Viral deubiquitinating proteases and the promising strategies of their inhibition.

Several viruses are now known to code for deubiquitinating proteases in their genomes. Ubiquitination is an essential post-translational modification of cellular substrates involved in many processes in the cell, including in innate immune signalling. This post-translational modification is regulated by the ubiquitin conjugation machinery, as well as various host deubiquitinating enzymes. The conjugation of ubiquitin chains to several innate immune related factors is often needed to induce downstream signalling, shaping the antiviral response. Viral deubiquitinating proteins, besides often having a primary function in the viral replication cycle by cleaving the viral polyprotein, are also able to cleave ubiquitin chains from such host substrates, in that way exerting a function in innate immune evasion. The presence of viral deubiquitinating enzymes has been firmly established for numerous animal-infecting viruses, such as some well-researched and clinically important nidoviruses, and their presence has now been confirmed in several plant viruses as well. Viral proteases in general have long been highlighted as promising drug targets, with a current focus on small molecule inhibitors. In this review, we will discuss the range of viral deubiquitinating proteases known to date, summarise the various avenues explored to inhibit such proteases and discuss novel strategies and models intended to inhibit and study these specific viral enzymes.

The Common Retention Practices Among Orthodontists from Different Countries.

Objective: To investigate the most common retention practices, factors influencing the retention protocol, and the differences among orthodontists regarding retention practices. Methods: An online validated questionnaire was anonymously sent to 3,000 orthodontic residents and clinicians. The survey consisted of 19 questions regarding the participants' demographics, prescribed retention appliances, factors affecting retention appliance choices, and adjunctive retention procedures. Descriptive statistics, Chi2 and Kendall's Tau-b tests were applied. Results: Five hundred fifty-five orthodontic residents and clinicians, 53.3% males and 46.7% females, completed the survey, indicating a response rate of 18.5%. Although participants' demographics, type of treatment and pre-treatment malocclusion influence the choice of retention protocols, thermoplastic retainers (TR) were the most popular retention regime for the maxillary arch for both adults (47.4%) and adolescents (42.3%). Bonded retainers (BR) were the favored option for the mandibular arch (44.9% of adults and 40.7% of adolescents). The degree of arch expansion (64.1%) and the degree of interdigitation (50.1%) after treatment were the most influential factors for the choice of the preferred type of retainers by the respondents. 68.6% of the participants thought professional retention guidelines would be useful. Conclusion: Thermoplastic retainers were the most common retention appliances for adults and adolescents in the maxilla. At the same time, BR was the most favored retainer in the mandibular arch, with clinical experience, practice setting, and malocclusion- and treatment-related factors influencing the type of the chosen appliance. The demographic differences and the uneven participation in the survey need to be considered while interpreting the findings of this study.

Deubiquitinating activity of SARS-CoV-2 papain-like protease does not influence virus replication or innate immune responses in vivo.

The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and deISGylating activities. To elucidate the role of these activities during SARS-CoV-2 infection, we introduced mutations that disrupt binding of PLpro to ubiquitin or ISG15. We identified several mutations that strongly reduced DUB activity of PLpro, without affecting viral polyprotein processing. In contrast, mutations that abrogated deISGylating activity also hampered viral polyprotein processing and when introduced into the virus these mutants were not viable. SARS-CoV-2 mutants exhibiting reduced DUB activity elicited a stronger interferon response in human lung cells. In a mouse model of severe disease, disruption of PLpro DUB activity did not affect lethality, virus replication, or innate immune responses in the lungs. This suggests that the DUB activity of SARS-CoV-2 PLpro is dispensable for virus replication and does not affect innate immune responses in vivo. Interestingly, the DUB mutant of SARS-CoV replicated to slightly lower titers in mice and elicited a diminished immune response early in infection, although lethality was unaffected. We previously showed that a MERS-CoV mutant deficient in DUB and deISGylating activity was strongly attenuated in mice. Here, we demonstrate that the role of PLpro DUB activity during infection can vary considerably between highly pathogenic coronaviruses. Therefore, careful considerations should be taken when developing pan-coronavirus antiviral strategies targeting PLpro.

Randomised study of a new inline respiratory function monitor (Juno) to improve mask seal and delivered ventilation with neonatal manikins.

