Endorsed midwife prescribing in Australia: "[For] the women, more than anything".

PROBLEM: Prescribing by Endorsed Midwives has existed in Australia for more than ten years. Significant barriers exist in the bureaucracy surrounding prescribing and state and territory legislation which further constrain midwives capacity to prescribe required medications. BACKGROUND: Current evidence indicates Endorsed Midwives improve timely access to medications and can experience both enablers and barriers to prescribing. AIM: To explore Endorsed Midwives' lived experiences of medication prescribing, including which medications are being prescribed, how this affects the women in their care, midwives' practice, and perspectives on the future of midwifery prescribing. METHODS: A descriptive qualitative approach was used. Data collection occurred through semi-structured interviews (n=10) of Endorsed Midwives from varied Australian practice contexts and locations. Data analysis followed Reflexive Thematic Analysis. FINDINGS: Four themes were developed: Medication prescription as essential healthcare; Prescribing optimises midwifery practice; External structures can both promote and inhibit the capacity to prescribe; The future of prescribing. DISCUSSION: Endorsed Midwife prescribing has the potential to positively impact women's maternity care and enable midwives to fulfil their scope of practice. However, limitations to prescribing need to be addressed to capitalise on these benefits. CONCLUSION: Significant reform of health service policy, state and territory legislation and further development of the Pharmaceutical Benefits Scheme are required to fully embrace and capitalise on the full scope of Endorsed Midwives in the Australian Healthcare system.

Endorsed midwives prescribing scheduled medicines in Australia: A scoping review.

PROBLEM: Despite 10 years of prescribing scheduled medicines by Endorsed Midwives, little is known about prescribing practices. BACKGROUND: Endorsed Midwives can prescribe scheduled medicines and have access to Medicare rebates to support service provision. Endorsed Midwives have the potential to improve access to medications for women, however, are met with barriers, including inconsistencies in state and national legislation. AIM: To search for what is published regarding Endorsed Midwife prescribing of scheduled medicines in Australia, report on the literature, synthesise the findings and discuss the results. METHODS: A scoping review utilising the Joanna Brigg's Institute methodology. A search of CINAHL, PubMed, Science Direct and Medline databases was conducted. Seven peer-reviewed articles were identified; three discussion papers, one literature review and three research papers, published between 2016 and 2023 in English. Qualitative content analysis was used to identify topic areas. FINDINGS: Four topic areas were identified: 1) Endorsed Midwives increase women's access to prescribed medications; 2) the Pharmaceutical Benefits Scheme is restrictive and diminishes midwifery prescribing; 3) medication prescribing depends on internal and external structures; 4) professional relationships support prescribing. DISCUSSION: The authority to prescribe augments Endorsed Midwives' practice, improves timely access to medications and enhances role satisfaction. The effective use of midwifery prescribing is hampered by barriers such as the Pharmaceutical Benefits Scheme, inappropriate medication formularies, and poorly designed health service policy. CONCLUSION: To fully utilise Endorsed Midwife prescribing in all settings of maternity care, further work is required to develop education, remove barriers, and demonstrate the safety and effectiveness of midwifery prescribing.

Biological Effects of HDAC Inhibitors Vary with Zinc Binding Group: Differential Effects on Zinc Bioavailability, ROS Production, and R175H p53 Mutant Protein Reactivation.

The coordination of zinc by histone deacetylase inhibitors (HDACi), altering the bioavailability of zinc to histone deacetylases (HDACs), is key to HDAC enzyme inhibition. However, the ability of zinc binding groups (ZBGs) to alter intracellular free Zn+2 levels, which may have far-reaching effects, has not been explored. Using two HDACis with different ZBGs, we documented shifts in intracellular free Zn+2 concentrations that correlate with subsequent ROS production. Next, we assayed refolding and reactivation of the R175H mutant p53 protein in vitro to provide greater biological context as the activity of this mutant depends on cellular zinc concentration. The data presented demonstrates the differential activity of HDACi in promoting R175H response element (RE) binding. After cells are treated with HDACi, there are differences in R175H mutant p53 refolding and reactivation, which may be related to treatments. Collectively, we show that HDACis with distinct ZBGs differentially impact the intracellular free Zn+2 concentration, ROS levels, and activity of R175H; therefore, HDACis may have significant activity independent of their ability to alter acetylation levels. Our results suggest a framework for reevaluating the role of zinc in the variable or off-target effects of HDACi, suggesting that the ZBGs of HDAC inhibitors may provide bioavailable zinc without the toxicity associated with zinc metallochaperones such as ZMC1.

