The enzymatic oxygen sensor cysteamine dioxygenase binds its protein substrates through their N-termini.

The non-heme iron-dependent dioxygenase 2-aminoethanethiol dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyses Nt-cys sulfinylation, which promotes O2-dependent proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signalling (RGS) 4 and 5, and the pro-inflammatory cytokine interleukin-32 (IL32), all of which exhibit cell and/or tissue specific expression patterns. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. Furthermore, the role of individual chemical groups, active site metal, amino acid composition and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and IL32. We demonstrate, using surface plasmon response (SPR) and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through 1H-15N heteronuclear single quantum coherence (15N-HSQC) nuclear magnetic resonance (NMR) titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation.

Protein SUMOylation promotes cAMP-independent EPAC1 activation.

Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.

Phase I Trial of GD2.CART Cells Augmented With Constitutive Interleukin-7 Receptor for Treatment of High-Grade Pediatric CNS Tumors.

PURPOSE: T cells modified with chimeric antigen receptors (CARTs) have demonstrated efficacy for hematologic malignancies; however, benefit for patients with CNS tumors has been limited. To enhance T cell activity against GD2+ CNS malignancies, we modified GD2-directed CART cells (GD2.CARTs) with a constitutively active interleukin (IL)-7 receptor (C7R-GD2.CARTs). METHODS: Patients age 1-21 years with H3K27-altered diffuse midline glioma (DMG) or other recurrent GD2-expressing CNS tumors were eligible for this phase I trial (ClinicalTrials.gov identifier: NCT04099797). All subjects received standard-of-care adjuvant radiation therapy or chemotherapy before study enrollment. The first treatment cohort received GD2.CARTs alone (1 × 107 cells/m2), and subsequent cohorts received C7R-GD2.CARTs at two dose levels (1 × 107 cells/m2; 3 × 107 cells/m2). Standard lymphodepletion with cyclophosphamide and fludarabine was included at all dose levels. RESULTS: Eleven patients (age 4-18 years) received therapy without dose-limiting toxicity. The GD2.CART cohort did not experience toxicity, but had disease progression after brief improvement of residual neurologic deficits (≤3 weeks). The C7R-GD2.CART cohort developed grade 1 tumor inflammation-associated neurotoxicity in seven of eight (88%) cases, controllable with anakinra. Cytokine release syndrome was observed in six of eight (75%, grade 1 in all but one patient) and associated with increased circulating IL-6 and IP-10 (P < .05). Patients receiving C7R-GD2.CARTs experienced temporary improvement from baseline neurologic deficits (range, 2 to >12 months), and seven of eight (88%) remained eligible for additional treatment cycles (range 2-4 cycles). Partial responses by iRANO criteria were observed in two of seven (29%) patients with DMG treated by C7R-GD2.CARTs. CONCLUSION: Intravenous GD2.CARTs with and without C7R were well tolerated. Patients treated with C7R-GD2.CARTs exhibited transient improvement of neurologic deficits and increased circulating cytokines/chemokines. Treatment with C7R-GD2.CARTs represents a novel approach warranting further investigation for children with these incurable CNS cancers.

The effectiveness of continuing professional development for the residential long-term care workforce: A systematic review.

OBJECTIVES: To investigate the effectiveness of continuing professional development in residential long-term care. DESIGN: Systematic review. DATA SOURCES: PubMed, Cumulative Index to Nursing & Allied Health (CINAHL), and Web of Science. REVIEW METHODS: Empirical studies published between 2003 and 2023 describing the effectiveness of continuing professional development in long-term care were selected according to PRISMA guidelines. The type, topic, and effectiveness of continuing professional development activities in long-term care were analysed, in addition to facilitators and barriers. The protocol of this review is registered in PROSPERO. RESULTS: A total of 155 studies were selected, including over 17,000 participants the majority of whom were nurses. The most common topics were 'dementia care' (n = 22; 14.2 %), and restraint use (n = 14; 9 %). The impact of continuing professional development was mainly evaluated in terms of 'participant satisfaction with continuing professional development' (n = 5; 3 %), 'staff knowledge' (n = 57; 37 %), 'staff competencies and skills' (n = 35; 23 %), 'resident outcomes' (n = 45; 29 %), and 'staff wellbeing' (n = 12; 8 %). A total of 64 (41 %) studies evaluated if impact of continuing professional development was sustained over time. 'Good organisation', 'a supportive learning environment', 'expressing personal preferences', and 'management support' were described as facilitators of continuing professional development. CONCLUSIONS: Increasing numbers of long-term care residents with complex health conditions require nurses with advanced skills, such as dementia care. To improve the effectiveness of continuing professional development, support from managers, who adopt relational leadership styles, is instrumental to integrate new knowledge and skills into practice. This needs to be linked to career progression, and consequently increase the attractiveness of working in the long-term care sector. This could meet the dual goal of improving outcomes for residents and nurses' job satisfaction.

