Voltage-calibrated, finely tunable protein assembly.

Neuronally triggered phosphorylation drives the calibrated and cyclable assembly of the reflectin signal transducing proteins, resulting in their fine tuning of colours reflected from specialized skin cells in squid for camouflage and communication. In close parallel to this physiological behaviour, we demonstrate for the first time that electrochemical reduction of reflectin A1, used as a surrogate for charge neutralization by phosphorylation, triggers voltage-calibrated, proportional and cyclable control of the size of the protein's assembly. Electrochemically triggered condensation, folding and assembly were simultaneously analysed using in situ dynamic light scattering, circular dichroism and UV absorbance spectroscopies. The correlation of assembly size with applied potential is probably linked to reflectin's mechanism of dynamic arrest, which is controlled by the extent of neuronally triggered charge neutralization and the corresponding fine tuning of colour in the biological system. This work opens a new perspective on electrically controlling and simultaneously observing reflectin assembly and, more broadly, provides access to manipulate, observe and electrokinetically control the formation of intermediates and conformational dynamics of macromolecular systems.

Building blocks of biofilms - an engaging and hands-on microbiology outreach activity for school children and the general public.

Biofilms are naturally occurring communities of micro-organisms, attached to a surface and often embedded in a matrix of self-produced polymeric substances. Biofilms are widely implicated in human infections, particularly on prostheses and medical implants. Such biofilms are difficult to eradicate, often leading to replacement of the prosthesis and resulting in a significant burden to healthcare. Here we present a fun and engaging interactive activity targeted toward primary school/early secondary school children, introducing the concept of natural and healthcare-associated biofilms, using dental plaque as an archetypal example. Dental plaque forms as a result of poor oral/dental hygiene, and develops according to a typical series of defined stages: attachment and adherence to the surface, followed by colonization and maturation of the biofilm structure, and eventually, dispersal. This activity uses dental disclosing tablets to visualize real biofilms (plaque) on the participants teeth, and uses interlocking building-blocks to represent microorganisms, where children build three-dimensional 'biofilms' of varying shapes and structural integrities. Each of the stages of development are discussed in detail, and after building the biofilms, balls of different shapes, sizes and weights can be used as 'antimicrobials' to disrupt the biofilm structure. The outcomes of the activity are to enhance knowledge and general understanding of biofilms; their ubiquitous presence in the natural environment, development, implications in healthcare, and challenges of treatment. The various 'antimicrobial' balls also provide a basis to introduce and discuss drug selection for infections, and the importance of using the correct antimicrobial for different infections to avoid development of resistance.

A new electrochemical method that mimics phosphorylation of the core tau peptide K18 enables kinetic and structural analysis of intermediates and assembly.

Tau protein's reversible assembly and binding of microtubules in brain neurons are regulated by charge-neutralizing phosphorylation, while its hyperphosphorylation drives the irreversible formation of cytotoxic filaments associated with neurodegenerative diseases. However, the structural changes that facilitate these diverse functions are unclear. Here, we analyzed K18, a core peptide of tau, using newly developed spectroelectrochemical instrumentation that enables electroreduction as a surrogate for charge neutralization by phosphorylation, with simultaneous, real-time quantitative analyses of the resulting conformational transitions and assembly. We observed a tipping point between behaviors that paralleled the transition between tau's physiologically required, reversible folding and assembly and the irreversibility of assemblies. The resulting rapidly electroassembled structures represent the first fibrillar tangles of K18 that have been formed in vitro at room temperature without using heparin or other charge-complementary anionic partners. These methods make it possible to (i) trigger and analyze in real time the early stages of conformational transitions and assembly without the need for preformed seeds, heterogenous coacervation, or crowding; (ii) kinetically resolve and potentially isolate never-before-seen early intermediates in these processes; and (iii) develop assays for additional factors and mechanisms that can direct the trajectory of assembly from physiologically benign and reversible to potentially pathological and irreversible structures. We anticipate wide applicability of these methods to other amyloidogenic systems and beyond.

Global developments in social prescribing.

