Enzymes for dermatological use.

Skin is the ultimate barrier between body and environment and prevents water loss and penetration of pathogens and toxins. Internal and external stressors, such as ultraviolet radiation (UVR), can damage skin integrity and lead to disorders. Therefore, skin health and skin ageing are important concerns and increased research from cosmetic and pharmaceutical sectors aims to improve skin conditions and provide new anti-ageing treatments. Biomolecules, compared to low molecular weight drugs and cosmetic ingredients, can offer high levels of specificity. Topically applied enzymes have been investigated to treat the adverse effects of sunlight, pollution and other external agents. Enzymes, with a diverse range of targets, present potential for dermatological use such as antioxidant enzymes, proteases and repairing enzymes. In this review, we discuss enzymes for dermatological applications and the challenges associated in this growing field.

Mechanistic aspects of IPTG (isopropylthio-ß-galactoside) transport across the cytoplasmic membrane of Escherichia coli-a rate limiting step in the induction of recombinant protein expression.

Coupling transcription of a cloned gene to the lac operon with induction by isopropylthio-ß-galactoside (IPTG) has been a favoured approach for recombinant protein expression using Escherichia coli as a heterologous host for more than six decades. Despite a wealth of experimental data gleaned over this period, a quantitative relationship between extracellular IPTG concentration and consequent levels of recombinant protein expression remains surprisingly elusive across a broad spectrum of experimental conditions. This is because gene expression under lac operon regulation is tightly correlated with intracellular IPTG concentration due to allosteric regulation of the lac repressor protein (lacY). An in-silico mathematical model established that uptake of IPTG across the cytoplasmic membrane of E. coli by simple diffusion was negligible. Conversely, lacY mediated active transport was a rapid process, taking only some seconds for internal and external IPTG concentrations to equalize. Optimizing kcat and KM parameters by targeted mutation of the galactoside binding site in lacY could be a future strategy to improve the performance of recombinant protein expression. For example, if kcat were reduced whilst KM was increased, active transport of IPTG across the cytoplasmic membrane would be reduced, thereby lessening the metabolic burden on the cell and expediating accumulation of recombinant protein. The computational model described herein is made freely available and is amenable to optimize recombinant protein expression in other heterologous hosts. ONE-SENTENCE SUMMARY: A computational model made freely available to optimize recombinant protein expression in Escherichia coli other heterologous hosts.

Dupilumab-Associated Sweet Syndrome.

Sweet syndrome (SS), also known as acute febrile neutrophilic dermatosis, is characterized by the abrupt appearance of edematous and erythematous papules, plaques, or nodules on the skin that have a distinct histopathologic appearance. Several subtypes of SS exist, including classic (also referred to as idiopathic) and drug induced. Although multiple medications have been implicated as causative agents, we present a rare case of SS caused by dupilumab, a monoclonal antibody therapy, used in the treatment of severe eosinophilic asthma and other conditions. Clinicians should be aware of this potential adverse reaction, as prompt recognition and treatment are essential.

Resolving Confusion Surrounding d-Ala-d-Ala Ligase Catalysis in Cyanobacterial Mycosporine-Like Amino Acid (MAA) Biosynthesis.

Mycosporine-like amino acids (MAAs) are natural UV-absorbing sunscreens that evolved in cyanobacteria and algae to palliate harmful effects from obligatory exposure to solar radiation. Multiple lines of evidence prove that in cyanobacteria all MAAs are derived from mycosporine-glycine, which is typically modified by an ATP-dependent ligase encoded by the gene mysD. The function of the mysD ligase has been experimentally described but haphazardly named based solely upon sequence similarity to the d-alanine-d-alanine ligase of bacterial peptidoglycan biosynthesis. Combining phylogeny and alpha-fold tertiary protein structure prediction unambiguously distinguished mysD from d-alanine-d-alanine ligase. The renaming of mysD to mycosporine-glycine-amine ligase (MG-amine ligase) using recognised enzymology rules of nomenclature is, therefore, proposed, and considers relaxed specificity for several different amino acid substrates. The evolutionary and ecological context of MG-amine ligase catalysis merits wider appreciation especially when considering exploiting cyanobacteria for biotechnology, for example, producing mixtures of MAAs with enhanced optical or antioxidant properties.

Phylogenetic and host-parasite relationship analyses of Henneguya caquetaia sp. nov (Myxosporea: Myxobolidae) infecting an Amazonian cichlid fish.

