CRISPR-based functional profiling of the Toxoplasma gondii genome during acute murine infection.

Examining host-pathogen interactions in animals can capture aspects of infection that are obscured in cell culture. Using CRISPR-based screens, we functionally profile the entire genome of the apicomplexan parasite Toxoplasma gondii during murine infection. Barcoded gRNAs enabled bottleneck detection and mapping of population structures within parasite lineages. Over 300 genes with previously unknown roles in infection were found to modulate parasite fitness in mice. Candidates span multiple axes of host-parasite interaction. Rhoptry Apical Surface Protein 1 was characterized as a mediator of host-cell tropism that facilitates repeated invasion attempts. GTP cyclohydrolase I was also required for fitness in mice and druggable through a repurposed compound, 2,4-diamino-6-hydroxypyrimidine. This compound synergized with pyrimethamine against T. gondii and malaria-causing Plasmodium falciparum parasites. This work represents a complete survey of an apicomplexan genome during infection of an animal host and points to novel interfaces of host-parasite interaction.

Children's surgery and the emergency, critical, and operative care resolution: Immediate actions to eliminate disparities in surgery, anesthesia, and perioperative care for all children.

Around 1.7 billion children lack access to surgical care worldwide. To reinvigorate the efforts to address these disparities and support work to address global challenges in surgery, anesthesia, emergency, and critical care, the World Health Assembly passed World Health Organization Resolution World Health Assembly 76.2: Integrated emergency, critical and operative care for universal health coverage and protection from health emergencies (ECO) in 2023. This resolution highlights the integral role of surgery, anesthesia, and perioperative care in health systems. However, understanding how best to operationalize this resolution is challenging. We review the ECO resolution and highlight points that the pediatric surgical and anesthesia community can leverage to advocate for its recommendations for operative care.

Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.

Clinical and prognostic differences in oropharyngeal squamous cell carcinoma in USA and Denmark, two HPV high-prevalence areas.

BACKGROUND: Uncertainty persists regarding clinical and treatment variations crucial to consider when comparing high human papillomavirus (HPV)-prevalence oropharyngeal squamous cell carcinoma (OPSCC) cohorts for accurate patient stratification and replicability of clinical trials across different geographical areas. METHODS: OPSCC patients were included from The University of Texas MD Anderson Cancer Center (UTMDACC), USA and from The University Hospital of Copenhagen, Denmark from 2015-2020, (n = 2484). Outcomes were 3-year overall survival (OS) and recurrence-free interval (RFI). Subgroup analyses were made for low-risk OPSCC patients (T1-2N0M0) and high-risk patients (UICC8 III-IV). RESULTS: There were significantly more HPV-positive (88.2 % vs. 63.1 %), males (89.4 % vs. 74.1 %), never-smokers (52.1 % vs. 23.7 %), lower UICC8-stage (I/II: 79.3 % vs. 68 %), and fewer patients treated with radiotherapy (RT) alone (14.8 % vs. 30.3 %) in the UTMDACC cohort. No difference in the adjusted OS was observed (hazard ratio [HR] 1.21, p = 0.23), but a significantly increased RFI HR was observed for the Copenhagen cohort (HR: 1.74, p = 0.003). Subgroup analyses of low- and high-risk patients revealed significant clinical and treatment differences. No difference in prognosis was observed for low-risk patients, but the prognosis for high-risk patients in the Copenhagen cohort was worse (OS HR 2.20, p = 0.004, RFI HR 2.80, p = 0.002). CONCLUSIONS: We identified significant differences in clinical characteristics, treatment modalities, and prognosis between a Northern European and Northern American OPSCC population. These differences are important to consider when comparing outcomes and for patient stratification in clinical trials, as reproducibility might be challenging.

The role of collaboration in educating the global pediatric anesthesia workforce.

An estimated 1.7 billion children and adolescents do not have access to safe and affordable surgical care, and the vast majority of these are located in low-middle-income countries (LMICs). Pediatric anesthesia, a specialized field that requires a diverse set of knowledge and skills, has seen various advancements over the years and has become well-established in upper-middle and high-income countries. However, in LMICs, due to a multitude of factors including severe workforce shortages, this has not been the case. Collaborations play a vital role in increasing the capacity of pediatric anesthesiology educators and training the pediatric anesthesia workforce. These efforts directly increase access for children who require surgical intervention. Collaboration models can be operationalized through bidirectional knowledge sharing, training, resource allocation, research and innovation, quality improvement, networking, and advocacy. This article aims to highlight a few of these collaborative efforts. Specifically, the role that the World Federation of Societies of Anaesthesiologists, the Safer Anesthesia from Education program, the Asian Society of Pediatric Anaesthesiologists, Pediatric Anesthesia Training in Africa, the Paediatric Anaesthesia Network New Zealand, the Safe Pediatric Anesthesia Network and two WhatsApp™ groups (global ped anesthesia and the Pediatric Difficult Intubation Collaborative) have played in improving anesthesiology care for children.