Social prescribing is an approach that aims to improve health and well-being. It connects individuals to non-clinical services and supports that address social needs, such as those related to loneliness, housing instability and mental health. At the person level, social prescribing can give individuals the knowledge, skills, motivation and confidence to manage their own health and well-being. At the society level, it can facilitate greater collaboration across health, social, and community sectors to promote integrated care and move beyond the traditional biomedical model of health. While the term social prescribing was first popularised in the UK, this practice has become more prevalent and widely publicised internationally over the last decade. This paper aims to illuminate the ways social prescribing has been conceptualised and implemented across 17 countries in Europe, Asia, Australia and North America. We draw from the 'Beyond the Building Blocks' framework to describe the essential inputs for adopting social prescribing into policy and practice, related to service delivery; social determinants and household production of health; workforce; leadership and governance; financing, community organisations and societal partnerships; health technology; and information, learning and accountability. Cross-cutting lessons can inform country and regional efforts to tailor social prescribing models to best support local needs.

Targeted control of pneumolysin production by a mobile genetic element in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen that can cause severe invasive diseases such as pneumonia, septicaemia and meningitis. Young children are at a particularly high risk, with an estimated 3-4 million cases of severe disease and between 300 000 and 500 000 deaths attributable to pneumococcal disease each year. The haemolytic toxin pneumolysin (Ply) is a primary virulence factor for this bacterium, yet despite its key role in pathogenesis, immune evasion and transmission, the regulation of Ply production is not well defined. Using a genome-wide association approach, we identified a large number of potential affectors of Ply activity, including a gene acquired horizontally on the antibiotic resistance-conferring Integrative and Conjugative Element (ICE) ICESp23FST81. This gene encodes a novel modular protein, ZomB, which has an N-terminal UvrD-like helicase domain followed by two Cas4-like domains with potent ATP-dependent nuclease activity. We found the regulatory effect of ZomB to be specific for the ply operon, potentially mediated by its high affinity for the BOX repeats encoded therein. Using a murine model of pneumococcal colonization, we further demonstrate that a ZomB mutant strain colonizes both the upper respiratory tract and lungs at higher levels when compared to the wild-type strain. While the antibiotic resistance-conferring aspects of ICESp23FST81 are often credited with contributing to the success of the S. pneumoniae lineages that acquire it, its ability to control the expression of a major virulence factor implicated in bacterial transmission is also likely to have played an important role.

Low Voltage Voltammetry Probes Proton Dissociation Equilibria of Amino Acids and Peptides.

Platinum-catalyzed electrochemical reduction of dissociable protons at low potentials was used to investigate proton dissociation equilibria of freely diffusing and peptide-incorporated charged amino acids. We first demonstrate with five charged essential amino acids and their analogs that the electrochemically induced deprotonation of each amino acid occurs at distinct formal reduction potential. Moreover, the observed direct reduction for all the charged species, excluding arginine, occurs at low potentials suitable for investigation under aqueous conditions (-0.4 to -0.9 V vs Ag/AgCl). The direct proton reduction was resolved via deconvolution of the observed differential pulse voltammogram (DPV) from background hydronium reduction and water electrolysis. A linear correlation was found between the formal reduction potentials and the pKa values of the dissociable protons hosted by various molecular moieties in the amino acids and their analogs and further verified with tripeptides. DPV of poly(l-lysine) decamer (Lys10) distinctively resolved the pKa values of the amino groups in the side chains and N-terminus, at a resolution not possible by conventional acid-base titration. This work demonstrates selective electrochemical titration of dissociable protons in charged amino acids in the free state and as residues in biomolecules, as well as the utility of DPV to indirectly interrogate local electrostatic environments that are essential to the stability and function of biomolecules.

Reversible electrochemical triggering and optical interrogation of polylysine α-helix formation.

Reversible electrochemical triggering of the random coil to α-helix conformational transition of polylysine (Lys10, Lys20, Lys50) was accomplished at a Pt electrode at potentials < |1| V vs. Ag/AgCl. Direct electroreduction of the N-terminus vs ε-amino groups in Lys sidechains, as well as hydronium reduction and electrolysis, could be easily distinguished and deconvolved using differential pulse voltammetry. Electrochemistry was coupled with in situ UV absorbance and circular dichroism spectroscopies to dynamically follow the evolution of α-helix formation at different potentials. Isotope experiments in H2O vs. D2O unequivocally confirm that direct electroreduction of ε-NH3+/ND3+ groups in Lys sidechains, rather than electrochemically generated pH gradient-induced deprotonation, leads to subsequent α-helix formation. The site-selective electrochemistry and optical methodologies presented herein can be generalized and extended to interrogate other protonation-sensitive biomolecular systems, and potentially provide access to early intermediates and control over the dynamic structural evolution of peptides and proteins.

