Current Practices in Clinical Supervision in Primary Care.

The purpose of this study was to examine current clinical supervision practices within primary care settings. We used a descriptive survey design, which blends quantitative and qualitative data, and examined the current state of clinical supervision practices and approaches in primary care and the type of training the behavioral health consultants received to provide supervision to pre-licensure level behavioral health trainees. Ninety-four participants completed the survey in 2022. Seventy-one percent of respondents felt they had adequate training to be an effective integrated behavioral health (IBH) supervisor; however, most training came from sources, such as workshops, continuing education, or supervision of supervision. Further efforts to establish universal competencies and formal training programs are needed to meet the growing need for IBH services in primary care.

Mental health symptoms in children and adolescents during COVID-19 in Australia.

OBJECTIVE: COVID-19 has led to disruptions to the lives of Australian families through social distancing, school closures, a temporary move to home-based online learning, and effective lockdown. Understanding the effects on child and adolescent mental health is important to inform policies to support communities as they continue to face the pandemic and future crises. This paper sought to report on mental health symptoms in Australian children and adolescents during the initial stages of the pandemic (May to November 2020) and to examine their association with child/family characteristics and exposure to the broad COVID-19 environment. METHODS: An online baseline survey was completed by 1327 parents and carers of Australian children aged 4 to 17 years. Parents/carers reported on their child's mental health using five measures, including emotional symptoms, conduct problems, hyperactivity/inattention, anxiety symptoms and depressive symptoms. Child/family characteristics and COVID-related variables were measured. RESULTS: Overall, 30.5%, 26.3% and 9.5% of our sample scored in the high to very high range for emotional symptoms, conduct problems and hyperactivity/inattention, respectively. Similarly, 20.2% and 20.4% of our sample scored in the clinical range for anxiety symptoms and depressive symptoms, respectively. A child's pre-existing mental health diagnosis, neurodevelopmental condition and chronic illness significantly predicted parent-reported child and adolescent mental health symptoms. Parental mental health symptoms, having a close contact with COVID-19 and applying for government financial assistance during COVID-19, were significantly associated with child and adolescent mental health symptoms. CONCLUSION: Our findings show that Australian children and adolescents experienced considerable levels of mental health symptoms during the initial phase of COVID-19. This highlights the need for targeted and effective support for affected youth, particularly for those with pre-existing vulnerabilities.

The use of constant observation with people with dementia in hospitals: a mixed-methods systematic review.

OBJECTIVES: Constant observation is used in hospitals with people with dementia to manage their safety. However, opportunities for proactive care are not consistently recognised or utilised. A systematic review of constant observation was conducted to understand measures of effectiveness and facilitators for person-centred approaches. METHOD: Electronic databases were searched between 2010 and 2022. Four reviewers completed screening, quality assessments and data extraction with 20% checked for consistency. Findings were presented through narrative synthesis (PROSPERO registration CRD42020221078). FINDINGS: Twenty-four studies were included. Non-registered staff without specific training were the main providers of constant observation. Assessments and processes clarifying the level of observation encouraged reviews that linked initiation and discontinuation to a patient's changing needs. Examples of person-centred care, derived from studies of volunteers or staff employed to provide activities, demonstrated meaningful engagement could reassure a person and improve their mood. Proactive approaches that anticipated distress were thought to reduce behaviours that carried a risk of harm but supporting evidence was lacking. CONCLUSION: Non-registered staff are limited by organisational efforts to reduce risk, leading to a focus on containment. Trained staff who are supported during constant observation can connect with patients, provide comfort and potentially reduce behaviours that carry a risk of harm.

A Comprehensive Literature Search of Digital Health Technology Use in Neurological Conditions: Review of Digital Tools to Promote Self-management and Support.