Revisiting Feeding Tube Utilization in Oropharynx Cancer: 6-Year Prospective Registry Analysis.

OBJECTIVE: Patients treated for oropharyngeal cancer (OPC) have historically demonstrated high feeding tube rates for decreased oral intake and malnutrition. We re-examined feeding tube practices in these patients. STUDY DESIGN: Retrospective analysis of prospective cohort from 2015 to 2021. SETTING: Single-institution NCI-Designated Comprehensive Cancer Center. METHODS: With IRB approval, patients with new oropharyngeal squamous cell cancer or (unknown primary with neck metastasis) were enrolled. Baseline swallowing was assessed via videofluoroscopy and Performance Status Scale for Head and Neck Cancer (PSSHN). G-tubes or nasogastric tubes (NGT) were placed for weight loss before, during, or after treatment. Prophylactic NGT were placed during transoral robotic surgery (TORS). Tube duration was censored at last disease-free follow-up. Multivariate regression was performed for G-tube placement (odds ratio [OR] [95% confidence interval [CI]) and removal (Cox hazard ratio, hazard ratio [HR] [95% CI]). RESULTS: Of 924 patients, most had stage I to II (81%), p16+ (89%), node-positive (88%) disease. Median follow-up was 2.6 years (interquartile range 1.5-3.9). Most (91%) received radiation/chemoradiation, and 16% received TORS. G-tube rate was 27% (5% after TORS). G-tube risk was increased with chemoradiation (OR 2.78 [1.87-4.22]) and decreased with TORS (OR 0.31 [0.15-0.57]) and PSSHN-Diet score ≥60 (OR 0.26 [0.15-0.45]). G-tube removal probability over time was lower for T3 to T4 tumors (HR 0.52 [0.38-0.71]) and higher for PSSHN-Diet score ≥60 (HR 1.65 [1.03-2.66]). CONCLUSIONS: In this modern cohort of patients treated for OPC, 27% received G-tubes-50% less than institutional rates 10 years ago. Patients with preserved baseline swallowing and/or those eligible for TORS may have lower G-tube risk and duration.

The initiative for medical equity and global health (IMEGH) resuscitation training program: A model for resuscitation training courses in Africa.

In high-income countries, outcomes following in hospital cardiac arrest have improved over the last two decades due to the introduction of rapid response teams, cardiac arrest teams, and advanced resuscitation training. However, in low-income countries, such as Rwanda, outcomes are still poor. This is due to multiple factors including lack of adequate resuscitation training, few trainers, and lack of equipment. To address this issue, the Initiative for Medical Equity and Global Health Equity (IMEGH), a training organization founded in 2018 by 5 local anesthesiologists has regularly taught resuscitation courses such as Basic Life Support, Advanced Cardiac Life Support, and Pediatric Advanced Life Support in hospitals throughout Rwanda. The aims of the organization include developing a sustainable model to offer context relevant resuscitation training courses, building a cadre of local instructors to teach on the courses, as well as engaging funding partners to help support the effort. From October 2018 until September 2022, 31 courses were run in 11 hospitals across Rwanda training 1,060 healthcare providers (mainly of non-physician anesthetists, nurses, midwives, and general practitioners). Ongoing challenges include lack of local protocols, inability to tracking resuscitation outcomes, and continued inaccessibility by many healthcare providers. Despite these challenges, the IMEGH program is an example of a successful context-relevant model and has potential to inform the design of resuscitation programs in other similar settings. This article describes the development of the IMEGH program, accomplishments as well as lessons learned, challenges, and next steps for expansion.

REV7 Monomer Is Unable to Participate in Double Strand Break Repair and Translesion Synthesis but Suppresses Mitotic Errors.