Immunotherapy and Systemic Treatment of Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinomas (cSCC) represent one of the most diagnosed non-melanoma skin cancers and its incidence is increasing globally. Whereas early stage and low risk cSCC is typically treated with surgery, and in some cases other localized therapeutic modalities, locally advanced or metastatic cSCC is a cause of significant morbidity and mortality that requires a different approach to therapy. Therapeutic attempts at treating advanced cSCC include a multi-disciplinary approach with considerations for surgery, radiation, and systemic therapies. In this review, we will discuss the various systemic therapies that have been trialed for advanced cSCC, beginning with the early cytotoxic and platinum-based agents as well as their corresponding limitations. We will then review the targeted approaches using EGFR inhibitors prior to discussing the more recent immunotherapeutics that have shown good tumor responses in this often-lethal disease.

Vulvar Primary Cutaneous CD8+ Aggressive Epidermotropic Cytotoxic T-Cell Lymphoma.

Cutaneous T-cell lymphomas may present with a clinical course that is incongruent with the associated histologic findings. Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma classically presents as an abrupt eruption of disseminated ulcerated annular plaques with aggressive behavior and a poor prognosis. Herein we describe a vulvar primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma with a locally aggressive clinical course that was strikingly responsive to radiation therapy. As aggressive therapy involving systemic chemotherapy is indicated for primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma, appropriate clinico-pathologic correlation is crucial for preventing potentially excessive or insufficient therapeutic intervention. Our case also highlights the pivotal role of both radiation therapy and infection control in the management of aggressive cutaneous vulvar lymphomas.

Post-acute COVID-19 associated with evidence of bystander T-cell activation and a recurring antibiotic-resistant bacterial pneumonia.

Here, we describe the case of a COVID-19 patient who developed recurring ventilator-associated pneumonia caused by Pseudomonas aeruginosa that acquired increasing levels of antimicrobial resistance (AMR) in response to treatment. Metagenomic analysis revealed the AMR genotype, while immunological analysis revealed massive and escalating levels of T-cell activation. These were both SARS-CoV-2 and P. aeruginosa specific, and bystander activated, which may have contributed to this patient's persistent symptoms and radiological changes.

Electrochemistry as a surrogate for protein phosphorylation: voltage-controlled assembly of reflectin A1.

Phosphorylation is among the most widely distributed mechanisms regulating the tunable structure and function of proteins in response to neuronal, hormonal and environmental signals. We demonstrate here that the low-voltage electrochemical reduction of histidine residues in reflectin A1, a protein that mediates the neuronal fine-tuning of colour reflected from skin cells for camouflage and communication in squids, acts as an in vitro surrogate for phosphorylation in vivo, driving the assembly previously shown to regulate its function. Using micro-drop voltammetry and a newly designed electrochemical cell integrated with an instrument measuring dynamic light scattering, we demonstrate selective reduction of the imidazolium side chains of histidine in monomers, oligopeptides and this complex protein in solution. The formal reduction potential of imidazolium proves readily distinguishable from those of hydronium and primary amines, allowing unequivocal confirmation of the direct and energetically selective deprotonation of histidine in the protein. The resulting 'electro-assembly' provides a new approach to probe, understand, and control the mechanisms that dynamically tune protein structure and function in normal physiology and disease. With its abilities to serve as a surrogate for phosphorylation and other mechanisms of charge neutralization, and to potentially isolate early intermediates in protein assembly, this method may be useful for analysing never-before-seen early intermediates in the phosphorylation-driven assembly of other proteins in normal physiology and disease.

Successful Mastectomy and Chemotherapy in a Patient with Breast Cancer and Active Generalized Pyoderma Gangrenosum.

INTRODUCTION: Pyoderma gangrenosum (PG) is a rare, ulcerating neutrophilic dermatosis often associated with inflammatory bowel disease, rheumatoid arthritis, and myeloproliferative disorders. The classic description of PG includes irregularly shaped ulcers with undermined edges with a gun-metal gray or violaceous hue. The etiology remains unclear but appears to be related to genetically predisposed dysregulation of the innate immune system. Diagnosis of PG can be difficult as it can present with symptoms similar to cutaneous infections including erythema, edema, ulceration, fever and leukocytosis. Surgical procedures are generally contraindicated in patients with PG due to the risk of pathergy, excessive cutaneous injury, or ulceration in response to trauma. CASE REPORT: The authors report the development of PG with the initiation of chemotherapy in a 46-year-old woman with breast cancer. The patient had a complicated clinical course after multiple surgical debridements due to an initial misdiagnosis of necrotizing fasciitis. The patient's rapid onset of post-procedural ulceration was consistent with the pathergy of PG. The diagnosis of PG was confirmed by skin biopsy, which revealed a diffuse neutrophilic infiltrate, and with the patient's negative cultures and response to steroids. The patient was treated with perioperative prednisone and intravenous immunoglobulin prior to a mastectomy for her breast cancer. The surgery was not complicated by pathergy. CONCLUSIONS: This unique case highlights the challenging aspects in the medical and perioperative management of active PG in a patient with breast cancer.