A Hybrid Epithelial to Mesenchymal Transition in Ex Vivo Cutaneous Squamous Cell Carcinoma Tissues.

While most cases of cutaneous squamous cell carcinoma (cSCC) are benign, invasive cSCC is associated with higher mortality and is often more difficult to treat. As such, understanding the factors that influence the progression of cSCC are important. Aggressive cancers metastasize through a series of evolutionary changes, collectively called the epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells transition to a highly mobile mesenchymal cell type with metastatic capacities. While changes in expression of TGF-ß, ZEB1, SNAI1, MMPs, vimentin, and E-cadherin are hallmarks of an EMT process occurring within cancer cells, including cSCC cells, EMT within tissues is not an "all or none" process. Using patient-derived cSCC and adjacent normal tissues, we show that cells within individual cSCC tumors are undergoing a hybrid EMT process, where there is variation in expression of EMT markers by cells within a tumor mass that may be facilitating invasion. Interestingly, cells along the outer edges of a tumor mass exhibit a more mesenchymal phenotype, with reduced E-cadherin, ß-catenin, and cytokeratin expression and increased vimentin expression. Conversely, cells in the center of a tumor mass retain a higher expression of the epithelial markers E-cadherin and cytokeratin and little to no expression of vimentin, a mesenchymal marker. We also detected inverse expression changes in the miR-200 family and the EMT-associated transcription factors ZEB1 and SNAI1, suggesting that cSCC EMT dynamics are regulated in a miRNA-dependent manner. These novel findings in cSCC tumors provide evidence of phenotypic plasticity of the EMT process occurring within patient tissues, and extend the characterization of a hybrid EMT program occurring within a tumor mass. This hybrid EMT program may be promoting both survival and invasiveness of the tumors. A better understanding of this hybrid EMT process may influence therapeutic strategies in more invasive disease.

Complement Factor H in cSCC: Evidence of a Link Between Sun Exposure and Immunosuppression in Skin Cancer Progression.

Cutaneous squamous cell carcinoma (cSCC) is a common form of skin cancer with an estimated 750,000 cases diagnosed annually in the United States. Most cases are successfully treated with a simple excision procedure, but ~5% of cases metastasize and have a 5-year survival rate of 25-45%. Thus, identification of biomarkers correlated to cSCC progression may be useful in the early identification of high-risk cSCC and in the development of new therapeutic strategies. This work investigates the role of complement factor H (CFH) in the development of cSCC. CFH is a regulatory component of the complement cascade which affects cell mediated immune responses and increases in complement proteins are associated with poor outcomes in multiple cancer types. We provide evidence that sun exposure may increase levels of CFH, suggesting an immunomodulatory role for CFH early in the development of cSCC. We then document increased levels of CFH in cSCC samples, compared to adjacent normal tissue (ANT) routinely excised in a dermatology clinic which, in paired samples, received the same level of sun exposure. We also provide evidence that levels of CFH are even greater in more advanced cases of cSCC. To provide a potential link between CFH and immune modulation, we assessed immune system function by measuring interferon gamma (IFN-γ) and FOXP3 in patient samples. IFN-γ levels were unchanged in cSCC relative to ANT which is consistent with an ineffective cell-mediated immune response. FOXP3 was used to assess prevalence of regulatory T cells within the tissues, indicating either a derailed or inhibitory immune response. Our data suggest that FOXP3 levels are higher in cSCC than in ANT. Our current working model is that increased CFH downstream of sun exposure is an early event in the development of cSCC as it interferes with proper immune surveillance and decreases the effectiveness of the immune response, and creates a more immunosuppressive environment, thus promoting cSCC progression.

HDAC inhibition induces EMT and alterations in cellular iron homeostasis to augment ferroptosis sensitivity in SW13 cells.