BACKGROUND: The use of digital health technology to promote and deliver postdiagnostic care in neurological conditions is becoming increasingly common. However, the range of digital tools available across different neurological conditions and how they facilitate self-management are unclear. OBJECTIVE: This review aims to identify digital tools that promote self-management in neurological conditions and to investigate their underlying functionality and salient clinical outcomes. METHODS: We conducted a search of 6 databases (ie, CINAHL, EMBASE, MEDLINE, PsycINFO, Web of Science, and the Cochrane Review) using free text and equivalent database-controlled vocabulary terms. RESULTS: We identified 27 published articles reporting 17 self-management digital tools. Multiple sclerosis (MS) had the highest number of digital tools followed by epilepsy, stroke, and headache and migraine with a similar number, and then pain. The majority were aimed at patients with a minority for carers. There were 5 broad categories of functionality promoting self-management: (1) knowledge and understanding; (2) behavior modification; (3) self-management support; (4) facilitating communication; and (5) recording condition characteristics. Salient clinical outcomes included improvements in self-management, self-efficacy, coping, depression, and fatigue. CONCLUSIONS: There now exist numerous digital tools to support user self-management, yet relatively few are described in the literature. More research is needed to investigate their use, effectiveness, and sustainability, as well as how this interacts with increasing disability, and their integration within formal neurological care environments.

Primary mental health prevention in partners of mothers with a major mental illness: SMS4Dads.

BACKGROUND: Mental health promotion and prevention with expecting and new fathers has historically been challenging. Approximately 10% of this population report experiencing depression in the post-partum period and 18% report experiencing anxiety. This population may be further at risk if their partner has a mental illness. OBJECTIVE: To assess if information provided by SMS may be a way to reach a vulnerable population of new fathers with partners who have a mental illness. METHOD: Twenty-three new and expecting fathers who have partners with a mental illness were engaged in a qualitative assessment of their experience with the SMS4Dads programme. RESULTS: This analysis showed that at risk new fathers appreciated the information received during the programme and reported acting on this, making changes in the way they interact with their children. CONCLUSION: The combination of timely, accurate and practical information delivered in a novel way, likely contributed to the reports of improved interactions within the family unit.

Treating Post-traumatic Stress Disorder with a Prolonged Exposure Protocol Within Primary Care Behavioral Health: A Case Example.

Posttraumatic stress disorder (PTSD) is a debilitating condition that impacts anywhere from 2 to 39% of primary care patients. Research suggests overall health, instances of hospitalizations, emergency room visits, and utilization of primary care services are impacted by a diagnosis of PTSD. Evidenced based treatments such as cognitive process therapy and prolonged exposure (PE) are available in specialty mental health but pose many barriers to treatment and implementation into primary care. This case study serves as the first known case example with an ethnic minority civilian, examining the treatment of PTSD within the Primary Care Behavioral Health Model using the brief (5 visits), PE protocol for primary care (PE-PC). PTSD was assessed using the PCL-5. Additional variables were assessed and tracked with the following tools: PHQ-9 (depressive symptoms), GAD-7 (anxiety symptoms), QLES-SF (quality of life), and the AAQ-2 (psychological flexibility) pre/post treatment, 6 months post-treatment and 9 months post-treatment. The patient reported clinically significant decreases in symptoms of PTSD, depression, and anxiety symptoms. Additionally, the patient's scores on quality of life and psychological flexibility improved. Brief, exposure-based treatment for PTSD can be delivered within the PCBH model. This treatment may result in improved quality of life and has the potential to reduce health care costs. This case encourages the treatment of PTSD within primary care, increasing access to care for patients. Future research is needed to further investigate this protocol in primary care with underserved, civilian populations and to explore patient attitudes toward brief treatment for PTSD in a primary care setting.

HLA-DQA1 and APOL1 as Risk Loci for Childhood-Onset Steroid-Sensitive and Steroid-Resistant Nephrotic Syndrome.

