Sterol 14-alpha demethylase (CYP51) activity in Leishmania donovani is likely dependent upon cytochrome P450 reductase 1.

Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of Leishmania donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.

Single nucleus DNA sequencing of flow sorted archived frozen and formalin fixed paraffin embedded solid tumors.

BACKGROUND: Archived samples, including frozen and formalin fixed paraffin embedded (FFPE) tissues, are a vast resource of clinically annotated materials for the application of high-definition genomics to improve patient management and provide a molecular basis for the delivery of personalized cancer therapeutics. Notably, FFPE tissues are stable, provide repeat sampling of tissues of interest, and can be stored indefinitely at ambient temperature. The development of single cell DNA sequencing (scDNA-seq) technologies provides an unparalleled opportunity for the study of tumor heterogeneity and the identification of often rare subclonal cell populations that drive tumor evolution and progression to advanced therapy resistant disease. However, major limitations to the use of archived tissues for scDNA-seq include the low yields of intact cells in the presence of high levels of subcellular debris in biopsies, and the highly variable quantity and quality of the DNA extracted from samples of interest. The latter is of high significance for the use of FFPE tissues due to the presence of DNA-protein crosslinks. In addition, many samples, notably tumors arising in solid tissues, contain admixtures of reactive stroma, inflammatory cells, and necrosis in immediate contact with tumor cells. RESULTS: To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived fresh frozen (FF) and FFPE tumor tissues. Our methods, which include isolation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching FF and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest. CONCLUSIONS: Our flow sorting based protocol provides intact nuclei suitable for snDNA-seq from archival FF and FFPE tissues. Furthermore, we have developed QC steps that optimize the preparation and selection of samples for deep single cell clonal profiling. Our data processing pipeline captures rare subclones in tumors with highly variable genomes based on variants in genes of interest.

Characterization of Morreton virus as an oncolytic virotherapy platform for liver cancers.

BACKGROUND: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). AIM: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. APPROACH AND RESULTS: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells. CONCLUSIONS: Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

Fever education for caregivers in the emergency room (The FEVER study)-an interventional trial.

BACKGROUND: Despite the vast majority of fevers representing benign self-limiting illnesses, caregiver anxiety regarding fever is high. Empowering caregivers with knowledge to safely and appropriately manage fever at home has the potential to reduce demands upon healthcare services. AIM: To improve caregiver knowledge about fever and its management in children via an educational intervention. METHODS: Caregivers of children over 6 months presenting with fever to a Paediatric Emergency Department were recruited. A pre-intervention survey was completed to ascertain caregiver knowledge about fever and its management. The intervention of (i) an infographic about fever, with (ii) a short video on fever was viewed. A post-intervention survey re-assessed knowledge. The primary outcome was the correct definition of fever as a temperature ≥38 °C. RESULTS: Caregivers (n = 51) who correctly defined fever increased from 41% (n = 21) pre-intervention to 94% (n = 48) post-intervention. There was a reduction in common misconceptions about fever, including a higher fever representing a more serious infection (76% vs. 8%). Caregivers reported they were less likely to seek emergency healthcare due to the height and nature of the fever alone. CONCLUSIONS: A simple brief educational intervention can rapidly increase caregiver knowledge about fever in children. There is a continuing need for clear, easily-accessible information for caregivers on this topic. IMPACT: Parental knowledge about fever and how to manage it in their children is low. A simple brief educational intervention can significantly increase caregiver knowledge about fever. A combined written and audiovisual approach is effective and well-received by parents. Educating caregivers has the potential to improve the management of childhood fever at home and to reduce the burden on healthcare services, as well as reduce unpleasant hospital visits for children and their caregivers.

Black and white: how good are clinicians at diagnosing elbow injuries from paediatric elbow radiographs alone?