Epithelial-to-mesenchymal transition (EMT) is an essential mechanism for development and wound healing, but in cancer it also mediates the progression and spread of aggressive tumors while increasing therapeutic resistance. Adoption of a mesenchymal state is also associated with increased iron uptake, but the relationship between EMT and the key regulators of cellular iron metabolism remains undefined. In this regard, the human adrenal cortical carcinoma SW13 cell line represents an invaluable research model as HDAC inhibitor treatment can convert them from an epithelial-like (SW13-) cell type to a mesenchymal-like (SW13+) subtype. In this study we establish SW13 cells as a model for exploring the link between iron and EMT. Increased iron accumulation following HDAC inhibitor mediated EMT is associated with decreased expression of the iron export protein ferroportin, enhanced ROS production, and reduced expression of antioxidant response genes. As availability of redox active iron and loss of lipid peroxide repair capacity are hallmarks of ferroptosis, a form of iron-mediated cell death, we next examined whether HDAC inhibitor treatment could augment ferroptosis sensitivity. Indeed, HDAC inhibitor treatment synergistically increased cell death following induction of ferroptosis. The exact mechanisms by which HDAC inhibition facilitates cell death following ferroptosis induction requires further study. As several HDAC inhibitors are already in use clinically for the treatment of certain cancer types, the findings from these studies have immediate implications for improving iron-targeted chemotherapeutic strategies.

Use of Amniotic Tissue-Derived Allografts Post-Mohs Micrographic Surgery: A Preliminary Study Assessing Wound Closure Rate.

INTRODUCTION: When closure is not feasible, Mohs micrographic surgical wounds typically are left to heal by secondary intention and require weeks to close. Amniotic tissue-derived allograft (ATDA) has proven successful in promoting wound closure in diabetic and refractory wounds, and it may be beneficial for patients who have undergone Mohs micrographic surgery. OBJECTIVE: The authors conducted a preliminary study to assess the efficacy of ATDA in speeding wound closure time and improving cosmetic outcomes in the specified patient population. MATERIALS AND METHODS: Patients received an injection of amniotic fluid, an overlay of amniotic membrane, or standard of care. Photographs of wounds taken at the time of treatment and at each subsequent visit were analyzed. RESULTS: The cosmetic outcome and time to wound closure appeared to be improved in patients treated with ATDA when compared with expected outcomes. Owing to small sample size, differences in initial defect size, and variety of body locations, the wound closure rate between treatment groups was not found to be significantly different with most comparisons. Statistical significance was seen, however, when normalized closure rates between membrane and control intervention were compared after outlier analysis (P = .0288). CONCLUSIONS: Data indicate that ATDA treatment may be beneficial and suggest that further investigation of the efficacy of ATDA to promote wound healing and improve cosmetic outcomes of post-Mohs surgical wounds is warranted. Future studies should be designed to match initial defect size and location between control and treatment groups.

Dual inhibition of TGFßR and ROCK reverses the epithelial to mesenchymal transition in collectively migrating zebrafish keratocytes.

There is a growing controversy about the role of the epithelial to mesenchymal transition (EMT) in the fibrosis associated with chronic disease. Recent studies suggest that it is not the EMT transcriptional program but differentiation of progenitor cells, response to chronic inflammation, or some combination of both which cause the appearance of fibroblasts and the production of the extracellular matrix. To address this issue, we study the EMT process in the zebrafish keratocytes which migrate from primary explants of epithelial tissue as these cells are both terminally differentiated and able to divide. To firmly place this EMT process in the context of other systems, we first demonstrate that the zebrafish keratocyte EMT process involves nuclear accumulation of twist and snail/slug transcription factors as part of a TGFßR-mediated EMT process. As assessed by the expression and localization of EMT transcription factors, the zebrafish keratocyte EMT process is reversed by the addition of Rho-activated kinase (ROCK) in combination with TGFßR inhibitors. The complete cycle of EMT to MET observed in this system links these in vitro results more closely to the process of wound healing in vivo. However, the absence of observable activation of EMT transcription factors when keratocytes are cultured on compliant substrata in a TGFß1-containing medium suggests that ROCK signaling, initiated by tension within the sheet, is an essential contributor to the EMT process. Most importantly, the requirement for ROCK activation by culturing on noncompliant substrata suggests that EMT in these terminally differentiated cells would not occur in vivo.

Journal data policies: Exploring how the understanding of editors and authors corresponds to the policies themselves.