BACKGROUND: Few data exist for the genetic variants underlying the risk for steroid-sensitive nephrotic syndrome (SSNS) in children. The objectives of this study were to evaluate HLA-DQA1 and APOL1 variants as risk factors for SSNS in African American children and use classic HLA antigen types and amino acid inference to refine the HLA-DQA1 association. STUDY DESIGN: Case-control study. SETTING & PARTICIPANTS: African American children with SSNS or steroid-resistant nephrotic syndrome (SRNS) were enrolled from Duke University and centers participating in the Midwest Pediatric Nephrology Consortium. FACTOR: Genetic variants in HLA-DQA1 (C34Y [rs1129740]; F41S [rs1071630]) and APOL1 high-risk alleles. OUTCOMES: SSNS and SRNS. MEASUREMENTS: Direct sequencing for the HLA-DQA1 and APOL1 variants in 115 African American children (65 with SSNS and 50 with SRNS). Imputation of classic HLA alleles and amino acids was done in 363 South Asian children. RESULTS: The 2 HLA-DQA1 variants were significantly associated with SSNS in African American children (C34Y: P=5.7 × 10-11; OR, 3.53; 95% CI, 2.33-5.42; F41S: P=1.2 × 10-13; OR, 4.08; 95% CI, 2.70-6.28), but not with SRNS (C34Y: P=0.6; F41S: P=0.2). APOL1 high-risk variants were not associated with SSNS (P=0.5) but showed significant associations with SRNS (P=1.04 × 10-7; OR, 4.17; 95% CI, 2.23-7.64). HLA-DQA1*0201, HLA-DQB1*0201, and HLA-DRB1*0701 were the classic HLA alleles with the most significant associations with SSNS risk. The most significantly associated amino acid positions were HLA-DQα1 56 and 76 (both P=2.8 × 10-7). Conditional analysis revealed that these variants most likely account for the observed association. LIMITATIONS: Modest sample size and limited statistical power to detect small to moderate effect sizes. Children studied may not be representative of all African American children in the United States. CONCLUSIONS: HLA-DQA1 is a risk locus for SSNS, but not SRNS, in African American children, consistent with its role in SSNS risk in children of European, Asian, and African ancestries. There is little evidence of a significant role for the APOL1 high-risk alleles in childhood SSNS in African American children. Refinement of the HLA-DQA1 association identified the critical classic HLA antigen types and amino acids of the HLA-DQ α1 molecule.

Review of complications associated with contact lenses from unregulated sources of supply.

OBJECTIVE: To review existing studies and case reports regarding complications associated with contact lenses (CLs) from unregulated sources of supply and to identify any relevant trends. METHODS: A systematic literature search was performed to locate publications concerning complications associated with CLs obtained from unregulated sources of supply. RESULTS: A total of 23 articles were identified that represent 70 individual cases. All 8 of the pre-2006 case reports originated from the United States and the United Kingdom, whereas from 2006 onwards, only 2 of the 15 reports came from these locations. Over-the-counter supply accounted for 73% (51/70) of cases, whereas 17% (12/70) were borrowed or shared lenses and 6% (4/70) lenses were obtained through the Internet. Nearly, three quarters of patients (30/42, 71%) waited longer than 48 hours after the onset of symptoms before seeking medical attention; 10 patients waited longer than a week, and 5 longer than a month. Microbial keratitis (MK) was reported in 43 (61%) patients, with permanent damage occurring in 72% (31/43) of patients followed to conclusion. Known risk factors associated for MK were present in all cases irrespective of whether the patients developed MK. CONCLUSIONS: There are various reasons to presume that the unregulated supply of CLs might result in the use of inappropriate lenses, increase the risk of poorer lens hygiene, and militate against the prompt treatment of any consequent complications. There is some indication that the introduction of regulations to control the supply of plano CLs has alleviated the level of complications.

Central vestibular disease in a blue and gold macaw (Ara ararauna) with cerebral infarction and hemorrhage.