The present study describes a new species of Henneguya infecting the ornamental fish Caquetaia spectabilis from the Brazilian Amazon. Fish specimens were collected where the Tapajós and Amazon rivers merge, municipality of Santarém in the State of Pará, Brazil. Infections were intense, with several plasmodia spread on the opercula, fins and eye. Phylogenetic characterization and host-parasite relationship studies of the new Henneguya species used a combination of small subunit ribosomal DNA (ssrDNA) and morphological (photonic and transmission electron microscopy) analyses. Plasmodia were white round to ellipsoidal measuring up to 1.8 mm. The myxospores body measured 20.5 ± 3.9 (15-27) in length, 7.9 µm (6.2-10.8) in width, 6.7 µm (6.0-7.6) in thickness, 20.5 µm (14.4-32.3) in caudal appendages length, and 40.6 µm (34.2-54.6) in total length. The two polar capsules were elongated and equal in size, measuring 4.3 µm (3.3-5.4) in length and 2.1 µm (1.3-2.8) in width. Histological analysis revealed the parasite development in connective tissues of the fins, eyes and opercula. The skin of the fins and opercula presented detachment of the epidermis, however, no inflamatory infiltrate was observed. In the eye were observed inflammatory infiltratate in the epithelium and stroma of the cornea. Ultrastructure analysis showed the connective tissue capsule composed by an inner cellular layer with fibroblasts and outer layer where collagen fibers arranged transversely yet interspersed by layers of fibers arranged longitudinally. Numerous invaginations and extensive pinocytotic channels were observed in the plasmodial membrane. A layer of microfilament-like microfilament-like material was observed in the ectoplasm area and along to the internal surface of the plasmodial membrane. Generative cells and early stages of sporogenesis were seen more internally. The ssrDNA based phylogeny showed the South American species grouped in two lineages and the new species arises in a well-sustained subclade as sister branch of the clade composed by Henneguya spp. parasites of cichlids fish.

Exploring the Relationship between Biosynthetic Gene Clusters and Constitutive Production of Mycosporine-like Amino Acids in Brazilian Cyanobacteria.

Cyanobacteria are oxygenic phototrophic prokaryotes that have evolved to produce ultraviolet-screening mycosporine-like amino acids (MAAs) to lessen harmful effects from obligatory exposure to solar UV radiation. The cyanobacterial MAA biosynthetic cluster is formed by a gene encoding 2-epi-5-epi-valiolone synthase (EVS) located immediately upstream from an O-methyltransferase (OMT) encoding gene, which together biosynthesize the expected MAA precursor 4-deoxygadusol. Accordingly, these genes are typically absent in non-producers. In this study, the relationship between gene cluster architecture and constitutive production of MAAs was evaluated in cyanobacteria isolated from various Brazilian biomes. Constitutive production of MAAs was only detected in strains where genes formed a co-linear cluster. Expectedly, this production was enhanced upon exposure of the strains to UV irradiance and by using distinct culture media. Constitutive production of MAAs was not detected in all other strains and, unexpectedly, production could not be induced by exposure to UV irradiation or changing growth media. Other photoprotection strategies which might be employed by these MAA non-producing strains are discussed. The evolutionary and ecological significance of gene order conservation warrants closer experimentation, which may provide a first insight into regulatory interactions of genes encoding enzymes for MAA biosynthesis.

Overview of the regulatory approval of tecovirimat intravenous formulation for treatment of smallpox: potential impact on smallpox outbreak response capabilities, and future tecovirimat development potential.

INTRODUCTION: Tecovirimat oral capsule formulation is approved in the US and Canada for treatment of smallpox and in the United Kingdom (UK) and European Union (EU) for treatment of multiple human orthopoxvirus diseases, including mpox. Smallpox is considered a serious threat, and there is currently an unprecedented global mpox outbreak. AREAS COVERED: A brief summary of the threat of smallpox, the threat of increasing mpox spread in endemic regions, and the unprecedented emergence of mpox into non-endemic regions is presented. The tecovirimat intravenous formulation clinical development program leading to USFDA approval for smallpox treatment is discussed. EXPERT OPINION: As of January 2023 tecovirimat is approved to treat mpox in the UK and EU. However, published clinical trial data evaluating tecovirimat efficacy and safety in mpox patients is pending. Increasing global prevalence of mpox highlights the potential benefits of a well-characterized, effective, and safe antiviral treatment for mpox infection. Ongoing trials in mpox patients may provide results supporting the use of tecovirimat to treat this disease. USFDA approval of tecovirimat for post-exposure prophylaxis in the event of a smallpox release, and the development of pediatric liquid formulations for patients under 13 kg, could provide additional public health benefits.