BACKGROUND: Respiratory function monitors (RFMs) have been used extensively in manikin and infant studies yet have not become the standard of training. We report the outcomes of a new portable, lightweight RFM, the Juno, designed to show mask leak and deflation tidal volume to assist in positive pressure ventilation (PPV) competency training using manikins. METHODS: Two leak-free manikins (preterm and term) were used. Participants provided PPV to manikins using two randomised devices, self-inflating bag (SIB) and T-piece resuscitator (TPR), with Juno display initially blinded then unblinded in four 90 s paired sequences, aiming for adequate chest wall rise and target minimal mask leak with appropriate target delivered volume when using the monitor. RESULTS: 49 experienced neonatal staff delivered 15 569 inflations to the term manikin and 14 580 inflations to the preterm. Comparing blinded to unblinded RFM display, there were significant reductions in all groups in the number of inflations out of target range volumes (preterm: SIB 22.6-6.6%, TPR 7.1-4.2% and term: SIB 54.8-37.8%, TPR 67.2-63.8%). The percentage of mask leak inflations >60% was reduced in preterm: SIB 20.7-7.2%, TPR 23.4-7.4% and in term: SIB 8.7-3.6%, TPR 23.5-6.2%). CONCLUSIONS: Using the Juno monitor during simulated resuscitation significantly improved mask leak and delivered ventilation among otherwise experienced staff using preterm and term manikins. The Juno is a novel RFM that may assist in teaching and self-assessment of resuscitation PPV technique.

Demonstrating the importance of porcine reproductive and respiratory syndrome virus papain-like protease 2 deubiquitinating activity in viral replication by structure-guided mutagenesis.

Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUB-specific mutations faces significant selective pressure to revert to wild-type virus in MARC-145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication.

Biochemical Insights into Imipenem Collateral Susceptibility Driven by ampC Mutations Conferring Ceftolozane/Tazobactam Resistance in Pseudomonas aeruginosa.

Several Pseudomonas aeruginosa AmpC mutants have emerged that exhibit enhanced activity against ceftazidime and ceftolozane, while also evading inhibition by avibactam. Interestingly, P. aeruginosa strains harboring these AmpC mutations fortuitously exhibit enhanced carbapenem susceptibility. This acquired susceptibility was investigated by comparing the degradation of imipenem by wild-type and cephalosporin-resistant AmpC. We show that cephalosporin-resistant AmpC enzymes lose their efficacy for hydrolyzing imipenem and suggest that this may be due to their increased flexibility and dynamics relative to the wild type.

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Increased phosphorylation of HexM improves lysosomal uptake and potential for managing GM2 gangliosidoses.

Tay-Sachs and Sandhoff diseases are genetic disorders resulting from mutations in HEXA or HEXB, which code for the α- and ß-subunits of the heterodimer ß-hexosaminidase A (HexA), respectively. Loss of HexA activity results in the accumulation of GM2 ganglioside (GM2) in neuronal lysosomes, culminating in neurodegeneration and death, often by age 4. Previously, we combined critical features of the α- and ß-subunits of HexA into a single subunit to create a homodimeric enzyme known as HexM. HexM is twice as active as HexA and degrades GM2 in vivo, making it a candidate for enzyme replacement therapy (ERT). Here we show HexM production is scalable to meet ERT requirements and we describe an approach that enhances its cellular uptake via co-expression with an engineered GlcNAc-1-phosphotransferase that highly phosphorylates lysosomal proteins. Further, we developed a HexA overexpression system and functionally compared the recombinant enzyme to HexM, revealing the kinetic differences between the enzymes. This study further advances HexM as an ERT candidate and provides a convenient system to produce HexA for comparative studies.

Mobile technology and delegated work in specialist community services: the EnComPaSS Integration project.

OBJECTIVES: The current UK healthcare workforce crisis is particularly severe in community services. A key limitation with traditional service-delivery models is the reliance on practitioners with levels of training and experience to enable them to operate independently. This paper describes a real-world evaluation of the implementation of digital health technology designed to provide remote, real-time support and task delegation in community palliative care services. It explores the ability of technology to support sustainable community workforce models and reports on key indicators of quality and efficiency. METHODS: The study was a mixed-methods, theory-driven evaluation, incorporating interviews, observations and analysis of routine data. The focus of this paper is the reporting of findings from pre-post implementation comparison and interrupted time series analysis. Data include community hospice service visits, hospital use by hospice patients and patient reported experiences. RESULTS: The digital health intervention allowed the service to include a more junior workforce (p<0.001, Cramer's V=0.241), requiring fewer joint visits (p<0.001, Cramer's V=0.087). No negative changes in hospitalisation were observed and patient reported experiences improved (p=0.023). Changes in hospital non-emergency bed days were inconclusive. However, emergency department admissions reduced significantly (-76.9 /month at 17 months, p=0.001). The cost per hour for visits reduced from £16.71 to £16.23 and annual savings of £135 153 are estimated for reduced emergency admissions. CONCLUSIONS: The evaluation demonstrates the value of digital innovation to support programmes of service redesign and begin to address the healthcare workforce crisis, while having a positive economic effect and indicating an improvement to patient experiences.