Integrating Phenotypic and Chemoproteomic Approaches to Identify Covalent Targets of Dietary Electrophiles in Platelets.

A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.

Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1ß/CXCR2 Axis in Generation of Metastasis-specific Neutrophils.

Neutrophils are a highly heterogeneous cellular population. However, a thorough examination of the different transcriptional neutrophil states between health and malignancy has not been performed. We utilized single-cell RNA sequencing of human and murine datasets, both publicly available and independently generated, to identify neutrophil transcriptomic subtypes and developmental lineages in health and malignancy. Datasets of lung, breast, and colorectal cancer were integrated to establish and validate neutrophil gene signatures. Pseudotime analysis was used to identify genes driving neutrophil development from health to cancer. Finally, ligand-receptor interactions and signaling pathways between neutrophils and other immune cell populations in primary colorectal cancer and metastatic colorectal cancer were investigated. We define two main neutrophil subtypes in primary tumors: an activated subtype sharing the transcriptomic signatures of healthy neutrophils; and a tumor-specific subtype. This signature is conserved in murine and human cancer, across different tumor types. In colorectal cancer metastases, neutrophils are more heterogeneous, exhibiting additional transcriptomic subtypes. Pseudotime analysis implicates IL1ß/CXCL8/CXCR2 axis in the progression of neutrophils from health to cancer and metastasis, with effects on T-cell effector function. Functional analysis of neutrophil-tumoroid cocultures and T-cell proliferation assays using orthotopic metastatic mouse models lacking Cxcr2 in neutrophils support our transcriptional analysis. We propose that the emergence of metastatic-specific neutrophil subtypes is driven by the IL1ß/CXCL8/CXCR2 axis, with the evolution of different transcriptomic signals that impair T-cell function at the metastatic site. Thus, a better understanding of neutrophil transcriptomic programming could optimize immunotherapeutic interventions into early and late interventions, targeting different neutrophil states. SIGNIFICANCE: We identify two recurring neutrophil populations and demonstrate their staged evolution from health to malignancy through the IL1ß/CXCL8/CXCR2 axis, allowing for immunotherapeutic neutrophil-targeting approaches to counteract immunosuppressive subtypes that emerge in metastasis.

Protein SUMOylation promotes cAMP-independent EPAC1 activation.

Exchange protein directly activated by cAMP (EPAC1) mediates the intracellular functions of a critical stress-response second messenger, cAMP. Herein, we report that EPAC1 is a cellular substrate of protein SUMOylation, a prevalent stress-response posttranslational modification. Site-specific mapping of SUMOylation by mass spectrometer leads to identifying K561 as a primary SUMOylation site in EPAC1. Sequence and site-directed mutagenesis analyses reveal a functional SUMO-interacting motif required for cellular SUMOylation of EPAC1. SUMO modification of EPAC1 mediates its heat shock-induced Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.

Biophysical and structural characterization of a multifunctional viral genome packaging motor.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid. It was proposed that bacteriophage λ terminase assembles as an anti-parallel dimer-of-dimers nuclease complex at the packaging initiation site. In contrast, all characterized packaging motors are composed of five terminase subunits bound to the procapsid in a parallel orientation. Here, we describe biophysical and structural characterization of the λ holoenzyme complex assembled in solution. Analytical ultracentrifugation, small angle X-ray scattering, and native mass spectrometry indicate that 5 subunits assemble a cone-shaped terminase complex. Classification of cryoEM images reveals starfish-like rings with skewed pentameric symmetry and one special subunit. We propose a model wherein nuclease domains of two subunits alternate between a dimeric head-to-head arrangement for genome maturation and a fully parallel arrangement during genome packaging. Given that genome packaging is strongly conserved in both prokaryotic and eukaryotic viruses, the results have broad biological implications.

Enhanced recovery in cranial surgery (ERACraS) - A single-centre quality improvement study.

