Stability of Fried Olive and Sunflower Oils Enriched with Thymbra capitata Essential Oil

The stability of olive and sunflower oils for domestic uses after frying cow steak or only heating were evaluated in the presence or absence of the carvacrol-rich essential oil of Thymbra capitata. The treatments consisted of sunflower and olive oils either enriched with 200 mg/l of T. capitata oil or without it, heating at 180°C for 20 min, or frying 100 g cow steak at the same temperature and for the same period of time. In all assays, acid, peroxide, and p-anisidine values were followed over time. The fatty acid profile was estimated before heating or frying as well as at the end of the experiment. The results showed that the type of fat as well as the type of treatment (frying or heating) was determinant for the acid, peroxide, and p-anisidine values found. The presence of the essential oil also demonstrated to affect those values depending on the type of the oil as well as on the type of the treatment (frying or heating). In contrast, the fatty acid profile did not change greatly

Assembly and structure of Lys³³-linked polyubiquitin reveals distinct conformations

Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys(33)-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys(33) chains by combining the HECT (homologous to the E6–AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys(33)- and Lys(11)-linked Ub chains. Intriguingly, the crystal structure of Lys(33)-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys(11)-linked diUb. In contrast, crystallographic analysis of Lys(33)-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys(33)-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys(33)-linked polyUb that will allow further characterization of this atypical chain type

A distinct mechanism of C-type inactivation in the Kv-like KcsA mutant E71V

C-type inactivation is of great physiological importance in voltage-activated K+ channels (Kv), but its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the bacterial K+ channel KcsA, whose selectivity filter constricts under inactivating conditions. However, the filter is highly sensitive to its molecular environment, which is different in Kv channels than in KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is substituted by a valine conserved in most Kv channels, suggesting that this side chain is a molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and MD simulations of the E71V KcsA mutant is undertaken to explore inactivation in this Kv-like construct. X-ray and ssNMR data show that the filter of the Kv-like mutant does not constrict under inactivating conditions. Rather, the filter adopts a conformation that is slightly narrowed and rigidified. On the other hand, MD simulations indicate that the constricted conformation can nonetheless be stably established in the mutant channel. Together, these findings suggest that the Kv-like KcsA mutant may be associated with different modes of C-type inactivation, showing that distinct filter environments entail distinct C-type inactivation mechanisms

Chickens Expressing IFIT5 Ameliorate Clinical Outcome and Pathology of Highly Pathogenic Avian Influenza and Velogenic Newcastle Disease Viruses

Innate antiviral immunity establishes first line of defense against invading pathogens through sensing their molecular structures such as viral RNA. This antiviral potential of innate immunity is mainly attributed to a myriad of IFN-stimulated genes (ISGs). Amongst well-characterized ISGs, we have previously shown that antiviral potential of chicken IFN-induced proteins with tetratricopeptides repeats 5 (chIFIT5) is determined by its interaction potential with 5′ppp containing viral RNA. Here, we generated transgenic chickens using avian sarcoma-leukosis virus (RCAS)-based gene transfer system that constitutively and stably express chIFIT5. The transgenic chickens infected with clinical dose (EID50 104 for HPAIV and 105 EID50 for vNDV) of high pathogenicity avian influenza virus (HPAIV; H5N1) or velogenic strain of Newcastle disease virus (vNDV; Genotype VII) showed marked resistance against infections. While transgenic chickens failed to sustain a lethal dose of these viruses (EID50 105 for HPAIV and 106 EID50 for vNDV), a delayed and lower level of clinical disease and mortality, reduced virus shedding and tissue damage was observed compared to non-transgenic control chickens. These observations suggest that stable expression of chIFIT5 alone is potentially insufficient in providing sterile protection against these highly virulent viruses; however, it is sufficient to ameliorate the clinical outcome of these RNA viruses. These findings propose the potential of innate immune genes in conferring genetic resistance in chickens against highly pathogenic and zoonotic viral pathogens causing sever disease in both animals and humans