The endopeptidase of the maize-affecting Marafivirus type member maize rayado fino virus doubles as a deubiquitinase.

Marafiviruses are capable of persistent infection in a range of plants that have importance to the agriculture and biofuel industries. Although the genomes of a few of these viruses have been studied in-depth, the composition and processing of the polyproteins produced from their main ORFs have not. The Marafivirus polyprotein consists of essential proteins that form the viral replicase, as well as structural proteins for virus assembly. It has been proposed that Marafiviruses code for cysteine proteases within their polyproteins, which act as endopeptidases to autocatalytically cleave the polyprotein into functional domains. Furthermore, it has also been suggested that Marafivirus endopeptidases may have deubiquitinating activity, which has been shown to enhance viral replication by downregulating viral protein degradation by the ubiquitin (Ub) proteasomal pathway as well as tampering with cell signaling associated with innate antiviral responses in other positive-sense ssRNA viruses. Here, we provide the first evidence of cysteine proteases from six different Marafiviruses that harbor deubiquitinating activity and reveal intragenus differences toward Ub linkage types. We also examine the structural basis of the endopeptidase/deubiquitinase from the Marafivirus type member, maize rayado fino virus. Structures of the enzyme alone and bound to Ub reveal marked structural rearrangements that occur upon binding of Ub and provide insights into substrate specificity and differences that set it apart from other viral cysteine proteases.

Characterization of the sorbitol dehydrogenase SmoS from Sinorhizobium meliloti 1021.

Sinorhizobium meliloti 1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol to fructose and tagatose, respectively, using NAD+ as a cofactor. The main objective of this study was to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon-optimized for heterologous expression in Escherichia coli BL21 (DE3) Gold cells and the protein was subsequently overexpressed and purified. Size-exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetramer. SmoS was crystallized, and crystals obtained in the absence of substrate diffracted to 2.1 Šresolution and those of a complex with sorbitol diffracted to 2.0 Šresolution. SmoS was characterized kinetically and shown to have a preference for sorbitol despite having a higher affinity for galactitol. Computational ligand-docking experiments suggest that tagatose binds the protein in a more energetically favourable complex than fructose, which is retained in the active site over a longer time frame following oxidation and reduces the rate of the reaction. These results supplement the inventory of biomolecules with potential for industrial applications and enhance the understanding of metabolism in the model organism S. meliloti.

T-piece resuscitators: can they provide safe ventilation in a low compliant newborn lung?

BACKGROUND: T-piece resuscitators (TPRs) are used for primary newborn resuscitation in birthing and emergency rooms worldwide. A recent study has shown spikes in peak inflation pressure (PIP) over set values with two brands of TPRs inbuilt into infant warmer/resuscitation platforms. We aimed to compare delivered ventilation between two TPR drivers with inflation pressure spikes to a standard handheld TPR in a low test lung compliance (Crs), leak-free bench test model. METHODS: A single operator provided positive pressure ventilation to a low compliance test lung model (Crs 0.2-1 mL/cmH2O) at set PIP of 15, 25, 35 and 40 cmH2O. Two TPR devices with known spikes (Draeger Resuscitaire, GE Panda) were compared with handheld Neopuff (NP). Recommended settings for positive end-expiratory pressure (5 cmH2O), inflation rate of 60/min and gas flow rate 10 L/min were used. RESULTS: 2293 inflations were analysed. Draeger and GE TPR drivers delivered higher mean PIP (Panda 18.9-49.5 cmH2O; Draeger 21.2-49.2 cmH2O and NP 14.8-39.9 cmH2O) compared with set PIP and tidal volumes (TVs) compared with the NP (Panda 2.9-7.8 mL; Draeger 3.8-8.1 mL; compared with NP 2.2-6.0 mL), outside the prespecified acceptable range (±10% of set PIP and ±10% TV compared with NP). CONCLUSION: The observed spike in PIP over set values with Draeger and GE Panda systems resulted in significantly higher delivered volumes compared with the NP with identical settings. Manufacturers need to address these differences. The effect on patient outcomes is unknown.

Frontrunners in the race to develop a SARS-CoV-2 vaccine.

Numerous studies continue to be published on the COVID-19 pandemic that is being caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Given the rapidly evolving global response to SARS-CoV-2, here we primarily review the leading COVID-19 vaccine strategies that are currently in Phase III clinical trials. Nonreplicating viral vector strategies, inactivated virus, recombinant protein subunit vaccines, and nucleic acid vaccine platforms are all being pursued in an effort to combat the infection. Preclinical and clinal trial results of these efforts are examined as well as the characteristics of each vaccine strategy from the humoral and cellular immune responses they stimulate, effects of any adjuvants used, and the potential risks associated with immunization such as antibody-dependent enhancement. A number of promising advancements have been made toward the development of multiple vaccine candidates. Preliminary data now emerging from phase III clinical trials show encouraging results for the protective efficacy and safety of at least 3 frontrunning candidates. There is hope that one or more will emerge as potent weapons to protect against SARS-CoV-2.

Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replicase expression.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1ß (nsp1ß) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1ß and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1ß and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1ß:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1ß within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1ß and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1ß and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

Determining the Electronic Structure of Paramagnetic Intermediates in membrane proteins: A high-resolution 2D 1H hyperfine sublevel correlation study of the redox-active tyrosines of photosystem II.

The photosynthetic reaction center, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive water oxidation. The four-electron water oxidation reaction occurs at the tetranuclear manganese­calcium-oxo (Mn4Ca-oxo) cluster that is present in the oxygen-evolving complex (OEC) of PSII. The water oxidation reaction is facilitated by proton-coupled electron transfer (PCET) at the redox-active tyrosine residue, YZ, in the OEC which is one of the two symmetric tyrosine residues, YZ and YD, in PSII. Although YZ and YD are chemically identical, their redox properties and reaction kinetics are very different. In the present study, we apply high-resolution two-dimensional (2D) 1H hyperfine sublevel correlation (HYSCORE) spectroscopy to determine the electronic structure of YZ and YD to understand better the functional tuning of PCET at each tyrosine. Most importantly, the 2D HYSCORE measurements that are described here are applicable for the study of paramagnetic cofactors in a wide variety of membrane-bound proteins.

HYSCORE and DFT Studies of Proton-Coupled Electron Transfer in a Bioinspired Artificial Photosynthetic Reaction Center.

The photosynthetic water-oxidation reaction is catalyzed by the oxygen-evolving complex in photosystem II (PSII) that comprises the Mn4CaO5 cluster, with participation of the redox-active tyrosine residue (YZ) and a hydrogen-bonded network of amino acids and water molecules. It has been proposed that the strong hydrogen bond between YZ and D1-His190 likely renders YZ kinetically and thermodynamically competent leading to highly efficient water oxidation. However, a detailed understanding of the proton-coupled electron transfer (PCET) at YZ remains elusive owing to the transient nature of its intermediate states involving YZ⋅. Herein, we employ a combination of high-resolution two-dimensional 14N hyperfine sublevel correlation spectroscopy and density functional theory methods to investigate a bioinspired artificial photosynthetic reaction center that mimics the PCET process involving the YZ residue of PSII. Our results underscore the importance of proximal water molecules and charge delocalization on the electronic structure of the artificial reaction center.

Adding Insult to Injury: Mechanistic Basis for How AmpC Mutations Allow Pseudomonas aeruginosa To Accelerate Cephalosporin Hydrolysis and Evade Avibactam.

Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide and notorious for its broad-spectrum resistance to antibiotics. A key mechanism that provides extensive resistance to ß-lactam antibiotics is the inducible expression of AmpC ß-lactamase. Recently, a number of clinical isolates expressing mutated forms of AmpC have been found to be clinically resistant to the antipseudomonal ß-lactam-ß-lactamase inhibitor (BLI) combinations ceftolozane-tazobactam and ceftazidime-avibactam. Here, we compare the enzymatic activity of wild-type (WT) AmpC from PAO1 to those of four of these reported AmpC mutants, bearing mutations E247K (a change of E to K at position 247), G183D, T96I, and ΔG229-E247 (a deletion from position 229 to 247), to gain detailed insights into how these mutations allow the circumvention of these clinically vital antibiotic-inhibitor combinations. We found that these mutations exert a 2-fold effect on the catalytic cycle of AmpC. First, they reduce the stability of the enzyme, thereby increasing its flexibility. This appears to increase the rate of deacylation of the enzyme-bound ß-lactam, resulting in greater catalytic efficiencies toward ceftolozane and ceftazidime. Second, these mutations reduce the affinity of avibactam for AmpC by increasing the apparent activation barrier of the enzyme acylation step. This does not influence the catalytic turnover of ceftolozane and ceftazidime significantly, as deacylation is the rate-limiting step for the breakdown of these antibiotic substrates. It is remarkable that these mutations enhance the catalytic efficiency of AmpC toward ceftolozane and ceftazidime while simultaneously reducing susceptibility to inhibition by avibactam. Knowledge gained from the molecular analysis of these and other AmpC resistance mutants will, we believe, aid in the design of ß-lactams and BLIs with reduced susceptibility to mutational resistance.