Despite the increase in the number of journals issuing data policies requiring authors to make data underlying reporting findings publicly available, authors do not always do so, and when they do, the data do not always meet standards of quality that allow others to verify or extend published results. This phenomenon suggests the need to consider the effectiveness of journal data policies to present and articulate transparency requirements, and how well they facilitate (or hinder) authors' ability to produce and provide access to data, code, and associated materials that meet quality standards for computational reproducibility. This article describes the results of a research study that examined the ability of journal-based data policies to: 1) effectively communicate transparency requirements to authors, and 2) enable authors to successfully meet policy requirements. To do this, we conducted a mixed-methods study that examined individual data policies alongside editors' and authors' interpretation of policy requirements to answer the following research questions. Survey responses from authors and editors along with results from a content analysis of data policies found discrepancies among editors' assertion of data policy requirements, authors' understanding of policy requirements, and the requirements stated in the policy language as written. We offer explanations for these discrepancies and offer recommendations for improving authors' understanding of policies and increasing the likelihood of policy compliance.

Integrating Agriculture and Health Research for Development: LCIRAH as an Interdisciplinary Programme to Address a Global Challenge.

The multiple burdens of persistent undernutrition and micronutrient deficiencies, along with the rapidly growing rates of overweight, obesity, and associated chronic diseases, are major challenges globally. The role of agriculture and the food system in meeting these challenges is very poorly understood. Achieving food security and addressing malnutrition in all its forms, a Sustainable Development Goal, requires an understanding of how changing food systems affect health outcomes and the development of new tools to design and evaluate interventions. An interinstitutional programme to address this interdisciplinary research challenge is described. Over the past seven years, the Leverhulme Centre for Integrative Research on Agriculture and Health has built a portfolio of successful and innovative research, trained a new cadre of interdisciplinary researchers in "Agri-Health," and built an international research community with a particular focus on strengthening research capacity in low- and middle-income countries. The evolution of this programme is described, and key factors contributing to its success are discussed that may be of general value in designing interdisciplinary research programmes directed at supporting global development goals.

Alterations in the glycome after HDAC inhibition impact oncogenic potential in epigenetically plastic SW13 cells.

BACKGROUND: Defects in the type and degree of cellular glycosylation impact oncogenesis on multiple levels. Although the type of glycosylation is determined by protein sequence encoded by the genome, the extent and modifications of glycosylation depends on the activity of biosynthetic enzymes and recent data suggests that the glycome is also subject to epigenetic regulation. This study focuses on the ability of HDAC inhibition to alter glycosylation and to lead to pro-oncogenic alterations in the glycome as assessed by metastatic potential and chemoresistance. METHODS: Epigenetically plastic SW13 adrenocortical carcinoma cells were treated with FK228, an HDAC inhibitor with high affinity for HDAC1 and, to a lesser extent, HDAC2. In comparing HDAC inhibitor treated and control cells, differential expression of glycome-related genes were assessed by microarray. Differential glycosylation was then assessed by lectin binding arrays and the ability of cellular proteins to bind to glycans was assessed by glycan binding arrays. Differential sensitivity to paclitaxel, proliferation, and MMP activity were also assessed. RESULTS: Treatment with FK228 alters expression of enzymes in the biosynthetic pathways for a large number of glycome related genes including enzymes in all major glycosylation pathways and several glycan binding proteins. 84% of these differentially expressed glycome-related genes are linked to cancer, some as prognostic markers and others contributing basic oncogenic functions such as metastasis or chemoresistance. Glycan binding proteins also appear to be differentially expressed as protein extracts from treated and untreated cells show differential binding to glycan arrays. The impact of differential mRNA expression of glycosylation enzymes was documented by differential lectin binding. However, the assessment of changes in the glycome is complicated by the fact that detection of differential glycosylation through lectin binding is dependent on the methods used to prepare samples as protein-rich lysates show different binding than fixed cells in several cases. Paralleling the alterations in the glycome, treatment of SW13 cells with FK228 increases metastatic potential and reduces sensitivity to paclitaxel. CONCLUSIONS: The glycome is substantially altered by HDAC inhibition and these changes may have far-reaching impacts on oncogenesis.

Effects of acupuncture on chronic idiopathic pruritus: an uncontrolled pilot study evaluating inflammatory changes with treatment.

Background Conduct a pilot study addressing the efficacy of acupuncture in the treatment of chronic idiopathic pruritus to aid in the design of a larger clinical trial. Routine laboratory tests to assess systemic inflammation in addition to subjective patient surveys were performed provide documentation of efficacy of treatment. Methods Patients with chronic pruritus who did not respond to standard treatment were recruited to participate. After exclusion of systemic or known reversible causes, each patient received up to 10 treatments which were performed approximately one week apart. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were measured before and after a series of acupuncture treatments to evaluate levels of inflammation and pre- and post-treatment surveys were conducted to evaluate levels of perceived itch. Results Only one of the ten patients in this study possessed an elevation of ESR before treatment. This patient's ESR value returned to normal range after treatment and this participant reported subjective relief of her pruritus. Conclusions Future studies on the efficacy of acupuncture in the treatment of chronic idiopathic pruritus should focus on those patients with measurable levels of inflammation at the initiation of the study or utilize alternative and more comprehensive values to monitor disease response.