OBJECTIVES: Paediatric trauma elbow radiographs are difficult to interpret and there is a potential for harm if misdiagnosed. The primary goal of this study was to assess the ability of healthcare professionals internationally to interpret paediatric trauma elbow radiographs from the radiograph alone by formulating the correct diagnosis. METHODS: This prospective international study was conducted online via the Free Open Access Medical Education platform, Don't Forget the Bubbles (DFTB, ISSN 2754-5407). Participants were recruited via the DFTB social media accounts between 17 August and 14 September 2021. Submissions that were incomplete or from participants who do not interpret paediatric elbow radiographs in their clinical practice were excluded. Participants completed an online survey of demographic data followed by interpreting 10 trauma-indicated elbow radiographs, by selecting multiple-choice options. The primary outcome was correct diagnosis. RESULTS: Participant responses from 18 countries were analysed, with most responses from the UK, Australia and Ireland. Participants had backgrounds in emergency medicine (EM), paediatric emergency medicine (PEM), general practice (GP) and paediatrics, with over 70% having 6+ years of postgraduate experience. 3180 radiographs were interpreted by 318 healthcare professionals. Only nine (2.8%) participants correctly diagnosed all 10. The mean number of radiographs correctly interpreted was 5.44 (SD 2.3). The mean number for those with 6+ years of experience was 6.02 (SD 2.2). On reviewing the normal radiograph, 158 (49.7%) overcalled injuries. Participants with EM or PEM background were equally likely to have more correct answers than those from paediatric or GP backgrounds. CONCLUSION: Globally, healthcare professional's success in correctly diagnosing paediatric elbow injuries from radiographs was suboptimal in this non-clinical exercise, despite capturing quite an experienced cohort of clinicians. This study has provided us with detailed baseline data to accurately assess the impact of interventions aimed at improving clinicians' interpretation of paediatric elbow radiographs in future studies.

Safety and efficacy of a nitrous oxide procedural sedation programme in a paediatric emergency department: a decade of outcomes.

BACKGROUND: Nitrous oxide (N2O) has multiple benefits in paediatric procedural sedation (PPS), but use is restricted by its limited analgesic properties. Analgesic potency could be increased by combining N2O and intranasal fentanyl (INF). We assessed safety and efficacy data from 10 years (2011-2021) of our N2O PPS programme. METHODS: Prospectively collected data from a sedation registry at a paediatric emergency department (PED) were reviewed. Total procedures performed with N2O alone or with INF, success rate, sedation depth and adverse events were determined. Contributing factors for these outcomes were assessed via regression analysis and compared between different N2O concentrations, N2O in combination with INF, and for physician versus nurse administered sedation. A post hoc analysis on factors associated with vomiting was also performed. RESULTS: 831 N2O procedural sedations were performed, 358 (43.1%) involved a combination INF and N2O. Nurses managed sedation in 728 (87.6%) cases. Median sedation depth on the University of Michigan Sedation Scale was 1 (IQR 1-2). Sedation was successful in 809 (97.4%) cases. Combination INF/N2O demonstrated higher median sedation scores (2 vs 1, p<0.001) and increased vomiting (RR 1.8, 95% CI 1.3 to 2.5), with no difference in sedation success compared with N2O alone. No serious adverse events (SAEs) were reported (desaturation, apnoea, aspiration, bradycardia or hypotension) regardless of N2O concentration or use of INF. 137 (16.5%) minor adverse events occurred. Vomiting occurred in 113 (13.6%) cases and was associated with higher concentrations of N2O and INF use, but not associated with fasting status. There were no differences in adverse events (RR 0.98, 95% CI 0.97 to 1.04) or success rates (RR 0.93, 95% CI 0.56 to 1.7) between physician provided and nurse provided sedation. CONCLUSION: N2O can provide effective PED PPS. No SAEs were recorded. INF may be an effective PPS adjunct but remains limited by increased rates of vomiting.

Autoimmune conditions and pancreatic cancer risk in older American adults.

Pancreatic cancer (PC) is highly fatal, and its incidence is increasing in the United States. Population-based registry studies suggest associations between a few autoimmune conditions and PC risk, albeit based on a relatively small number of cases. We conducted a population-based, nested case-control study to examine the associations between autoimmune conditions and PC risk within the Surveillance, Epidemiology, and End Results Program (SEER)-Medicare population. Incident primary malignant PC cases (n = 80 074) were adults ≥66 years and diagnosed between 1992 and 2015. Controls (n = 320 296) were alive at the time cases were diagnosed and frequency-matched to cases (4:1 ratio) by age, sex, and year of diagnosis. We used multivariable-adjusted, unconditional logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for 45 autoimmune conditions identified from Medicare claims. Eight autoimmune conditions including ankylosing spondylitis (OR = 1.45; 95% CI: 1.14-1.84), Graves' disease (OR = 1.18; 95% CI: 1.03-1.34), localized scleroderma (OR = 1.27; 95% CI: 1.06-1.52), pernicious anemia (OR = 1.08; 95% CI: 1.02-1.14), primary sclerosing cholangitis (OR = 1.37; 95% CI: 1.18-1.59), pure red cell aplasia (OR = 1.31; 95% CI: 1.16-1.47), type 1 diabetes (OR = 1.11; 95% CI: 1.07-1.15), and ulcerative colitis (OR = 1.18; 95% CI: 1.07-1.31) were associated with increased PC risk (false discovery rate-adjusted P values <.10). In subtype analyses, these conditions were associated with pancreatic ductal adenocarcinoma, whereas only ulcerative colitis was associated with pancreatic neuroendocrine tumors. Our results support the hypothesis that autoimmune conditions may play a role in PC development.