Rev7 is a regulatory protein with roles in translesion synthesis (TLS), double strand break (DSB) repair, replication fork protection, and cell cycle regulation. Rev7 forms a homodimer in vitro using its HORMA (Hop, Rev7, Mad2) domain; however, the functional importance of Rev7 dimerization has been incompletely understood. We analyzed the functional properties of cells expressing either wild-type mouse Rev7 or Rev7K44A/R124A/A135D, a mutant that cannot dimerize. The expression of wild-type Rev7, but not the mutant, rescued the sensitivity of Rev7-/- cells to X-rays and several alkylating agents and reversed the olaparib resistance phenotype of Rev7-/- cells. Using a novel fluorescent host-cell reactivation assay, we found that Rev7K44A/R124A/A135D is unable to promote gap-filling TLS opposite an abasic site analog. The Rev7 dimerization interface is also required for shieldin function, as both Rev7-/- cells and Rev7-/- cells expressing Rev7K44A/R124A/A135D exhibit decreased proficiency in rejoining some types of double strand breaks, as well as increased homologous recombination. Interestingly, Rev7K44A/R124A/A135D retains some function in cell cycle regulation, as it maintains an interaction with Ras-related nuclear protein (Ran) and partially rescues the formation of micronuclei. The mutant Rev7 also rescues the G2/M accumulation observed in Rev7-/- cells but does not affect progression through mitosis following nocodazole release. We conclude that while Rev7 dimerization is required for its roles in TLS, DSB repair, and regulation of the anaphase promoting complex, dimerization is at least partially dispensable for promoting mitotic spindle assembly through its interaction with Ran.

The Capnography Project.

Capnography is an essential tool used in the monitoring of patients during anesthesia and in critical care which, while required in most high-income countries, is unavailable in many low- and middle-income countries. Launched in 2020, the Smile Train-Lifebox Capnography Project aimed to find a "capnography solution" for resource-poor settings. The project was specifically interested in a capnography device that would meet the needs of the Smile Train partner hospitals to help monitor children requiring airway or cleft surgery. Project advisory and technical groups were formed and included representation from anesthesia practitioners from a balanced representation from all level of income countries, technical experts in capnography, and representatives from the Global Capnography Project (GCAP), the University of California at San Francisco Center for Health Equity in Surgery & Anesthesia (CHESA), and the World Federation of Societies of Anaesthesiologists (WFSA). Built upon the WFSA minimum capnometer specifications, a human centered design approach was used to develop a Target Product Profile. Seven manufacturers submitted 13 devices for consideration and 3 devices were selected for the testing phase. Each of these devices was evaluated for build quality, and clinical and usability performance. Based on the findings from the overall testing process, a combined capnography and pulse oximetry device by Zug Medical Systems was chosen. To accompany the new Smile Train-Lifebox capnograph, an international team of experienced anesthesiologists and educators came together to develop the necessary education materials. These materials were piloted in Ethiopia, subsequently modified, and endorsed by the education team. The device is now ready for distribution, with the accompanying education package, to the Smile Train network and beyond. In addition, a study is being planned to measure the impact of capnography introduction into operating rooms in resource-constrained settings.

Delivery of Safe Pediatric Anesthesia Care in the First 8000 days: Realities, Challenges, and Solutions in Low- and Middle-Income Countries.

BACKGROUND: Worldwide, perioperative mortality has declined over the past 50 years, but the reduction is skewed toward high-income countries (HICs). Currently, pediatric perioperative mortality is much higher in low- and middle-income countries (LMICs) compared to HICs, despite studied cohorts being predominantly low-risk. These disparities must be studied and addressed. METHODS: A narrative review of the literature was undertaken to identify contributing factors and potential knowledge gaps. Interventions aimed at alleviating the outcomes disparities are discussed, and recommendations are made for future directions. RESULTS AND CONCLUSIONS: There is a lack of adequately trained pediatric anesthesia providers in LMICs, and the number must be bolstered by making such training available. Essential anesthesia medications and equipment, in pediatric-appropriate sizes, are often not available; neither are essential infrastructure items. Perioperative staff are underprepared for emergent situations that may arise and simulation training may help to ameliorate this. The global anesthesia community has implemented several solutions to address these issues. The World Federation of Societies of Anaesthesiologists (WFSA) and Global Initiative for Children's Surgery have published standards that outline essential items for the provision of safe perioperative pediatric care. Several short educational courses have been developed and introduced in LMICs that either specifically address pediatric patients, or contain a pediatric component. The WFSA also maintains a collection of discrete tutorials for educational purposes. Finally, in Africa, large-scale, prospective data collection is underway to examine pediatric perioperative outcomes. More work needs to be done, though, to improve perioperative outcomes for pediatric patients in LMICs.