Simultaneous Deletion of Virulence Factors and Insertion of Antigens into the Infectious Laryngotracheitis Virus Using NHEJ-CRISPR/Cas9 and Cre–Lox System for Construction of a Stable Vaccine Vector

Infectious laryngotracheitis virus (ILTV) is a promising vaccine vector due to its heterologous gene accommodation capabilities, low pathogenicity, and potential to induce cellular and humoral arms of immunity. Owing to these characteristics, different gene-deletion versions of ILTVs have been successfully deployed as a vector platform for the development of recombinant vaccines against multiple avian viruses using conventional recombination methods, which are tedious, time-demanding, and error-prone. Here, we applied a versatile, and customisable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 accompanied with Cre–Lox system to simultaneously delete virulence factors and to insert foreign genes in the ILTV genome. Using this pipeline, we successfully deleted thymidine kinase (TK) and unique short 4 (US4) genes and inserted fusion (F) gene of the Newcastle disease virus without adversely affecting ILTV replication and expression of the F protein. Taken together, the proposed approach offers novel tools to attenuate (by deletion of virulence factor) and to generate multivalent (by insertion of heterologous genes) vaccine vectors to protect chickens against pathogens of poultry and public health importance

A Scalable Topical Vectored Vaccine Candidate Against SARS-CoV-2

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) caused an ongoing unprecedented global public health crises of coronavirus disease in 2019 (CoVID-19). The precipitously increased death rates, its impact on livelihood and trembling economies warrant the urgent development of a SARS-CoV-2 vaccine which would be safe, efficacious and scalable. Owing to unavailability of the vaccine, we propose a de novo synthesized avian orthoavulavirus 1 (AOaV-1)-based topical respiratory vaccine candidate against CoVID-19. Avirulent strain of AOaV-1 was engineered to express full length spike (S) glycoprotein which is highly neutralizing and a major protective antigen of the SARS-CoV-2. Broad-scale in vitro characterization of a recombinant vaccine candidate demonstrated efficient co-expression of the hemagglutinin-neuraminidase (HN) of AOaV-1 and S protein of SARS-CoV-2, and comparable replication kinetics were observed in a cell culture model. The recombinant vaccine candidate virus actively replicated and spread within cells independently of exogenous trypsin. Interestingly, incorporation of S protein of SARS-CoV-2 into the recombinant AOaV-1 particles attributed the sensitivity to anti-SARS-CoV-2 antiserum and more prominently to anti-AOaV-1 antiserum. Finally, our results demonstrated that the recombinant vaccine vector stably expressed S protein after multiple propagations in chicken embryonated eggs, and this expression did not significantly impact the in vitro growth characteristics of the recombinant. Taken together, the presented respiratory vaccine candidate is highly attenuated in primates per se, safe and lacking pre-existing immunity in human, and carries the potential for accelerated vaccine development against CoVID-19 for clinical studies

K29-selective ubiquitin binding domain reveals structural basis of specificity and heterotypic nature of K29 polyubiquitin

Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages

Structural topological analysis of spike proteins of SARS-CoV-2 variants of concern highlight distinctive amino acid substitution patterns