Point-of-Care Platform for Diagnosis of Venous Thrombosis by Simultaneous Detection of Thrombin Generation and D-Dimer in Human Plasma.

Venous thromboembolism (VTE) refers to a blood clot that starts in a vein. The risk of developing VTE is highest after major surgery or a major injury, or when someone has heart failure, cancer, or infectious disease (e.g., COVID-19). Without prompt treatment to break up clots and prevent more from forming, VTE can restrict or block blood flow and oxygen, which can damage the body tissue or organs. VTE can occur without any obvious signs, and imaging technologies are used. Alternatively rapid measurement of thrombin generation (TG) and D-dimer could be used to make a fast, portable, and easy-to-use diagnostic platform for VTE. Here, we have demonstrated a diagnostic sensing platform with the ability of simultaneous detection of TG and D-dimer in human plasma. Modifications were made to both the assay protocols to eliminate the need for sample dilution and incubation steps. Using a substantially reduced sample volume, the measurement results show comparable performance to the gold standard method. Our platform is able to deliver accurate and cost-effective results for both TG and D-dimer assays when using undiluted plasma in under 15 min. The assays presented are therefore a good candidate technology for use in a point-of-care platform to diagnose VTE.

Respective roles of non-pharmaceutical interventions in bronchiolitis outbreaks: an interrupted time-series analysis based on a multinational surveillance system.

BACKGROUND: Bronchiolitis is a major source of morbimortality among young children worldwide. Non-pharmaceutical interventions (NPIs) implemented to reduce the spread of severe acute respiratory syndrome coronavirus 2 may have had an important impact on bronchiolitis outbreaks, as well as major societal consequences. Discriminating between their respective impacts would help define optimal public health strategies against bronchiolitis. We aimed to assess the respective impact of each NPI on bronchiolitis outbreaks in 14 European countries. METHODS: We conducted a quasi-experimental interrupted time-series analysis based on a multicentre international study. All children diagnosed with bronchiolitis presenting to the paediatric emergency department of one of 27 centres from January 2018 to March 2021 were included. We assessed the association between each NPI and change in the bronchiolitis trend over time by seasonally adjusted multivariable quasi-Poisson regression modelling. RESULTS: In total, 42 916 children were included. We observed an overall cumulative 78% (95% CI -100- -54%; p<0.0001) reduction in bronchiolitis cases following NPI implementation. The decrease varied between countries from -97% (95% CI -100- -47%; p=0.0005) to -36% (95% CI -79-7%; p=0.105). Full lockdown (incidence rate ratio (IRR) 0.21 (95% CI 0.14-0.30); p<0.001), secondary school closure (IRR 0.33 (95% CI 0.20-0.52); p<0.0001), wearing a mask indoors (IRR 0.49 (95% CI 0.25-0.94); p=0.034) and teleworking (IRR 0.55 (95% CI 0.31-0.97); p=0.038) were independently associated with reducing bronchiolitis. CONCLUSIONS: Several NPIs were associated with a reduction of bronchiolitis outbreaks, including full lockdown, school closure, teleworking and facial masking. Some of these public health interventions may be considered to further reduce the global burden of bronchiolitis.

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline.

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.

Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential sensitivity to metabolic inhibition.

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

Synergistic combination of cytotoxic chemotherapy and cyclin-dependent kinase 4/6 inhibitors in biliary tract cancers.