Pediatric procedural sedation in African clinical settings: A mixed methods study of African providers' sedation practices.

Background: Little is known about the practice of pediatric procedural sedation in Africa, despite being incredibly useful to the emergency care of children. This study describes the clinical experiences of African medical providers who use pediatric procedural sedation, including clinical indications, medications, adverse events, training, clinical guideline use, and comfort level. The goals of this study are to describe pediatric sedation practices in resource-limited settings in Africa and identify potential barriers to the provision of safe pediatric sedation. Methods: This mixed methods study describes the pediatric procedural sedation practices of African providers using semi-structured interviews. Purposive sampling was used to identify key informants working in African resource-limited settings across a broad geographic, economic, and professional range. Quantitative data about provider background and sedation practices were collected concurrently with qualitative data about perceived barriers to pediatric procedural sedation and suggestions to improve the practice of pediatric sedation in their settings. All interviews were transcribed, coded, and analyzed for major themes. Results: Thirty-eight key informants participated, representing 19 countries and the specialties of Anesthesia, Surgery, Pediatrics, Critical Care, Emergency Medicine, and General Practice. The most common indication for pediatric sedation was imaging (42%), the most common medication used was ketamine (92%), and hypoxia was the most common adverse event (61%). Despite 92% of key informants stating that pediatric procedural sedation was critical to their practice, only half reported feeling adequately trained. The three major qualitative themes regarding barriers to safe pediatric sedation in their settings were: lack of resources, lack of education, and lack of standardization across sites and providers. Conclusions: The results of this study suggest that training specialized pediatric sedation teams, creating portable "pediatric sedation kits," and producing locally relevant pediatric sedation guidelines may help reduce current barriers to the provision of safe pediatric sedation in resource-limited African settings.

Virtual Human-Delivered Interviews for Patients With Chronic Pain: Feasibility, Acceptability, and a Pilot Randomized Trial of Standard Medical, Psychosocial, and Educational Interviews.

OBJECTIVE: Seminal advances in virtual human (VH) technology have introduced highly interactive, computer-animated VH interviewers. Their utility for aiding in chronic pain care is unknown. We developed three interactive telehealth VH interviews-a standard pain-focused, a psychosocial risk factor, and a pain psychology and neuroscience educational interview. We then conducted a preliminary investigation of their feasibility, acceptability, and efficacy. We also experimentally compared a human and a computer-generated VH voice. METHODS: Patients ( N = 94, age = 22-78 years) with chronic musculoskeletal pain were randomly assigned to the standard ( n = 31), psychosocial ( n = 34), or educational ( n = 29) VH interview and one of the two VH voices. Acceptability ratings included patient satisfaction and expectations/evaluations of the VH interview. Outcomes assessed at baseline and about 1-month postinterview were pain intensity, interference, emotional distress, pain catastrophizing, and readiness for pain self-management. Linear mixed-effects models were used to test between- and within-condition effects. RESULTS: Acceptability ratings showed that satisfaction with the VH and telehealth format was generally high, with no condition differences. Study attrition was low ( n = 5). Intent-to-treat-analyses showed that, compared with the standard interview, the psychosocial interview yielded a significantly greater reduction in pain interference ( p = .049, d = 0.43) and a marginally greater reduction in pain intensity ( p = .054, d = 0.36), whereas the educational interview led to a marginally greater yet nonsignificant increase in readiness for change ( p = .095, d = 0.24), as well as several significant improvements within-condition. Results did not differ by VH voice. CONCLUSIONS: Interactive VH interviewers hold promise for improving chronic pain care, including probing for psychosocial risk factors and providing pain-related education.

Evolution of cisplatin resistance through coordinated metabolic reprogramming of the cellular reductive state.