A 24-year-old female blue and gold macaw (Ara ararauna) was presented for an acute onset of left head tilt. On examination, the macaw was dehydrated and had a 120-degree left head tilt, decreased proprioception of the left pelvic limb, and intermittent vertical nystagmus. Results of hematologic testing and biochemical analysis revealed severe leukocytosis with lymphopenia and heterophilia and a high uric acid concentration. Radiographs showed bilateral intertarsal joint osteoarthritis and a healed ulnar fracture. Magnetic resonance imaging of the brain revealed focal T2 and fluid-attenuated inversion recovery hyperintense lesions in the right cerebral hemisphere and in the midbrain. The midbrain lesion showed susceptibility artifact on the T2* sequence, suggesting hemorrhage. In the T2* sequence, iron accumulation (as seen with hemorrhage) distorts the magnetic signal, resulting in the production of a susceptibility artifact, which can then be visualized as a region of hypointensity. The bird was hospitalized but died despite intensive care. Necropsy revealed multiple cerebral vascular lesions including an acute cerebral infarct, a ruptured midbrain aneurysm, and multifocal systemic atherosclerosis. To our knowledge, this is the first report of a cerebral aneurysm in a bird. This report correlates the clinical presentation, imaging, and histopathologic findings in a macaw with central vestibular disease and demonstrates how advanced imaging techniques can identify hemorrhagic lesions through the T2* sequence.

Tropine exacerbates the ventilatory depressant actions of fentanyl in freely-moving rats.

Our lab is investigating the efficacy profiles of tropine analogs against opioid-induced respiratory depression. The companion manuscript reports that the cell-permeant tropeine, tropine ester (Ibutropin), produces a rapid and sustained reversal of the deleterious actions of fentanyl on breathing, alveolar-arterial (A-a) gradient (i.e., index of alveolar gas exchange), and arterial blood-gas (ABG) chemistry in freely-moving male Sprague Dawley rats, while not compromising fentanyl analgesia. We report here that in contrast to Ibutropin, the injection of the parent molecule, tropine (200 µmol/kg, IV), worsens the adverse actions of fentanyl (75 µg/kg, IV) on ventilatory parameters (e.g., frequency of breathing, tidal volume, minute ventilation, peak inspiratory and expiratory flows, and inspiratory and expiratory drives), A-a gradient, ABG chemistry (e.g., pH, pCO2, pO2, and sO2), and sedation (i.e., the righting reflex), while not affecting fentanyl antinociception (i.e., the tail-flick latency) in freely-moving male Sprague Dawley rats. These data suggest that tropine augments opioid receptor-induced signaling events that mediate the actions of fentanyl on breathing and alveolar gas exchange. The opposite effects of Ibutropin and tropine may result from the ability of Ibutropin to readily enter peripheral and central cells. Of direct relevance is that tropine, resulting from the hydrolysis of Ibutropin, would combat the Ibutropin-induced reversal of the adverse effects of fentanyl. Because numerous drug classes, such as cocaine, atropine, and neuromuscular blocking drugs contain a tropine moiety, it is possible that their hydrolysis to tropine has unexpected/unintended consequences. Indeed, others have found that tropine exerts the same behavioral profile as cocaine upon central administration. Together, these data add valuable information about the pharmacological properties of tropine.

Cell cycle specific, differentially tagged ribosomal proteins to measure phase specific transcriptomes from asynchronously cycling cells.

Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including RNAseq, survey the steady state gene expression across the cell cycle and are inherently limited by their inability to resolve dynamic gene regulatory networks. Single cell RNAseq (scRNAseq) can identify different cell cycle transcriptomes if enough cycling cells are present, however some cells are not amenable to scRNAseq. Therefore, we merged two powerful strategies, the CDT1 and GMNN degrons used in Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) cell cycle sensors and the ribosomal protein epitope tagging used in RiboTrap/Tag technologies to isolate cell cycle phase-specific mRNA for sequencing. The resulting cell cycle dependent, tagged ribosomal proteins (ccTaggedRP) were differentially expressed during the cell cycle, had similar subcellular locations as endogenous ribosomal proteins, incorporated into ribosomes and polysomes, and facilitated the recovery of cell cycle phase-specific RNA for sequencing. ccTaggedRP has broad applications to investigate phase-specific gene expression in complex cell populations.

L-cysteine ethylester reverses the adverse effects of morphine on breathing and arterial blood-gas chemistry while minimally affecting antinociception in unanesthetized rats.