BACKGROUND AND AIMS: Biliary tract cancers (BTCs) are uncommon, but highly lethal, gastrointestinal malignancies. Gemcitabine/cisplatin is a standard-of-care systemic therapy, but has a modest impact on survival and harbors toxicities, including myelosuppression, nephropathy, neuropathy, and ototoxicity. Whereas BTCs are characterized by aberrations activating the cyclinD1/cyclin-dependent kinase (CDK)4/6/CDK inhibitor 2a/retinoblastoma pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident preclinical efficacy, and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the antitumor efficacy of CDK4/6 inhibitors by the combination with chemotherapy regimens, but their mechanism of action remains elusive. APPROACH AND RESULTS: Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity, and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy attributable to autophagy-induced resistance. Notably, triplet therapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated ribonucleotide reductase catalytic subunit M1 reduction, resulting in sensitization to gemcitabine. CONCLUSIONS: As such, these data provide robust preclinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.

Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei.

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.

Presentations of children to emergency departments across Europe and the COVID-19 pandemic: A multinational observational study.

BACKGROUND: During the initial phase of the Coronavirus Disease 2019 (COVID-19) pandemic, reduced numbers of acutely ill or injured children presented to emergency departments (EDs). Concerns were raised about the potential for delayed and more severe presentations and an increase in diagnoses such as diabetic ketoacidosis and mental health issues. This multinational observational study aimed to study the number of children presenting to EDs across Europe during the early COVID-19 pandemic and factors influencing this and to investigate changes in severity of illness and diagnoses. METHODS AND FINDINGS: Routine health data were extracted retrospectively from electronic patient records of children aged 18 years and under, presenting to 38 EDs in 16 European countries for the period January 2018 to May 2020, using predefined and standardized data domains. Observed and predicted numbers of ED attendances were calculated for the period February 2020 to May 2020. Poisson models and incidence rate ratios (IRRs), using predicted counts for each site as offset to adjust for case-mix differences, were used to compare age groups, diagnoses, and outcomes. Reductions in pediatric ED attendances, hospital admissions, and high triage urgencies were seen in all participating sites. ED attendances were relatively higher in countries with lower SARS-CoV-2 prevalence (IRR 2.26, 95% CI 1.90 to 2.70, p < 0.001) and in children aged <12 months (12 to <24 months IRR 0.86, 95% CI 0.84 to 0.89; 2 to <5 years IRR 0.80, 95% CI 0.78 to 0.82; 5 to <12 years IRR 0.68, 95% CI 0.67 to 0.70; 12 to 18 years IRR 0.72, 95% CI 0.70 to 0.74; versus age <12 months as reference group, p < 0.001). The lowering of pediatric intensive care admissions was not as great as that of general admissions (IRR 1.30, 95% CI 1.16 to 1.45, p < 0.001). Lower triage urgencies were reduced more than higher triage urgencies (urgent triage IRR 1.10, 95% CI 1.08 to 1.12; emergent and very urgent triage IRR 1.53, 95% CI 1.49 to 1.57; versus nonurgent triage category, p < 0.001). Reductions were highest and sustained throughout the study period for children with communicable infectious diseases. The main limitation was the retrospective nature of the study, using routine clinical data from a wide range of European hospitals and health systems. CONCLUSIONS: Reductions in ED attendances were seen across Europe during the first COVID-19 lockdown period. More severely ill children continued to attend hospital more frequently compared to those with minor injuries and illnesses, although absolute numbers fell. TRIAL REGISTRATION: ISRCTN91495258 https://www.isrctn.com/ISRCTN91495258.

Diminazene resistance in Trypanosoma congolense is not caused by reduced transport capacity but associated with reduced mitochondrial membrane potential.

Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross-resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogs DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3 H]-diminazene was slow with low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene-resistant Trypanosoma brucei brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3 H]-diminazene transport studies, whole-genome sequencing, and RNA-seq found no major changes in diminazene uptake, folate transporter sequence, or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential Ψm. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in Ψm it is unclear whether this is the primary cause of the resistance.

Genomic landscape of Epstein-Barr virus-positive extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue.