A scoping review to compare and contrast quality assurance aspects of l-asparaginase biosimilars.

l-asparaginase is a first-line medicine used for the treatment of acute lymphoblastic leukemia. Differing quality of marketed l-asparaginase biosimilars has been reported to adversely influence treatment outcomes. Herein, the quality of l-asparaginase biosimilars intended for clinical use was reviewed in sight of quality assurance parameters using English and Chinese language database searching, which provided information for possible improvements to the manufacture of this medicine. Ten articles met inclusion criteria, and quality attributes that measured potency, specific activity, purity and host cell proteins (HCPs) were identified. Biosimilars manufactured in high-income countries represented good quality in all aspects. Biosimilars manufactured in high-middle/middle-income countries, however, suggested poorer quality control particularly over removal of HCPs. Future work should now focus on establishing pharmacopeia monographs to establish equivalent quality assurance for l-asparaginase biosimilars manufactured between countries. Standardization of the quality profile, analytical methods and the limits of critical quality parameters, are essential to ensure appropriated efficacy and safety of clinical grade l-asparaginase.

Australian doctors are more engaged than UK doctors: why is this the case?

BACKGROUND: Despite reports highlighting the need for greater medical engagement and the benefits of being widely understood, very little information is available on the status of medical engagement in Australia, and how this compares to the UK. Answering this question will no doubt assist training bodies, curriculum designers and policy makers better understand relevant issues. METHODS: The medical engagement questionnaire (MES) was emailed to all medical staff working at 159 UK National Health Service Trusts and 18 health service organisations in Australia. The questionnaire consists of 30 predetermined items seeking responses using a 5-point Likert scale. RESULTS: Overall, doctors in the Australian dataset are slightly more engaged, or more positive, than their UK colleagues. Good interpersonal relationships was the only variable that UK doctors scored more positively than their Australian counterparts. At the lower end of the responses, that is the least engaged, we found this even more apparent. Where doctors in Australia are less disengaged, that is still more positive than the UK colleagues. CONCLUSION: While the profiles of medical engagement vary at the sites and also across the MES and subscales, the data illustrate that overall doctors in Australia feel valued and empowered, and they have purpose and direction and work in a collaborate culture. At the most disengaged end of the scale, Australian doctors are markedly less disengaged than their UK counterparts. There may be numerous factors that influence and change how engaged doctors are in both countries. The most prominent of these are appear to be working conditions and lifestyle, driven by funding and other economics issues. This research is likely to be of great interest to regulators and training bodies in both countries.

Factors that influence and change medical engagement in Australian not for profit hospitals.

PURPOSE: The study aims to assess medical engagement levels at two teaching hospitals and a 500 bed private hospital in two states operated by the same health care provider and to describe individual and organisational factors that influence and change medical engagement. DESIGN/METHODOLOGY/APPROACH: A survey was emailed to all junior and senior medical staff, seeking responses to 30 pre-determined items. The survey used a valid and reliable instrument which provided an overall index of medical engagement. Qualitative data were also collected by including an open ended question. FINDINGS: Doctors (n = 810) working at all sites are in the top 20-40 percentile when compared to Australia and the United Kingdom. Two sites in one state were in the highest relative engagement band with the other being in the high relative range when compared to the (UK) and the medium relative band when compared to sites in Australia. Senior doctors working at all three were less engaged on feeling valued and empowered, when compared to having purpose and direction or working in a collaborative culture. This appears to be related to work satisfaction and whether they feel encouraged to develop their skills and progress their careers. Junior doctors at 1 site are much less engaged than colleagues working at another. Since their formal training pathways are identical the informal training experience appears to be an engagement factor. ORIGINALITY/VALUE: Despite medical engagement being recognised as crucial, little is known about individual and organisational factors that support doctors to be engaged, particularly for juniors and in the private sector.

Integration of a community-based harm reduction program into a safety net hospital: a qualitative study.