L-cysteine ethylester (L-CYSee) is a membrane-permeable analogue of L-cysteine with a variety of pharmacological effects. The purpose of this study was to determine the effects of L-CYSee on morphine-induced changes in ventilation, arterial-blood gas (ABG) chemistry, Alveolar-arterial (A-a) gradient (i.e., a measure of the index of alveolar gas-exchange), antinociception and sedation in male Sprague Dawley rats. An injection of morphine (10 mg/kg, IV) produced adverse effects on breathing, including sustained decreases in minute ventilation. L-CYSee (500 µmol/kg, IV) given 15 min later immediately reversed the actions of morphine. Another injection of L-CYSee (500 µmol/kg, IV) after 15 min elicited more pronounced excitatory ventilatory responses. L-CYSee (250 or 500 µmol/kg, IV) elicited a rapid and prolonged reversal of the actions of morphine (10 mg/kg, IV) on ABG chemistry (pH, pCO2, pO2, sO2) and A-a gradient. L-serine ethylester (an oxygen atom replaces the sulfur; 500 µmol/kg, IV), was ineffective in all studies. L-CYSee (500 µmol/kg, IV) did not alter morphine (10 mg/kg, IV)-induced sedation, but slightly reduced the overall duration of morphine (5 or 10 mg/kg, IV)-induced analgesia. In summary, L-CYSee rapidly overcame the effects of morphine on breathing and alveolar gas-exchange, while not affecting morphine sedation or early-stage analgesia. The mechanisms by which L-CYSee modulates morphine depression of breathing are unknown, but appear to require thiol-dependent processes.

Mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade response in melanoma.

The mitochondrial genome (mtDNA) encodes essential machinery for oxidative phosphorylation and metabolic homeostasis. Tumor mtDNA is among the most somatically mutated regions of the cancer genome, but whether these mutations impact tumor biology is debated. We engineered truncating mutations of the mtDNA-encoded complex I gene, Mt-Nd5, into several murine models of melanoma. These mutations promoted a Warburg-like metabolic shift that reshaped tumor microenvironments in both mice and humans, consistently eliciting an anti-tumor immune response characterized by loss of resident neutrophils. Tumors bearing mtDNA mutations were sensitized to checkpoint blockade in a neutrophil-dependent manner, with induction of redox imbalance being sufficient to induce this effect in mtDNA wild-type tumors. Patient lesions bearing >50% mtDNA mutation heteroplasmy demonstrated a response rate to checkpoint blockade that was improved by ~2.5-fold over mtDNA wild-type cancer. These data nominate mtDNA mutations as functional regulators of cancer metabolism and tumor biology, with potential for therapeutic exploitation and treatment stratification.

Proteotoxic stress response in atherosclerotic cardiovascular disease: Emerging role of heat shock factor 1.

Atherosclerosis is a major risk factor for cardiovascular diseases. Hypercholesterolemia has been both clinically and experimentally linked to cardiovascular disease and is involved in the initiation of atherosclerosis. Heat shock factor 1 (HSF1) is involved in the control of atherosclerosis. HSF1 is a critical transcriptional factor of the proteotoxic stress response that regulates the production of heat shock proteins (HSPs) and other important activities such as lipid metabolism. Recently, HSF1 is reported to directly interact with and inhibit AMP-activated protein kinase (AMPK) to promote lipogenesis and cholesterol synthesis. This review highlights roles of HSF1 and HSPs in critical metabolic pathways of atherosclerosis, including lipogenesis and proteome homeostasis.

Chemotaxis: Dendritic cells as trendsetters of the immune response.

A new study reports that dendritic cells actively shape the CCL19 chemokine gradient to which they respond and that the chemokine receptor CCR7 both senses CCL19 and mediates its internalisation. Generation of local changes in chemokines allows coordination of movement over longer distances than previous models could explain.

Systematic Literature Review of Real-World Effectiveness Results Data for First-Line Ibrutinib in Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma.