Since the onset of pandemic in 2019, SARS-CoV-2 has diverged into numerous variants driven by antigenic and infectivity-oriented selection. Some variants have accumulated fitness-enhancing mutations, evaded immunity and spread despite global vaccination campaigns. The spike (S) glycoprotein of SARS-CoV-2 demonstrated the greatest immunogenicity and amino acid substitution diversity owing to its importance in the interaction with human angiotensin receptor 2 (hACE2). The S protein consistently emerges as an amino acid substitution (AAS) hotspot in all six lineages, however, in Omicron this enrichment is significantly higher. This study attempts to design and validate a method of mapping S-protein substitution profile across variants to identify the conserved and AAS regions. A substitution matrix was created based on publicly available databases, and the substitution localization was illustrated on a cryo-electron microscopy generated S-protein model. Our analyses indicated that the diversity of N-terminal (NTD) and receptor-binding (RBD) domains exceeded that of any other regions but still contained extended low substitution density regions particularly considering significantly broader substitution profiles of Omicron BA.2 and BA.4/5. Finally, the substitution matrix was compared to a random sample alignment of variant sequences, revealing discrepancies. Therefore, it was suggested to improve matrix accuracy by processing a large number of S-protein sequences using an automated algorithm. Several critical immunogenic and receptor-interacting residues were identified in the conserved regions within NTD and RBD. In conclusion, the structural and topological analysis of S proteins of SARS-CoV-2 variants highlight distinctive amino acid substitution patterns which may be foundational in predicting future variants

Structural and functional insights into non-structural proteins of coronaviruses

Coronaviruses (CoVs) are causing a number of human and animal diseases because of their zoonotic nature such as Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). These viruses can infect respiratory, gastrointestinal, hepatic and central nervous systems of human, livestock, birds, bat, mouse, and many wild animals. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerging respiratory virus and is causing CoVID-19 with high morbidity and considerable mortality. All CoVs belong to the order Nidovirales, family Coronaviridae, are enveloped positive-sense RNA viruses, characterised by club-like spikes on their surfaces and large RNA genome with a distinctive replication strategy. Coronavirus have the largest RNA genomes (~26–32 kilobases) and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. Non-structural proteins (nsp) 7–16 are cleaved from two large replicase polyproteins and guide the replication and processing of coronavirus RNA. Coronavirus replicase has more or less universal activities, such as RNA polymerase (nsp 12) and helicase (nsp 13), as well as a variety of unusual or even special mRNA capping (nsp 14, nsp 16) and fidelity regulation (nsp 14) domains. Besides that, several smaller subunits (nsp 7– nsp 10) serve as essential cofactors for these enzymes and contribute to the emerging “nsp interactome.” In spite of the significant progress in studying coronaviruses structural and functional properties, there is an urgent need to understand the coronaviruses evolutionary success that will be helpful to develop enhanced control strategies. Therefore, it is crucial to understand the structure, function, and interactions of coronaviruses RNA synthesizing machinery and their replication strategies. © 202

Engineered protein G variants for multifunctional antibody-based assemblies

We have developed a portfolio of antibody-based modules that can be prefabricated as standalone units and snapped together in plug-and-play fashion to create uniquely powerful multifunctional assemblies. The basic building blocks are derived from multiple pairs of native and modified Fab scaffolds and protein G (PG) variants engineered by phage display to introduce high pair-wise specificity. The variety of possible Fab-PG pairings provides a highly orthogonal system that can be exploited to perform challenging cell biology operations in a straightforward manner. The simplest manifestation allows multiplexed antigen detection using PG variants fused to fluorescently labeled SNAP-tags. Moreover, Fabs can be readily attached to a PG-Fc dimer module which acts as the core unit to produce plug-and-play IgG-like assemblies, and the utility can be further expanded to produce bispecific analogs using the “knobs into holes” strategy. These core PG-Fc dimer modules can be made and stored in bulk to produce off-the-shelf customized IgG entities in minutes, not days or weeks by just adding a Fab with the desired antigen specificity. In another application, the bispecific modalities form the building block for fabricating potent bispecific T-cell engagers (BiTEs), demonstrating their efficacy in cancer cell-killing assays. Additionally, the system can be adapted to include commercial antibodies as building blocks, greatly increasing the target space. Crystal structure analysis reveals that a few strategically positioned interactions engender the specificity between the Fab-PG variant pairs, requiring minimal changes to match the scaffolds for different possible combinations. This plug-and-play platform offers a user-friendly and versatile approach to enhance the functionality of antibody-based reagents in cell biology research