Epigenetic Regulation of the Biosynthesis & Enzymatic Modification of Heparan Sulfate Proteoglycans: Implications for Tumorigenesis and Cancer Biomarkers.

Emerging evidence suggests that the enzymes in the biosynthetic pathway for the synthesis of heparan sulfate moieties of heparan sulfate proteoglycans (HSPGs) are epigenetically regulated at many levels. As the exact composition of the heparan sulfate portion of the resulting HSPG molecules is critical to the broad spectrum of biological processes involved in oncogenesis, the epigenetic regulation of heparan sulfate biosynthesis has far-reaching effects on many cellular activities related to cancer progression. Given the current focus on developing new anti-cancer therapeutics focused on epigenetic targets, it is important to understand the effects that these emerging therapeutics may have on the synthesis of HSPGs as alterations in HSPG composition may have profound and unanticipated effects. As an introduction, this review will briefly summarize the variety of important roles which HSPGs play in a wide-spectrum of cancer-related cellular and physiological functions and then describe the biosynthesis of the heparan sulfate chains of HSPGs, including how alterations observed in cancer cells serve as potential biomarkers. This review will then focus on detailing the multiple levels of epigenetic regulation of the enzymes in the heparan sulfate synthesis pathway with a particular focus on regulation by miRNA and effects of epigenetic therapies on HSPGs. We will also explore the use of lectins to detect differences in heparan sulfate composition and preview their potential diagnostic and prognostic use in the clinic.

Establishment of a Clinic-based Biorepository.

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is expected that there will be a parallel demand for cutaneous tumor samples for biomedical research studies. Tissue availability, however, is limited due the cost of establishing a biorepository and the lack of protocols available for obtaining clinical samples that do not interfere with clinical operations. A protocol was established to collect and process cutaneous tumor and associated blood and saliva samples that has minimal impact on routine clinical procedures on the date of a Mohs surgery. Tumor samples are collected and processed from patients undergoing their first layer of Mohs surgery for biopsy-proven cutaneous malignancies by the Mohs histotechnologist. Adjacent normal tissue is collected at the time of surgical closure. Additional samples that may be collected are whole-blood and buccal swabs. By utilizing tissue samples that are normally discarded, a biorepository was generated that offers several key advantages by being based in the clinic versus the laboratory setting. These include a wide range of collected samples; access to de-identified patient records, including pathology reports; and, for the typical donor, access to additional samples during follow-up visits.

HDAC Inhibitors as Epigenetic Regulators of the Immune System: Impacts on Cancer Therapy and Inflammatory Diseases.

Histone deacetylase (HDAC) inhibitors are powerful epigenetic regulators that have enormous therapeutic potential and have pleiotropic effects at the cellular and systemic levels. To date, HDAC inhibitors are used clinically for a wide variety of disorders ranging from hematopoietic malignancies to psychiatric disorders, are known to have anti-inflammatory properties, and are in clinical trials for several other diseases. In addition to influencing gene expression, HDAC enzymes also function as part of large, multisubunit complexes which have many nonhistone targets, alter signaling at the cellular and systemic levels, and result in divergent and cell-type specific effects. Thus, the effects of HDAC inhibitor treatment are too intricate to completely understand with current knowledge but the ability of HDAC inhibitors to modulate the immune system presents intriguing therapeutic possibilities. This review will explore the complexity of HDAC inhibitor treatment at the cellular and systemic levels and suggest strategies for effective use of HDAC inhibitors in biomedical research, focusing on the ability of HDAC inhibitors to modulate the immune system. The possibility of combining the documented anticancer effects and newly emerging immunomodulatory effects of HDAC inhibitors represents a promising new combinatorial therapeutic approach for HDAC inhibitor treatments.

Epigenetically maintained SW13+ and SW13- subtypes have different oncogenic potential and convert with HDAC1 inhibition.