BACKGROUND: Cisplatin (CDDP) is a mainstay treatment for advanced head and neck squamous cell carcinomas (HNSCC) despite a high frequency of innate and acquired resistance. We hypothesised that tumours acquire CDDP resistance through an enhanced reductive state dependent on metabolic rewiring. METHODS: To validate this model and understand how an adaptive metabolic programme might be imprinted, we performed an integrated analysis of CDDP-resistant HNSCC clones from multiple genomic backgrounds by whole-exome sequencing, RNA-seq, mass spectrometry, steady state and flux metabolomics. RESULTS: Inactivating KEAP1 mutations or reductions in KEAP1 RNA correlated with Nrf2 activation in CDDP-resistant cells, which functionally contributed to resistance. Proteomics identified elevation of downstream Nrf2 targets and the enrichment of enzymes involved in generation of biomass and reducing equivalents, metabolism of glucose, glutathione, NAD(P), and oxoacids. This was accompanied by biochemical and metabolic evidence of an enhanced reductive state dependent on coordinated glucose and glutamine catabolism, associated with reduced energy production and proliferation, despite normal mitochondrial structure and function. CONCLUSIONS: Our analysis identified coordinated metabolic changes associated with CDDP resistance that may provide new therapeutic avenues through targeting of these convergent pathways.

Functional profiling of the Toxoplasma genome during acute mouse infection.

Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR-based screens to functionally profile the entire genome of the model apicomplexan parasite Toxoplasma gondii during mouse infection. Barcoded gRNAs were used to track mutant parasite lineages, enabling detection of bottlenecks and mapping of population structures. We uncovered over 300 genes that modulate parasite fitness in mice with previously unknown roles in infection. These candidates span multiple axes of host-parasite interaction, including determinants of tropism, host organelle remodeling, and metabolic rewiring. We mechanistically characterized three novel candidates, including GTP cyclohydrolase I, against which a small-molecule inhibitor could be repurposed as an antiparasitic compound. This compound exhibited antiparasitic activity against T. gondii and Plasmodium falciparum, the most lethal agent of malaria. Taken together, we present the first complete survey of an apicomplexan genome during infection of an animal host, and point to novel interfaces of host-parasite interaction that may offer new avenues for treatment.

Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy.

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates PTEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

The retention and bioavailability of phytochemicals in the manufacturing of baked snacks.

There is a growing body of evidence supporting the role that phytochemicals play in reducing the risk of various chronic diseases. Although there has been a rise in health products marketed as being "supergrains," "superfood," or advertising their abundance in antioxidants, these food items are often limited to powdered blends, dried fruit, nuts, or seeds, rarely intercepting the market of baked snacks. This is in part due to the still limited understanding of the impact that different industrial processes have on phytochemicals in a complex food matrix and their corresponding bioavailability. This review brings together the current data on how various industrial dehydration processes influence the retention and bioaccessibility of phytochemicals in baked snacks. It considers the interplay of molecules in an intricate snack matrix, limitations of conventional technologies, and constraints with consumer acceptance preventing wider utilization of novel technologies. Furthermore, the review takes a holistic approach, encompassing each stage of production-discussing the potential for inclusion of by-products to promote a circular economy and the proposal for a shift in agriculture toward biofortification or tailored growing of crops for their nutritional and post-harvest attributes.

Pediatric procedural sedation in African clinical settings: A mixed methods study of African providers' sedation practices

Background: Little is known about the practice of pediatric procedural sedation in Africa, despite being incredibly useful to the emergency care of children. This study describes the clinical experiences of African medical providers who use pediatric proceduralsedation, including clinical indications, medications, adverse events, training, clinical guideline use, and comfort level. The goals of this study are to describe pediatric sedation practices in resource-limited settings in Africa and identify potential barriers to the provision of safe pediatric sedation. Methods: This mixed methods study describes the pediatric procedural sedation practices of African providers using semi-structured interviews. Purposive sampling was used to identify key informants working in African resource-limited settings across a broad geographic, economic, and professional range. Quantitative data about provider background and sedation practices were collected concurrently with qualitative data about perceived barriers to pediatric procedural sedation and suggestions to improve the practice of pediatric sedation in their settings. All interviews were transcribed, coded, and analyzed for major themes. Results: Thirty-eight key informants participated, representing 19 countries and the specialties of Anesthesia, Surgery, Pediatrics, Critical Care, Emergency Medicine, and General Practice. The most common indication for pediatric sedation was imaging (42%), the most common medication used was ketamine (92%), and hypoxia was the most common adverse event (61%). Despite 92% of key informants stating that pediatric procedural sedation was critical to their practice, only half reported feeling adequately trained. The three major qualitative themes regarding barriers to safe pediatric sedation in their settings were: lack of resources, lack of education, and lack of standardization across sites and providers. Conclusions: The results of this study suggest that training specialized pediatric sedation teams, creating portable "pediatric sedation kits," and producing locally relevant pediatric sedation guidelines may help reduce current barriers to the provision of safe pediatric sedation in resource-limited African settings.