BACKGROUND: Community-based harm reduction programs reduce morbidity and mortality associated with drug use. While hospital-based inpatient addiction consult services can also improve outcomes for patients using drugs, inpatient clinical care is often focused on acute withdrawal and the medical management of substance use disorders. There has been limited exploration of the integration of community-based harm reduction programs into the hospital setting. We conducted a qualitative study to describe provider perspectives on the implementation of a harm reduction in-reach program. METHODS: We conducted 24 semi-structured interviews with providers from three different primary work sites within a safety net hospital in Boston, MA, in 2021. Interviews explored perceived facilitators and barriers to the implementation of the harm reduction in-reach program in the hospital setting and solicited recommendations for potential improvements to the harm reduction in-reach program. Interviews were analyzed using an inductive approach that incorporated principles of grounded theory methodology to identify prevailing themes. RESULTS: Twenty-four participants were interviewed from the harm reduction in-reach program, inpatient addiction consult service, and the hospital observation unit. Thematic analysis revealed seven major themes and multiple facilitators and barriers to the implementation of the harm reduction in-reach program. Participants highlighted the impact of power differences within the medical hierarchy on inter-team communication and clinical care, the persistence of addiction-related stigma, the importance of coordination and role delineation between care team members, and the benefits of a streamlined referral process. CONCLUSIONS: Harm reduction programs offer accessible, patient-centered, low-barrier care to patients using drugs. The integration of community-based harm reduction programs into the inpatient setting is a unique opportunity to bridge inpatient and outpatient care and expand the provision of harm reduction services. TRIAL REGISTRATION: Not applicable.

Structural and functional diversity of asparaginases: Overview and recommendations for a revised nomenclature.

Asparaginases (ASNases) are a large and structurally diverse group of enzymes ubiquitous amongst archaea, bacteria and eukaryotes, that catalyze hydrolysis of asparagine to aspartate and ammonia. Bacterial ASNases are important biopharmaceuticals for the treatment of acute lymphoblastic leukemia, although some patients experience adverse allergic side effects during treatment with these protein therapeutics. ASNases are currently divided into three families: plant-type ASNases, Rhizobium etli-type ASNases and bacterial-type ASNases. This system is outdated as both bacterial-type and plant-type families also include archaeal, bacterial and eukaryotic enzymes, each with their own distinct characteristics. Herein, phylogenetic studies allied to tertiary structural analyses are described with the aim of proposing a revised and more robust classification system that considers the biochemical diversity of ASNases. Accordingly, based on distinct peptide domains, phylogenetic data, structural analysis and functional characteristics, we recommend that ASNases now be divided into three new distinct classes containing subgroups according to structural and functional aspects. Using this new classification scheme, 25 ASNases were identified as candidates for future new lead discovery.

Recruitment of toxin-like proteins with ancestral venom function supports endoparasitic lifestyles of Myxozoa.

Cnidarians are the oldest lineage of venomous animals and use nematocysts to discharge toxins. Whether venom toxins have been recruited to support parasitic lifestyles in the Endocnidozoa (Myxozoa + Polypodium) is, however, unknown. To examine this issue we variously employed transcriptomic, proteomic, associated molecular phylogenies, and localisation studies on representative primitive and derived myxozoans (Malacosporea and Myxosporea, respectively), Polypodium hydriforme, and the free-living staurozoan Calvadosia cruxmelitensis. Our transcriptomics and proteomics analyses provide evidence for expression and translation of venom toxin homologs in myxozoans. Phylogenetic placement of Kunitz type serine protease inhibitors and phospholipase A2 enzymes reveals modification of toxins inherited from ancestral free-living cnidarian toxins, and that venom diversity is reduced in myxozoans concordant with their reduced genome sizes. Various phylogenetic analyses of the Kunitz-type toxin family in Endocnidozoa suggested lineage-specific gene duplications, which offers a possible mechanism for enhancing toxin diversification. Toxin localisation in the malacosporean Buddenbrockia plumatellae substantiates toxin translation and thus illustrates a repurposing of toxin function for endoparasite development and interactions with hosts, rather than for prey capture or defence. Whether myxozoan venom candidates are expressed in transmission stages (e.g. in nematocysts or secretory vesicles) requires further investigation.

The Australian and New Zealand Clinician Educator Network (ANZCEN) Unconference: What's an unconference and how can it develop communities of practice?

The Australian and New Zealand Clinician Educator Network (ANZCEN) is a collaborative interprofessional group developed to promote the development of education in critical care healthcare practice. In November 2018, 45 critical care practitioners met at the first ANZCEN Unconference. In an unconference, the participants drive the agenda, and learning occurs from the active process of engaging in a community of practice. The aim of this unconference was to develop an innovative approach to learning through a collaborative framework with interprofessional representation across critical care specialties. Four key themes were identified in the unconference as drivers of interprofessional critical care educational priorities: interprofessional learning, workplace learning, faculty development, research, and scholarship. In this discussion paper, we describe our experiences organizing, participating in, and evaluating an unconference, and we examine its usefulness as a medium for promoting the interprofessional learning agenda in critical care. We hope that the processes outlined in this discussion paper will provide a useful resource for other clinicians who are considering developing an unconference. Finally, we argue that the unconference offers a unique and important model for future education of critical care practitioners where the emphasis on collaboration and communication through interprofessional learning and practice will be required to improve health outcomes and promote a patient-centered model of care.