BACKGROUND: The BRM and BRG1 tumor suppressor genes are mutually exclusive ATPase subunits of the SWI/SNF chromatin remodeling complex. The human adrenal carcinoma SW13 cell line can switch between a subtype which expresses these subunits, SW13+, and one that expresses neither subunit, SW13-. Loss of BRM expression occurs post-transcriptionally and can be restored via histone deacetylase (HDAC) inhibition. However, most previously used HDAC inhibitors are toxic and broad-spectrum, providing little insight into the mechanism of the switch between subtypes. In this work, we explore the mechanisms of HDAC inhibition in promoting subtype switching and further characterize the oncogenic potential of the two epigenetically distinct SW13 subtypes. METHODS: SW13 subtype morphology, chemotaxis, growth rates, and gene expression were assessed by standard immunofluorescence, transwell, growth, and qPCR assays. Metastatic potential was measured by anchorage-independent growth and MMP activity. The efficacy of HDAC inhibitors in inducing subtype switching was determined by immunofluorescence and qPCR. Histone modifications were assessed by western blot. RESULTS: Treatment of SW13- cells with HDAC1 inhibitors most effectively promotes re-expression of BRM and VIM, characteristic of the SW13+ phenotype. During treatment, hyperacetylation of histone residues and hypertrimethylation of H3K4 is pronounced. Furthermore, histone modification enzymes, including HDACs and KDM5C, are differentially expressed during treatment but several features of this differential expression pattern differs from that seen in the SW13- and SW13+ subtypes. As the SW13- subtype is more proliferative while the SW13+ subtype is more metastatic, treatment with HDACi increases the metastatic potential of SW13 cells while restoring expression of the BRM tumor suppressor. CONCLUSIONS: When compared to the SW13- subtype, SW13+ cells have restored BRM expression, increased metastatic capacity, and significantly different expression of a variety of chromatin remodeling factors including those involved with histone acetylation and methylation. These data are consistent with a multistep mechanism of SW13- to SW13+ conversion and subtype stabilization: histone hypermodification results in the altered expression of chromatin remodeling factors and chromatin epigenetic enzymes and the re-expression of BRM which results in restoration of SWI/SNF complex function and leads to changes in chromatin structure and gene expression that stabilize the SW13+ phenotype.

Zebrafish keratocyte explants to study collective cell migration and reepithelialization in cutaneous wound healing.

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Homogeneous bispecifics by disulfide bridging.

We report on a chemical platform to generate site-specific, homogeneous, antibody-antibody conjugates by targeting and bridging disulfide bonds. A bispecific antibody construct was produced in good yield through simple reduction and bridging of antibody fragment disulfide bonds, using a readily synthesized bis-dibromomaleimide cross-linker. Binding activity of antibodies was maintained, and in vitro binding of target antigens was observed. This technology is demonstrated through linking scFv and Fab antibody fragments, showing its potential for the construction of a diverse range of bispecifics.

Collective cell migration of primary zebrafish keratocytes.

Fish keratocytes are an established model in single cell motility but little is known about their collective migration. Initially, sheets migrate from the scale at ~145 µm/h but over the course of 24h the rate of leading edge advance decreases to ~23 µm/h. During this period, leader cells retain their ability to migrate rapidly when released from the sheet and follower cell area increases. After the addition of RGD peptide, leader cell lamellae are lost, altering migratory forces within the sheet, resulting in rapid retraction. Leader and follower cell states interconvert within minutes with changes in cell-cell adhesions. Leader cells migrate as single cells when they detach from the leading edge and single cells appear to become leader cells if they rejoin the sheet. Follower cells rapidly establish leader cell morphology during closing of holes formed during sheet expansion and revert to follower cell morphology after hole-closure. Inhibition of Rho associated kinase releases leader cells and halts advancement of the leading edge suggesting an important role for the intercellular actomyosin cable at the leading edge. In addition, the presence of the stationary scale orients direction of sheet migration which is characterized by a more uniform advance of the leading edge than in some cell line systems. These data establish fish keratocyte explant cultures as a collective cell migration system and suggest that cell-cell interactions determine the role of keratocytes within the migrating sheet.

A mild synthesis of N-functionalised bromomaleimides, thiomaleimides and bromopyridazinediones.

Bromomaleimides are useful building blocks in synthesis and powerful reagents for the selective chemical modification of proteins. A mild new synthesis of these reagents is described, along with the convenient transferability of the approach to dithiomaleimides and bromopyridazinediones.