Reflectin Proteins Bind and Reorganize Synthetic Phospholipid Vesicles.

The reflectin proteins have been extensively studied for their role in reflectance in cephalopods. In the recently evolved Loliginid squids, these proteins and the structural color they regulate are dynamically tunable, enhancing their effectiveness for camouflage and communication. In these species, the reflectins are found in highest concentrations within the structurally tunable, membrane enclosed, periodically stacked lamellae of subcellular Bragg reflectors and in the intracellular vesicles of specialized skin cells known as iridocytes and leuocophores, respectively. To better understand the interactions between the reflectins and the membrane structures that encompass them, we analyzed the interactions of two purified reflectins with synthetic phospholipid membrane vesicles similar in composition to cellular membranes, using confocal fluorescence microscopy and dynamic light scattering. The purified recombinant reflectins were found to drive multivalent vesicle agglomeration in a ratio-dependent and saturable manner. Extensive proteolytic digestion terminated with PMSF of the reflectin A1-vesicle complexes triggered energetic membrane rearrangement, resulting in vesicle fusion, fission, and tubulation. This behavior contrasted markedly with that of vesicles complexed with reflectin C, from which PMSF-terminated proteolysis only released the original size vesicles. Clues to the basis for this difference, residing in significant differences between the structures of the two reflectins, led to the suggestion that specific reflectin-membrane interactions may play a role in the ontogenetic formation, long-term maintenance, and/or dynamic behavior of their biophotonically active host membrane nanostructures. Similar energetic remodeling has been associated with osmotic stress in other membrane systems, suggesting a path to reconstitution of the biophotonic system in vitro.

Perioperative management of pyoderma gangrenosum.

Pyoderma gangrenosum (PG) classically presents with an acute inflammatory stage, characterized by rapid evolution of painful ulcerations. The pathergy associated with PG lesions complicates disease management. Although PG is commonly treated with immunosuppression, some patients have refractory noninflammatory ulcers. In this subpopulation, there are case reports of successful surgical treatment. However, there is no consensus on optimal perioperative treatment for patients with PG undergoing surgery of any kind, PG related or otherwise. Therefore, we conducted a comprehensive literature review describing perioperative management practices and risk factors that may predict response to surgical intervention. We identified 126 cases of surgical intervention in patients with active PG; among these, only 16.7% experienced postoperative disease progression. No perioperative treatments or clinical risk factors were identified as statistically significant predictors of disease recurrence. Although limited by case series design and publication bias, this study is a valuable means of hypothesis generation for this rare condition.

Calibration between trigger and color: Neutralization of a genetically encoded coulombic switch and dynamic arrest precisely tune reflectin assembly.

Reflectin proteins are widely distributed in reflective structures in cephalopods. However, only in loliginid squids are they and the subwavelength photonic structures they control dynamically tunable, driving changes in skin color for camouflage and communication. The reflectins are block copolymers with repeated canonical domains interspersed with cationic linkers. Neurotransmitter-activated signal transduction culminates in catalytic phosphorylation of the tunable reflectins' cationic linkers; the resulting charge neutralization overcomes coulombic repulsion to progressively allow condensation, folding, and assembly into multimeric spheres of tunable well-defined size and low polydispersity. Here, we used dynamic light scattering, transmission EM, CD, atomic force microscopy, and fluorimetry to analyze the structural transitions of reflectins A1 and A2. We also analyzed the assembly behavior of phosphomimetic, deletion, and other mutants in conjunction with pH titration as an in vitro surrogate of phosphorylation. Our experiments uncovered a previously unsuspected, precisely predictive relationship between the extent of neutralization of a reflectin's net charge density and the size of resulting multimeric protein assemblies of narrow polydispersity. Comparisons of mutants revealed that this sensitivity to neutralization resides in the linkers and is spatially distributed along the protein. Imaging of large particles and analysis of sequence composition suggested that assembly may proceed through a dynamically arrested liquid-liquid phase-separated intermediate. Intriguingly, it is this dynamic arrest that enables the observed fine-tuning by charge and the resulting calibration between neuronal trigger and color in the squid. These results offer insights into the basis of reflectin-based biophotonics, opening paths for the design of new materials with tunable properties.