BACKGROUND: Enhanced Recovery After Surgery (ERAS) is a well-established, protocol-driven, evidence-based approach to peri-operative care. ERAS protocols have been used to improve patient morbidity and mortality outcomes in various surgical specialties. More recently, it has been introduced to neurosurgery. Our aim was to establish an Enhanced Recovery After Cranial Surgery (ERACraS) protocol for patients as part of a quality improvement project (QIP) with the intention of reducing hospital length of stay (HLOS). METHODS: This QIP was carried out in the Department of Neurosciences (DCN), Edinburgh, over two four-month periods. A total of 40 patients over 18 years of age undergoing elective craniotomy surgery under a sole neurosurgeon were invited to take part in this QIP. Subsequently, data was retrospectively collected through our institution's online documentation system. RESULTS: 19 patients received conventional perioperative care (pre-ERACraS group) during December 2021-March 2022, and 21 received care according to the novel ERACraS (ERACraS group) during June-September 2022. Regarding supra-tentorial surgery, there was a reduction of 73% in HLOS in the ERACraS group. No change was observed in infra-tentorial surgery. Overall, the ERACraS protocol reduced HLOS by 50% in cranial surgery. CONCLUSION: The QIP data from ERACraS in our unit has shown that implementing ERAS protocols is feasible. A reduction in HLOS has implications for patient morbidity, mortality, and quality of care. We endeavour to collect long-term data by collaborating with neurosurgical units across the UK and Ireland to validate its feasibility and sustainability as part of a major QIP in neurosurgical practice. This can be potentially adopted by neurosurgical centres across the globe in a safe and sustained manner.

The experience and attitudes of long-term care workers with teaching and learning modalities for the delivery of continuing professional development activities: a mixed-methods study.

AIM: to develop a better understanding of the attitudes and experiences of healthcare workers and managers with face-to-face, online asynchronous (pre-recorded), and online synchronous (live) modalities for Continuing Professional Development (CPD) working in the Long-Term Care (LTC) sector. BACKGROUND: The recent global pandemic significantly interrupted the delivery and organisation of education and training for healthcare professionals internationally. As a result of the restrictions, healthcare professionals' access to CPD was limited, consequentially education and training activities were mostly delivered online. Research into the experiences of healthcare workers with the various teaching and learning modalities in the LTC setting is limited. DESIGN: An explanatory mixed methods study using an exploratory sequential design. METHODS: A two-phase study conducted from January 2022 to September 2022, in the Republic of Ireland. In phase one, researchers conducted four focus groups and in phase two 168 participants completed a survey to explore the results of phase one. RESULTS: From the focus groups interviews five themes emerged regarding participants' experiences, attitudes and preferences with the three modalities of CPD education and training in the field of LTC: 1) Flexibility, 2) Networking, 3) Resources and Support, 4) Engaging and meaningful learning, and 5) Balancing online and face-to-face learning through Blended Learning. Results from the survey found the preferred modality was face to face (n = 54, 32.1%), followed very closely by blended learning (n = 51, 30.4%). Most of the respondents reported that synchronous online CPD education was convenient, flexible, offers the opportunity to interact with peers, and that its quality depends on educators' skills. The majority of respondents (n = 155, 92.3%) declared that they would require support in the workplace to implement their new knowledge and skills. CONCLUSIONS: This study revealed the significance participants place on 'engagement' when taking part in education and training. Engagement was described as a key factor to improve the delivery of CPD in the LTC setting. In addition, regardless of the mode of delivery, participants reported that they need to be supported in the workplace to implement their new knowledge and skills. This requires the support and endorsement of employers and managers, who could ensure more protected time for learning, technical support and championing facilitators and mentors in the workplace to enhance the translation of new knowledge into clinical practice. TWEETABLE ABSTRACT: In LTC settings, face-to-face CPD is the preferred mode of delivery. Although synchronous online CPD is very convenient, educators must be able to engage learners. LTC workers need support by managers to implement their new knowledge and skills in their workplace.

Modulation of limbic resting-state networks by subthalamic nucleus deep brain stimulation.