Epstein-Barr virus (EBV)-positive extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT lymphomas) were initially described in solid organ transplant recipients, and, more recently, in other immunodeficiency settings. The overall prevalence of EBV-positive MALT lymphomas has not been established, and little is known with respect to their genomic characteristics. Eight EBV-positive MALT lymphomas were identified, including 1 case found after screening a series of 88 consecutive MALT lymphomas with EBER in situ hybridization (1%). The genomic landscape was assessed in 7 of the 8 cases with a targeted high throughput sequencing panel and array comparative genomic hybridization. Results were compared to published data for MALT lymphomas. Of the 8 cases, 6 occurred post-transplant, 1 in the setting of primary immunodeficiency, and 1 case was age-related. Single pathogenic/likely pathogenic mutations were identified in 4 of 7 cases, including mutations in IRF8, BRAF, TNFAIP3, and SMARCA4. Other than TNFAIP3, these genes are mutated in <3% of EBV-negative MALT lymphomas. Copy number abnormalities were identified in 6 of 7 cases with a median of 6 gains and 2 losses per case, including 4 cases with gains in regions encompassing several IRF family or interacting genes (IRF2BP2, IRF2, and IRF4). There was no evidence of trisomies of chromosomes 3 or 18. In summary, EBV-positive MALT lymphomas are rare and, like other MALT lymphomas, are usually genetically non-complex. Conversely, while EBV-negative MALT lymphomas typically show mutational abnormalities in the NF-κB pathway, other than the 1 TNFAIP3-mutated case, no other NF-κB pathway mutations were identified in the EBV-positive cases. EBV-positive MALT lymphomas often have either mutations or copy number abnormalities in IRF family or interacting genes, suggesting that this pathway may play a role in these lymphomas.

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

FGFR2-IIIb Expression by Immunohistochemistry Has High Specificity in Cholangiocarcinoma with FGFR2 Genomic Alterations.

BACKGROUND: FGFR2 genomic alterations are observed in 10-20% of cholangiocarcinoma (CCA). Although FGFR2 fusions are an important actionable target, FGFR2 protein expression has not been thoroughly characterized. AIMS: To evaluate FGFR2 protein expression in cholangiocarcinoma harboring FGFR2 genomic alterations. METHODS: FGFR2 protein expression was evaluated in 99 CCA cases with two different antibodies. FGFR2 genomic alterations were confirmed via next-generating sequencing (NGS) or FISH. Primary objective was to determine the specificity and sensitivity of FGFR2 immunohistochemistry staining for detecting FGFR2 genomic alterations. Secondary objectives included overall FGFR2 immunohistochemistry staining in CCA patients, and evaluation of whether FGFR2 expression correlates with clinical outcomes including overall survival (OS), progression-free survival (PFS), and time-to-tumor recurrence (TTR). RESULTS: Immunohistochemistry staining with two antibodies against FGFR2, FPR2-D, and clone 98706 showed high accuracy (78.7% and 91.9%) and specificity (82.9% and 97.7%), and moderate sensitivity (53.9% and 57.1%), respectively, when compared with the standard methods for detecting FGFR2 genomic alterations. In a median follow-up of 72 months, there were no statistically significant differences in OS, PFS, and TTR, for patients with positive or negative FGFR2 staining. CONCLUSION: FGFR2 protein expression by immunohistochemistry has high specificity and therefore could be used to imply the presence of FGFR2 genomic alterations in the context of a positive test. In the case of a negative test, NGS or FISH would be necessary to ascertain cases with FGFR2 genomic alterations.

A Bibliometric Analysis of Global Pediatric Emergency Medicine Research Networks.

OBJECTIVES: During the last 3 decades newly formed pediatric emergency medicine (PEM) research networks have been publishing research. A desire of these networks is to produce and disseminate research to improve patient health and outcomes. The aims of the study were to quantitatively analyze and compare the literature by PEM research networks globally through numeric and visual bibliometrics. METHODS: A bibliometric analysis of articles published from 1994 to 2019 (26 years) by authors from PEM research networks globally were retrieved using PubMed, Web of Science (Thompson Reuters), and accessing individual research network databases. Bibliometric analysis was performed utilizing Web of Science, VOSviewer, and Dimensions. Research was quantified to ascertain the number of articles, related articles, citations, and Altmetric attention score. RESULTS: A total of 493 articles were published across 9 research networks in 3 decades. Pediatric Emergency Care Applied Research Network produced the most articles, citations, and h-index of all networks. We identified 3 main groupings of productive authors across the networks who collaborate globally. The sex of the first author was female in 46% of publications, and the corresponding author(s) was female in 45%. A nonsignificant moderate positive correlation between the number of years publishing and the number of publications was identified. There was nonsignificant moderate negative association between the number of countries in a network and total publications per annum. CONCLUSIONS: This study is the first bibliometric analysis of publications from PEM research networks that collaborate globally. Exploring the relationships of numerical bibliometric indicators and visualizations of productivity will benefit the understanding of the generation, reach, and dissemination of PEM research within the global research community.

Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes.

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.