Molecular community profiling of the bacterial microbiota associated with denture-related stomatitis.

Denture-associated stomatitis (DS) affects over two-thirds of denture-wearers. DS presents as erythema of the palatal mucosa in areas where denture-surface associated polymicrobial biofilms containing the fungus Candida albicans exist. The contribution of the oral bacterial microbiota toward the infection is unknown. Therefore, this study characterised the bacterial microbiota of sites within the oral cavity to identify potential associations with occurrence of DS. Denture-wearing patients were recruited (denture stomatitis (DS) n = 8; non-denture stomatitis (NoDS) n = 11) and the oral bacterial microbiota of the tongue, palate and denture-fitting surface was characterised using next-generation sequencing. Operational taxonomic units (OTUs) were identified to bacterial genera and species, and presence/absence and relative abundances were examined. A significant (P = 0.007) decrease in the number of OTUs and thus, diversity of the microbiota was observed in tongue samples of DS patients (vs non-DS). The microbiota of denture-fitting surfaces and palatal mucosae were similar. Large differences in the abundance of bacterial genera and species were observed at each sample site, and unique presence/absence of bacteria was noted. Presence/absence and relative abundance of specific bacteria associated with DS warrants further in vitro and in vivo evaluation, particularly as our previous work has shown C. albicans virulence factor modulation by oral bacteria.

Hidradenitis suppurativa associated with sorafenib initiation.

Sorafenib is a multi-kinase inhibitor approved for the treatment of renal cell and hepatocellular carcinoma. Adverse cutaneous reactions are a very common side effect of the medication. We report the development of hidradenitis suppurativa (HS) in a patient after initiation of treatment with sorafenib. HS is marked by recurrent deep painful nodules, fluctuant abscesses, and draining sinus tracts most frequently occurring in the groin and axilla. To our knowledge, sorafenib-induced HS in the axillary and inguinal skin folds has not been previously reported.

Mechanisms of Inflammation in Neutrophil-Mediated Skin Diseases.

Neutrophil-mediated skin diseases, originally named neutrophilic dermatoses (NDs), are a group of conditions due to an altered neutrophil recruitment and activation, characterized by polymorphic cutaneous manifestations with possible internal organ involvement. Although a number of diseases are included in this setting, the two prototypic forms are pyoderma gangrenosum (PG) and Sweet's syndrome (SS) which usually present with skin ulcers and plaque-type lesions, respectively. They have central features significantly overlapping with autoinflammatory conditions which manifest as repeated episodes of tissue inflammation. However, in contrast to appropriate inflammatory responses to insults or to autoimmune disease, there is an absence of identifiable pathogens, autoantibodies, or autoreactive lymphocytes. The recognition of monogenic autoinflammatory diseases which can present with NDs has led to study several genes involved in autoinflammation in NDs. Based on discovering of a number of mutations involving different autoinflammatory genes, neutrophil-mediated skin diseases are nowadays regarded as a spectrum of polygenic autoinflammatory conditions. Although disease mechanisms have not yet been completely elucidated, NDs are recognized as diseases involving dysfunctional cellular signaling mediated by pathways mainly related to inflammasome and IL-1 with the contributory role of IL-17 and other effector molecules. The precise elucidation of the above-mentioned pathologic mechanisms may pave the way to tailored treatments for patients with different neutrophil-mediated skin diseases.

A Rare Variant of Penile Squamous Cell Carcinoma in a Man with Paraplegia.

Verrucous carcinoma (VC) is a rare variant of squamous cell carcinoma (SCC). It is described as a low grade, slow growing, locally infiltrative neoplasm that accounts for 3%-8% of penile SCCs. Here we report a case of destructive VC of the glans penis in a paraplegic man resulting in a hypospadias from the tip of the glans to the corona. Histology demonstrated exophytic squamous epithelial proliferation with characteristic round, pushing borders. In situ hybridization was positive for both low-risk and high-risk strains of human papillomavirus.