Beyond the established effects of subthalamic nucleus deep brain stimulation (STN-DBS) in reducing motor symptoms in Parkinson's disease, recent evidence has highlighted the effect on non-motor symptoms. However, the impact of STN-DBS on disseminated networks remains unclear. This study aimed to perform a quantitative evaluation of network-specific modulation induced by STN-DBS using Leading Eigenvector Dynamics Analysis (LEiDA). We calculated the occupancy of resting-state networks (RSNs) in functional MRI data from 10 patients with Parkinson's disease implanted with STN-DBS and statistically compared between ON and OFF conditions. STN-DBS was found to specifically modulate the occupancy of networks overlapping with limbic RSNs. STN-DBS significantly increased the occupancy of an orbitofrontal limbic subsystem with respect to both DBS OFF (p = 0.0057) and 49 age-matched healthy controls (p = 0.0033). Occupancy of a diffuse limbic RSN was increased with STN-DBS OFF when compared with healthy controls (p = 0.021), but not when STN-DBS was ON, which indicates rebalancing of this network. These results highlight the modulatory effect of STN-DBS on components of the limbic system, particularly within the orbitofrontal cortex, a structure associated with reward processing. These results reinforce the value of quantitative biomarkers of RSN activity in evaluating the disseminated impact of brain stimulation techniques and the personalization of therapeutic strategies.

Structural basis for cloverleaf RNA-initiated viral genome replication.

The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5' end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CDpro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CDpro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem-loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.

Examination of PTSD symptom networks over the course of cognitive processing therapy.

OBJECTIVES: Cognitive processing therapy (CPT) is an evidence-based psychotherapy for posttraumatic stress disorder (PTSD); however, little is known about how interrelationships between PTSD symptoms change over the course of treatment. The current study examined baseline, midtreatment, and posttreatment PTSD symptom networks during CPT for PTSD. METHOD: Adults with PTSD (n = 107) received 12 sessions of CPT as part of a randomized trial. Self-reported PTSD symptoms were assessed at pretreatment, midtreatment, and posttreatment, and network analysis was used to examine the interrelationships between symptoms at these three timepoints. Linear regression was conducted to examine whether any baseline symptoms or midpoint symptoms predicted overall treatment change. RESULTS: In the baseline PTSD network, feelings of detachment and feeling upset at reminders of the trauma were central to the symptom network. These symptoms were no longer central at midtreatment, possibly suggesting that CPT quickly reduces the importance of these symptoms. These findings were consistent with regression results that, after accounting for multiple comparisons, high baseline scores of feeling upset at trauma reminders predicted later treatment change. At the conclusion of treatment, strong negative emotions were the most central symptom and may be most important in maintaining or lowering other PTSD symptoms at the conclusion of treatment. CONCLUSIONS: Though replication is necessary, these findings offer insights into identifying which symptoms may be most predictive of treatment outcomes and the course by which CPT reduces PTSD symptoms. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Effects of electron-donating ability of binding sites on coordination number: the interactions of a cyclic Schiff base with copper ions.

The stepwise addition of Cu2+ ions to the nonplanar cyclic Schiff base 5,9,14,18-tetramethyl-1,4,10,13-tetraazacyclooctadeca-5,8,14,17-tetraene-7,16-dione (H4daaden, C18H28N4O2), yields a one-end-open dinuclear copper chelate. The pyridine adduct of the dinuclear copper chelate, namely, [µ-6,11-dimethyl-7,10-diazahexadeca-5,11-diene-2,4,13,15-tetraolato(4-)](pyridine)dicopper(II), [Cu2(C16H20N2O4)(C5H5N)], was characterized by single-crystal X-ray crystallography. The two CuII atoms of the copper chelate display different coordination modes, i.e. inner-N2O2 and outer-O2O2. The Cu atom which is bonded in the outer-O2O2 mode is axially bonded to a pyridine molecule, which suggests that the electron-donating ability of the O2O2 site to the Cu atom is poor. As a result, the O2O2-bonded Cu atom has a coordination number of five, showing square-bipyramidal geometry around the Cu atom. The N2O2-coordinated site provides sufficient electron density to the other Cu atom to be stabilized with a coordination number of four, showing square-planar geometry around the Cu atom. The electron-donating ability of the ligand coordination sites plays a key role in determining the coordination number of the Cu atoms of the dicopper chelate.

Cannabis use and suicide risk among Gulf War veterans.

Cannabis use has been indicated as a risk factor for suicide in veterans. This study of Gulf War veterans tested the relationship between self-report past year cannabis use and (a) past year suicidal ideation and (b) risk for suicidal behavior. Data were from a national sample (N = 1126) of Gulf War veterans. Logistic regression models indicated cannabis use was associated with past year suicidal ideation and elevated risk for suicidal behavior, independent of key covariates. In corroboration with research on other military populations, this study indicates a potentially concerning association between cannabis use and suicide risk in Gulf War veterans.