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We aim to develop an in vivohematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. In vivoHSC transduction/selection resulted in stable serum eCD4-Ig levels of ∼100 μg/mL (mice) and >20 μg/mL (rhesus) with half maximal inhibitory concentrations (IC50s) of 1 μg/mL measured by an HIV neutralization assay. After simian-human-immunodeficiency virus D (SHIV.D) challenge of rhesus macaques injected with HDAd-eCD4-Ig or a control HDAd5/35++ vector, peak plasma viral load levels were ∼50-fold lower in the eCD4-Ig-expressing animal. Furthermore, the viral load was lower in tissues with the highest eCD4-Ig expression, specifically the spleen and lymph nodes. SHIV.D challenge triggered a selective expansion of transduced CD4+CCR5+cells, thereby increasing serum eCD4-Ig levels. The latter, however, broke immune tolerance and triggered anti-eCD4-Ig antibody responses, which could have contributed to the inability to eliminate SHIV.D. Our data will guide us in the improvement of the in vivoapproach. Clearly, our conclusions need to be validated in larger animal cohorts.

ObjectivesNo randomised controlled trials have been conducted for breakthrough pain in paediatric palliative care and there are currently no standardised outcome measures. The DIPPER study aims to establish the feasibility of conducting a prospective randomised controlled trial comparing oral and transmucosal administration of opioids for breakthrough pain. The aim of the current study was to achieve consensus on design aspects for a small-scale prospective study to inform a future randomised controlled trial of oral morphine, the current first-line treatment, versus transmucosal diamorphine.MethodsThe nominal group technique was used to achieve consensus on best practice for mode of administration, dose regimen and a range of suitable pain intensity outcome measures for transmucosal diamorphine in children and young people with breakthrough pain. An expert panel of ten clinicians in paediatric palliative care and three parent representatives participated. Consensus was achieved when agreement was reached and no further comments from participants were forthcoming.ResultsThe panel favoured the buccal route of administration, with dosing according to the recommendations in the Association for Paediatric Palliative Medicine formulary (fifth Edition, 2020). The verbal Numerical Rating Scale was selected to measure pain in children 8 years old and older, the Faces Pain Scale-Revised for children between 4 and 8 years old, and Face, Legs, Activity, Cry and Consolability (FLACC)/FLACC-Revised as the observational tools.ConclusionsThe nominal group technique allowed consensus to be reached for a small-scale, prospective, cohort study and provided information to inform the design of a randomised controlled trial.

Vascular surgery is facing an impending workforce shortage as the population ages and the demand for vascular surgical services increases. The integrated vascular surgery residency (0+5) paradigm is well-established and provides a mechanism to increase the number of board-certified vascular surgeons. Recruitment of medical students to these programs has proven challenging with unfilled positions in each of the past 2 years. The aim of this study is to explore factors that influence medical students’ interest in vascular surgery and their decision to ultimately pursue a career in the field.

Background Breakthrough pain is common in life-limiting conditions and at end-of-life. Despite over 30 years of study, there is little consensus regarding the definition and characteristics of breakthrough pain.Objective This study aims to update and expand a 2010 systematic review by Haugen and colleagues to identify (1) all definitions of breakthrough pain and (2) all descriptions and classifications of breakthrough pain reported by patients, caregivers, clinicians, and experts.Design This rapid systematic review followed the Cochrane Rapid Review Methods Group guidelines. A protocol is published on PROSPERO (CRD42019155583).Data sources CINAHL, MEDLINE, PsycINFO, and the Web of Science were searched for breakthrough pain terms from the inception dates of each database to 26th August 2022.Results We identified 65 studies that included data on breakthrough pain definitions, descriptions, or classifications from patients (n= 30), clinicians (n= 6), and experts (n= 29), but none with data from caregivers. Most experts proposed that breakthrough pain was a sudden, severe, brief pain occurring in patients with adequately controlled mild-moderate background pain. However, definitions varied and there was no consensus. Pain characteristics were broadly similar across studies though temporal factors varied widely. Experts classified breakthrough pain into nociceptive, neuropathic, visceral, somatic, or mixed types. Patients with breakthrough pain commonly experienced depression, anxiety, and interference with daily life.Conclusions Despite ongoing efforts, there is still no consensus on the definition of breakthrough pain. A compromise is needed on breakthrough pain nomenclature to collect reliable incidence and prevalence data and to inform further refinement of the construct.

•Using WU-106/AMD3100 mobilization, in vivo transduction of long-term persisting HSCs in SCD mice can be done within 2 hours.•Compared with G-CSF/mobilization, the WU-106/AMD3100-based approach is safer and more efficient for phenotypic correction of the disease in mice.

There is a paucity of evidence on the role, use, benefit and challenges of artificial nutrition and hydration (ANH) in children at end of life. Parents express the difficulty they face with making the decision to withdraw ANH. Decision-making on the role of ANH in an individual child requires careful multidisciplinary team deliberation and clear goals of care with children and families. Four paediatric palliative care specialist centres reviewed the current literature and developed consensus guidelines on ANH at end of life. These guidelines seek to provide a practical approach to clinical decision-making on the role of ANH in a child or young person entering the end-of-life phase.