BACKGROUND AND OBJECTIVE: Ibrutinib, an oral Bruton's tyrosine kinase inhibitor, has demonstrated efficacy as a first-line treatment for chronic lymphocytic leukemia in multiple, phase III, randomized clinical trials. This systematic literature review assessed the clinical effectiveness of ibrutinib in the first-line treatment of chronic lymphocytic leukemia in real-world clinical settings. METHODS: MEDLINE, EMBASE, and relevant conference websites were searched for articles published in the USA from 1 January, 2014 to 30 June, 2020. Overall survival, progression-free survival, overall response rate, and time to next treatment were summarized. RESULTS: This analysis included a total of 12 publications representing data from 112 to 2033 patients from community and academic centers, and the multicenter informCLL registry. Patients were predominantly male (60-99%) with a median age range from 62 to 77 years, and included those with high-risk genomic features (del[17p]: 21-33%; del[11q]: 33%; and unmutated immunoglobulin heavy chain variable gene: 59%). Real-world effectiveness with ibrutinib complemented the efficacy demonstrated in randomized clinical trials. Across various studies, the 12-month overall survival rates ranged from 95% to 96%; 18-month overall survival rates were similarly high (91%). Twelve-month progression-free survival rates ranged from 89% to 93%, and the overall response rate ranged from 71% to 90% across four studies. In the studies that reported time to next treatment, 91% and 87% of patients treated with first-line ibrutinib did not initiate new treatment at 12 months and 24 months, respectively. CONCLUSIONS: This systematic literature review confirms the benefit of ibrutinib as a first-line treatment in patients with chronic lymphocytic leukemia in real-world clinical settings and is consistent with results from randomized clinical trials, including in patients with high-risk genomic features.

Comparison of Radiography and Computed Tomography for Evaluation of Third Carpal Bone Fractures in Horses.

Radiographs underestimate the extent of bone injury in horses with third carpal bone (C3) fractures (Fx). We aimed to describe bone pathologies identified using computed tomography (CT) and compare the diagnostic value of digital radiography (DR) and CT in horses with C3 Fx. CT images of 15 racehorses with C3 Fx and 10 controls were reviewed (Part 1) then DR and CT images of 26 racehorses (24 Thoroughbred, 2 Standardbred) with C3 Fx (Part 2) were evaluated. Agreement on fracture geometry and concomitant bone lesions was tested between DR and CT using the kappa statistic (Part 2). For agreement analysis, 38 limbs were used (27 Fx carpi from 26 horses and 11 contralateral carpi). Intermodality agreement was good for recognition of displacement, fair for comminution, articular surface bone loss and osseous fragmentation, and poor-slight for recognition of whether the Fx was complete, additional fissures and lucencies. CT provides more detailed information than DR regarding bone pathology and fracture configuration in horses with C3 fracture. Correlation of CT findings with clinical information and outcome needs to be explored; however, the more accurate diagnosis possible with CT is likely valuable when deciding on the most appropriate management and for surgical planning.

Effectiveness and cost-effectiveness of an electronic mindfulness-based intervention to improve maternal mental health in the peripartum: study protocol for a randomised controlled trial.

BACKGROUND: Perinatal women are highly vulnerable to developing mental health issues and particularly susceptible to a recurrence of psychiatric illness. Poor mental health during the perinatal period can have long-term impacts on the physical and psychiatric health of both mother and child. A potentially useful strategy to improve women's mental health is through a mobile application teaching mindfulness, an evidence-based technique helping individuals focus on the present moment. METHODS: A mixed method, prospective randomised controlled trial. The study group comprise women aged 18 years and over, who are attending the public and private maternity clinics at Mater Mothers' Hospital. A sample of 360 prenatal women will be randomised into the intervention group (with the use of the mindfulness app) or usual care. Participants will remain in the study for 11 months and will be assessed at four timepoints for changes in postnatal depression, mother-infant bonding, and quality of life. A cost-effectiveness evaluation will also be conducted using quality-adjusted life year (QALY) calculations. A random selection of intervention participants will be invited to attend focus groups to give feedback on the mindfulness app. DISCUSSION: Previous studies have found mindfulness interventions can reduce stress, anxiety, depression, and sleep disturbances in a prenatal population. The risks of the intervention are low, but could be of significant benefit for women who are unable to attend face-to-face appointments due to geographical, financial, or time barriers; during endemic or pandemic scenarios; or due to health or mobility issues. TRIAL REGISTRATION: This study was approved by the Mater Misericordiae Human Research Ethics Committee (83,589). Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12622001581752 ( https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=385107&isReview=true ). Registered on 22 Dec. 2022.

