C. muridarum proteins in the C. muridarum-exosome preparation including the major outer membrane protein and the polymorphic membrane proteins. Vaccination with C. muridarum-exosomes elicited robust humoral and cell-mediated immune responses to C. muridarum elementary bodies. Following vaccination, mice were challenged intranasally with C. muridarum. Compared to the negative controls, mice immunized with C. muridarum-exosomes were significantly protected as measured by changes in body weight, lungs’ weight, and number of inclusion forming units recovered from lungs. This is the first report, of a vaccine formulated with Chlamydia exosomes, shown to elicit protection against a challenge.

A method for synthesis of nickel nanoparticles in a magnetic nickel phthalocyanine anions matrix has been developed. The method is based on intercalation of potassium atoms to the nickel phthalocyanine (NiPc) polycrystalline powder at 300°C. The structure of (K2NiPc) was investigated by using high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD) and X-ray absorption fine structure (XAFS) spectroscopes. Magnetic properties were studied by SQUID magnetometry and magnetic resonances methods. It is revealed that the resultant compound contains of 1 wt% Ni nanoparticles with the average size of 15 nm. The measured values of the magnetization and absorption of the ferromagnetic resonance considerably exceed the magnetism which can be attributed to metallic Ni nanoparticles. The obtained results indicate the presence of room temperature molecular ferromagnetism caused by anionic molecules of NiPc.

Data for the vapor-phase doping (300°C) of nickel phthalocyanine (NiPc) by sodium taken in different concentrations (x), as well as structural analysis data for Na x = 0.2NiPc, Na x = 1NiPc, and Na x = 3NiPc samples, have been reported. The structure of the samples and their atomic configuration versus the doping level have been studied by transmission electron microscopy, Raman scattering, X-ray diffraction, X-ray absorption spectroscopy, and extended X-ray absorption fine structure (EXAFS) spectroscopy. The structural parameters of Ni–N, Ni–C, and Ni–Ni bonds have been determined, and it has been found that, at a low level of doping by sodium, local structural distortions are observed in some molecules of the NiPc matrix near nickel atoms. The fraction of these molecules grows as the doping level rises from x = 0.2 to x = 1.0. It has been shown that doping changes the oscillation mode of light atoms, which indicates a rise in the electron concentration on five- and six-membered rings. At a high level of sodium doping (x = 3.0), nickel nanoparticles with a mean size of 20 nm and molecule decomposition products have been observed in the NiPc matrix. It has been found that the fraction of nickel atoms in the Na x = 3NiPc nanoparticles as estimated from EXAFS data is sufficient for the room-temperature magnetic properties of the samples to persist for a long time.

The mitochondrial and chloroplast mRNAs of the majority of land plants are modified through cytidine to uridine (C-to-U) RNA editing. Previously, forward and reverse genetic screens demonstrated a requirement for pentatricopeptide repeat (PPR) proteins for RNA editing. Moreover, chloroplast editing factors OZ1, RIP2, RIP9 and ORRM1 were identified in co-immunoprecipitation (co-IP) experiments, albeit the minimal complex sufficient for editing activity was never deduced. The current study focuses on isolated, intact complexes that are capable of editing distinct sites. Peak editing activity for four sites was discovered in size-exclusion chromatography (SEC) fractions ≥ 670 kDa, while fractions estimated to be approximately 413 kDa exhibited the greatest ability to convert a substrate containing the editing site rps14 C80. RNA content peaked in the ≥ 670 kDa fraction. Treatment of active chloroplast extracts with RNase A abolished the relationship of editing activity with high-MW fractions, suggesting a structural RNA component in native complexes. By immunoblotting, RIP9, OTP86, OZ1 and ORRM1 were shown to be present in active gel filtration fractions, though OZ1 and ORRM1 were mainly found in low-MW inactive fractions. Active editing factor complexes were affinity-purified using anti-RIP9 antibodies, and orthologs to putative Arabidopsis thaliana RNA editing factor PPR proteins, RIP2, RIP9, RIP1, OZ1, ORRM1 and ISE2 were identified via mass spectrometry. Western blots from co-IP studies revealed the mutual association of OTP86 and OZ1 with native RIP9 complexes. Thus, RIP9 complexes were discovered to be highly associated with C-to-U RNA editing activity and other editing factors indicative of their critical role in vascular plant editosomes.