An overview of tecovirimat for smallpox treatment and expanded anti-orthopoxvirus applications.

INTRODUCTION: Tecovirimat (TPOXX®; ST-246) was approved for the treatment of symptomatic smallpox by the USFDA in July of 2018 and has been stockpiled by the US government for use in a smallpox outbreak. While there has not been a reported case of smallpox since 1978 it is still considered a serious bioterrorism threat. AREAS COVERED: A brief history of smallpox from its proposed origins as a human disease through its eradication in the late 20th century is presented. The current smallpox threat and the current public health response plans are described. The discovery, and development of tecovirimat through NDA submission and subsequent approval for treatment of smallpox are discussed. Google Scholar and PubMed were searched over all available dates for relevant publications. EXPERT OPINION: Approval of tecovirimat to treat smallpox represents an important milestone in biosecurity preparedness. Incorporating tecovirimat into the CDC smallpox response plan, development of pediatric liquid and intravenous formulations, and approval for post-exposure prophylaxis would provide additional health security benefit.Tecovirimat shows broad efficacy against orthopoxviruses in vitro and in vivo and could be developed for use against emerging orthopoxvirus diseases such as monkeypox, vaccination-associated adverse events, and side effects of vaccinia oncolytic virus therapy.

Epidemiology of Polypodium hydriforme in American Paddlefish.

Polypodium hydriforme is a parasitic cnidarian that develops within the eggs of acipenseriform fish in the Old and New Worlds. Currently regarded as monotypic, P. hydriforme has been studied largely in the context of caviar production in Russian sturgeon species. We report the first robust epidemiological study of P. hydriforme in North American acipenseriform fish. We sampled infection prevalences (in 2017 and 2018) and intensities (in 2017) during annual surveys of American Paddlefish, Polyodon spathula, caught during spawning migration in north-eastern Oklahoma. Egg masses were characterized for the presence and intensity of P. hydriforme infection. Prevalences were similar in 2017 and 2018 (49% and 45%, respectively). Generally, a small number of eggs were infected per egg mass, but a few were heavily infected. Longer, heavier and older fish are more likely to be infected and to harbour more severe infections. In addition, infection is linked to decreases in roe fat weight independently of fish length, weight, age or roe weight. Infection thus diminishes Paddlefish energy reserves (roe fat) which could in turn impact host fitness. Our results raise questions about the impacts of infection on caviar production and Paddlefish conservation and suggest insights on infection dynamics and parasite strategies.

Horizontal transfer of a natterin-like toxin encoding gene within the holobiont of the reef building coral Acropora digitifera (Cnidaria: Anthozoa: Scleractinia) and across multiple animal linages.

Phylogenetic evidence is provided for horizontal transfer of a natterin-like toxin encoding gene from fungi into the genome of the coral Acropora digitifera. Sequencing analysis of the coral tissues supported that a fungal taxon predicted to be the most likely gene donor was represented in the coral microbiome. Further bioinformatics data suggested widespread recruitment of the natterin-like gene into venomous terrestrial invertebrates, and repositioning of this gene to non-toxic functions in non-venomous teleost fish.

Reciprocal transplantation of the heterotrophic coral Tubastraea coccinea (Scleractinia: Dendrophylliidae) between distinct habitats did not alter its venom toxin composition.

Tubastraea coccinea is an azooxanthellate coral species recorded in the Indian and Atlantic oceans and is presently widespread in the southwestern Atlantic with an alien status for Brazil. T. coccinea outcompete other native coral species by using a varied repertoire of biological traits. For example, T. coccinea has evolved potent venom capable of immobilizing and digesting zooplankton prey. Diversification and modification of venom toxins can provide potential adaptive benefits to individual fitness, yet acquired alteration of venom composition in cnidarians is poorly understood as the adaptive flexibility affecting toxin composition in these ancient lineages has been largely ignored. We used quantitative high-throughput proteomics to detect changes in toxin expression in clonal fragments of specimens collected and interchanged from two environmentally distinct and geographically separate study sites. Unexpectedly, despite global changes in protein expression, there were no changes in the composition and abundance of toxins from coral fragments recovered from either site, and following clonal transplantation between sites. There were also no apparent changes to the cnidome (cnidae) and gross skeletal or soft tissue morphologies of the specimens. These results suggest that the conserved toxin complexity of T. coccinea co-evolved with innovation of the venom delivery system, and its morphological development and phenotypic expression are not modulated by habitat pressures over short periods of time. The adaptive response of the venom trait to specific predatory regimes, however, necessitates further consideration.