Tumour mitochondrial DNA mutations drive aerobic glycolysis to enhance checkpoint blockade.

The mitochondrial genome encodes essential machinery for respiration and metabolic homeostasis but is paradoxically among the most common targets of somatic mutation in the cancer genome, with truncating mutations in respiratory complex I genes being most over-represented1. While mitochondrial DNA (mtDNA) mutations have been associated with both improved and worsened prognoses in several tumour lineages1-3, whether these mutations are drivers or exert any functional effect on tumour biology remains controversial. Here we discovered that complex I-encoding mtDNA mutations are sufficient to remodel the tumour immune landscape and therapeutic resistance to immune checkpoint blockade. Using mtDNA base editing technology4 we engineered recurrent truncating mutations in the mtDNA-encoded complex I gene, Mt-Nd5, into murine models of melanoma. Mechanistically, these mutations promoted utilisation of pyruvate as a terminal electron acceptor and increased glycolytic flux without major effects on oxygen consumption, driven by an over-reduced NAD pool and NADH shuttling between GAPDH and MDH1, mediating a Warburg-like metabolic shift. In turn, without modifying tumour growth, this altered cancer cell-intrinsic metabolism reshaped the tumour microenvironment in both mice and humans, promoting an anti-tumour immune response characterised by loss of resident neutrophils. This subsequently sensitised tumours bearing high mtDNA mutant heteroplasmy to immune checkpoint blockade, with phenocopy of key metabolic changes being sufficient to mediate this effect. Strikingly, patient lesions bearing >50% mtDNA mutation heteroplasmy also demonstrated a >2.5-fold improved response rate to checkpoint inhibitor blockade. Taken together these data nominate mtDNA mutations as functional regulators of cancer metabolism and tumour biology, with potential for therapeutic exploitation and treatment stratification.

D-cysteine ethyl ester and D-cystine dimethyl ester reverse the deleterious effects of morphine on arterial blood-gas chemistry and Alveolar-arterial gradient in anesthetized rats.

We determined whether intravenous injections of the membrane-permeable ventilatory stimulants, D-cysteine ethyl ester (ethyl (2 S)- 2-amino-3-sulfanylpropanoate) (D-CYSee) and D-cystine dimethyl ester (methyl (2 S)- 2-amino-3-[[(2 S)- 2-amino-3-methoxy-3-oxopropyl]disulfanyl] propanoate) (D-CYSdime), could overcome the deleterious actions of intravenous morphine on arterial blood pH, pCO2, pO2 and sO2, and Alveolar-arterial (A-a) gradient (i.e., the measure of exchange of gases in the lungs) in Sprague Dawley rats anesthetized with isoflurane. Injection of morphine (2 mg/kg, IV) caused pronounced reductions in pH, pO2 and sO2 accompanied by elevations in pCO2, all which are suggestive of diminished ventilation, and elevations in A-a gradient, which suggests a mismatch of ventilation-perfusion. Subsequent boluses of D-cysteine ethyl ester (2 ×100 µmol/kg, IV) or D-cystine dimethyl ester (2 ×50 µmol/kg, IV) rapidly reversed of the negative actions of morphine on pH, pCO2, pO2 and sO2, and A-a gradient. Similar injections of D-cysteine (2 ×100 µmol/kg, IV) were without effect, whereas injections of D-cystine (2 ×50 µmol/kg, IV) produced a modest reversal. Our data show that D-cysteine ethyl ester and D-cystine dimethyl ester readily overcome the deleterious effects of morphine on arterial blood gas (ABG) chemistry and A-a gradient by mechanisms that may depend upon their ability to rapidly enter cells. As a result of their known ability to enter the brain, lungs, muscles of the chest wall, and most likely the major peripheral chemoreceptors (i.e., carotid bodies), the effects of the thiolesters on changes in ABG chemistry and A-a gradient elicited by morphine likely involve central and peripheral mechanisms. We are employing target prediction methods to identify an array of in vitro and in vivo methods to test potential functional proteins by which D-CYSee and D-CYSdime modulate the effects of morphine on breathing.