Overexpression and activation of c-Jun N-terminal kinases (JNKs) have been observed in multiple cancer cell lines and tumor samples. Various JNK isoforms have been reported to promote lung and liver cancer, as well as keratinocyte transformation, suggesting an important role of JNK signaling in promoting tumor development. However, there are three JNK isoforms, and it is unclear how each individual isoform, especially the ubiquitously expressed JNK1 and JNK2, functions in melanoma. Our previous study found that C116S mutations in both JNK1 and JNK2 rendered them insensitive to the covalent pan-JNK inhibitor JNK-IN-8 while retaining kinase activity. To delineate the specific roles of JNK1 and JNK2 in melanoma cell proliferation and invasiveness, we expressed the wild type (WT) and C116S mutants in melanoma cell lines and used JNK-IN-8 to enable chemical–genetic dissection of JNK1 and JNK2 activity. We found that the JNK2C116Sallele consistently enhanced colony proliferation and cell invasiveness in the presence of JNK-IN-8. When cells individually expressing WT or C116S JNK1/2 were subcutaneously implanted into immunodeficient mice, we again found that bypass of JNK-IN-8-mediated inhibition of JNK signaling by expression of JNK2C116Sspecifically resulted in enhanced tumor growth in vivo. In addition, we observed a high level of JNK pathway activation in some human BRAF inhibitor (BRAFi) resistant melanoma cell lines relative to their BRAFi sensitive isogenic counterparts. JNK-IN-8 significantly enhanced the response to dabrafenib in resistant cells overexpressing JNK1WT, JNK2WT, and JNK1C116Sbut had no effect on cells expressing JNK2C116S, suggesting that JNK2 signaling is also crucial for BRAFi resistance in a subset of melanomas. Collectively, our data show that JNK2 activity is specifically required for melanoma cell proliferation, invasiveness, and BRAFi resistance and that this activity is most important in the context of JNK1 suppression, thus providing a compelling rationale for the development of JNK2 selective inhibitors as a potential therapy for the treatment of melanoma.

Ken Kelly is president of Kelly Roofing & Energy Saving Solutions, headquartered in Naples, Fla. He tried to find one person to handle his print media, website, social media and [...]

Priority setting for healthcare research is as important as conducting the research itself because rigorous and systematic processes of priority setting can make an important contribution to the quality of research. This project aimed to prioritise clinical therapeutic uncertainties in paediatric pain and palliative care in order to encourage and inform the future research agenda and raise the profile of paediatric pain and palliative care in the United Kingdom. Clinical therapeutic uncertainties were identified and transformed into patient, intervention, comparison and outcome (PICO) format and prioritised using a modified Nominal Group Technique. Members of the Clinical Studies Group in Pain and Palliative Care within National Institute for Health Research (NIHR) Clinical Research Network (CRN)-Children took part in the prioritisation exercise. There were 11 clinically active professionals spanning across a wide range of paediatric disciplines and one parent representative. The top three research priorities related to establishing the safety and efficacy of (1) gabapentin in the management of chronic pain with neuropathic characteristics, (2) intravenous non-steroidal anti-inflammatory drugs in the management of post-operative pain in pre-schoolers and (3) different opioid formulations in the management of acute pain in children while at home. Questions about the long-term effect of psychological interventions in the management of chronic pain and various pharmacological interventions to improve pain and symptom management in palliative care were among the ‘top 10’ priorities. The results of prioritisation were included in the UK Database of Uncertainties about the Effects of Treatments (DUETS) database. Increased awareness of priorities and priority-setting processes should encourage clinicians and other stakeholders to engage in such exercises in the future.

•Correction of the sickle-cell mutation and disease phenotypes is achieved by in vivo HSC transduction with vectorized prime editors.•Our approach for in vivo HSC prime editing that does not require HSC transplantation and myeloablation should simplify HSC gene therapy.

Context:The mechanism of action of metformin remains unclear. Given the regulation of the cortisol-regenerating enzyme 11βhydroxysteroid dehydrogenase 1 (11βHSD1) by insulin and the limited efficacy of selective 11βHSD1 inhibitors to lower blood glucose when co-prescribed with metformin, we hypothesized that metformin reduces 11βHSD1 activity.Objective:To determine whether metformin regulates 11βHSD1 activity in vivo in obese men with and without type 2 diabetes mellitus.Design:Double-blind, randomized, placebo-controlled, crossover study.Setting:A hospital clinical research facility.Participants:Eight obese nondiabetic (OND) men and eight obese men with type 2 diabetes (ODM).Intervention:Participants received 28 days of metformin (1 g twice daily), placebo, or (in the ODM group) gliclazide (80 mg twice daily) in random order. A deuterated cortisol infusion at the end of each phase measured cortisol regeneration by 11βHSD1. Oral cortisone was given to measure hepatic 11βHSD1 activity in the ODM group. The effect of metformin on 11βHSD1 was also assessed in human hepatocytes and Simpson-Golabi-Behmel syndrome adipocytes.Main Outcome Measures:The effect of metformin on whole-body and hepatic 11βHSD1 activity.Results:Whole-body 11βHSD1 activity was approximately 25% higher in the ODM group than the OND group. Metformin increased whole-body cortisol regeneration by 11βHSD1 in both groups compared with placebo and gliclazide and tended to increase hepatic 11βHSD1 activity. In vitro, metformin did not increase 11βHSD1 activity in hepatocytes or adipocytes.Conclusions:Metformin increases whole-body cortisol generation by 11βHSD1 probably through an indirect mechanism, potentially offsetting other metabolic benefits of metformin. Co-prescription with metformin should provide a greater target for selective 11βHSD1 inhibitors.

Step-by-step directions for casting metal animal figures using the lost-wax method are offered. The project requires advanced skills.

An interview with artist Anna Anderson from Churubusco, Indiana is presented. Topics discussed include her assignment of making self-portrait as an inspiration for drawing, her development of funny faces using acrylic paints and taking their photographs, working process of outlining faces and coloring them and her advice for similar aspiring artists to use their power of observation.