Acute response to pathogens in the early human placenta at single-cell resolution.

The placenta is a selective maternal-fetal barrier that provides nourishment and protection from infections. However, certain pathogens can attach to and even cross the placenta, causing pregnancy complications with potential lifelong impacts on the child's health. Here, we profiled at the single-cell level the placental responses to three pathogens associated with intrauterine complications-Plasmodium falciparum, Listeria monocytogenes, and Toxoplasma gondii. We found that upon exposure to the pathogens, all placental lineages trigger inflammatory responses that may compromise placental function. Additionally, we characterized the responses of fetal macrophages known as Hofbauer cells (HBCs) to each pathogen and propose that they are the probable niche for T. gondii. Finally, we revealed how P. falciparum adapts to the placental microenvironment by modulating protein export into the host erythrocyte and nutrient uptake pathways. Altogether, we have defined the cellular networks and signaling pathways mediating acute placental inflammatory responses that could contribute to pregnancy complications.

Quinoxaline-Based Anti-Schistosomal Compounds Have Potent Anti-Malarial Activity.

The human pathogens Plasmodium and Schistosoma are each responsible for over 200 million infections annually, being particularly problematic in low- and middle-income countries. There is a pressing need for new drug targets for these diseases, driven by emergence of drug-resistance in Plasmodium and the overall dearth of new drug targets for Schistosoma. Here, we explored the opportunity for pathogen-hopping by evaluating a series of quinoxaline-based anti-schistosomal compounds for activity against P. falciparum. We identified compounds with low nanomolar potency against 3D7 and multidrug-resistant strains. Evolution of resistance using a mutator P. falciparum line revealed a low propensity for resistance. Only one of the series, compound 22, yielded resistance mutations, including point mutations in a non-essential putative hydrolase pfqrp1, as well as copy-number amplification of a phospholipid-translocating ATPase, pfatp2, a potential target. Notably, independently generated CRISPR-edited mutants in pfqrp1 also showed resistance to compound 22 and a related analogue. Moreover, previous lines with pfatp2 copy-number variations were similarly less susceptible to challenge with the new compounds. Finally, we examined whether the predicted hydrolase activity of PfQRP1 underlies its mechanism of resistance, showing that both mutation of the putative catalytic triad and a more severe loss of function mutation elicited resistance. Collectively, we describe a compound series with potent activity against two important pathogens and their potential target in P. falciparum.

Technical Considerations in Laparoscopic-Assisted Endoscopic Retrograde Cholangiography.

The use of endoscopic retrograde cholangiography (ERCP) for diagnostic and therapeutic interventions on the pancreaticobiliary system has steadily increased, but the standard approach through the oropharynx is prohibited after Roux-en-Y (RYGB) gastric bypass surgery. Laparoscopic access to the gastric remnant allows for the completion of ERCP using the standard side-viewing duodenoscope to facilitate the completion of standard and advanced endoscopic maneuvers. Here, we describe our experience with the technical aspects of safe and effective performance of laparoscopic-assisted ERCP.

hERG, Plasmodium Life Cycle, and Cross Resistance Profiling of New Azabenzimidazole Analogues of Astemizole.

Toward addressing the cardiotoxicity liability associated with the antimalarial drug astemizole (AST, hERG IC50 = 0.0042 µM) and its derivatives, we designed and synthesized analogues based on compound 1 (Pf NF54 IC50 = 0.012 µM; hERG IC50 = 0.63 µM), our previously identified 3-trifluoromethyl-1,2,4-oxadiazole AST analogue. Compound 11 retained in vitro multistage antiplasmodium activity (ABS PfNF54 IC50 = 0.017 µM; gametocytes PfiGc/PfLGc IC50 = 1.24/1.39 µM, and liver-stage PbHepG2 IC50 = 2.30 µM), good microsomal metabolic stability (MLM CLint < 11 µL·min-1·mg-1, EH < 0.33), and solubility (150 µM). It shows a ∼6-fold and >6000-fold higher selectivity against human ether-á-go-go-related gene higher selectively potential over hERG relative to 1 and AST, respectively. Despite the excellent in vitro antiplasmodium activity profile, in vivo efficacy in the Plasmodium berghei mouse infection model was diminished, attributable to suboptimal oral bioavailability (F = 14.9%) at 10 mg·kg-1 resulting from poor permeability (log D7.4 = -0.82). No cross-resistance was observed against 44 common Pf mutant lines, suggesting activity via a novel mechanism of action.

Three-dimensional domain identification in a single hexagonal manganite nanocrystal.

The three-dimensional domain structure of ferroelectric materials significantly influences their properties. The ferroelectric domain structure of improper multiferroics, such as YMnO3, is driven by a non-ferroelectric order parameter, leading to unique hexagonal vortex patterns and topologically protected domain walls. Characterizing the three-dimensional structure of these domains and domain walls has been elusive, however, due to a lack of suitable imaging techniques. Here, we present a multi-peak Bragg coherent x-ray diffraction imaging determination of the domain structure in single YMnO3 nanocrystals. We resolve two ferroelectric domains separated by a domain wall and confirm that the primary atomic displacements occur along the crystallographic c-axis. Correlation with atomistic simulations confirms the Mexican hat symmetry model of domain formation, identifying two domains with opposite ferroelectric polarization and adjacent trimerization, manifesting in a clockwise arrangement around the hat's brim.

High-definition transcranial direct current stimulation desynchronizes refractory status epilepticus.

Recently, we showed that high-definition transcranial direct current stimulation (hd-tDCS) can acutely reduce epileptic spike rates during and after stimulation in refractory status epilepticus (RSE), with a greater likelihood of patient discharge from the intensive care unit compared to historical controls. We investigate whether electroencephalographic (EEG) desynchronization during hd-tDCS can help account for observed anti-epileptic effects. Defining desynchronization as greater power in higher frequencies such as above 30 â€‹Hz ("gamma") and lesser power in frequency bands lower than 30 â€‹Hz, we analyzed 27 EEG sessions from 10 RSE patients who had received 20-minute session(s) of 2-milliamperes of transcranial direct current custom-targeted at the epileptic focus as previously determined by a clinical EEGer monitoring the EEG in real-time. During hd-tDCS, median relative power change over the EEG electrode chains in which power changes were maximal was +4.84%, -5.25%, -1.88%, -1.94%, and +4.99% for respective delta, theta, alpha, beta, and gamma frequency bands in the bipolar longitudinal montage (p â€‹= â€‹0.0001); and +4.13%, -5.44%, -1.81%, -3.23%, and +5.41% in the referential Laplacian montage (p â€‹= â€‹0.0012). After hd-tDCS, median relative power changes reversed over the EEG electrode chains in which power changes were maximal: -2.74%, +4.20%, +1.74%, +1.75%, and -4.68% for the respective delta, theta, alpha, beta, and gamma frequency bands in the bipolar longitudinal montage (p â€‹= â€‹0.0001); and +1.59%, +5.07%, +1.74%, +2.40%, and -5.12% in the referential Laplacian montage (p â€‹= â€‹0.0004). These findings are consistent with EEG desynchronization through theta-alpha-beta-gamma bands during hd-tDCS, helping account for the efficacy of hd-tDCS as an emerging novel anti-epileptic therapy against RSE.

Schreckstoff: It takes two to panic.

Schreckstoff (fear substance) is an alarm signal released by injured fish that induces a fear response. Its chemical nature has long been debated. A new study finds that zebrafish Schreckstoff is composed of at least three components, two of which elicit the fear response only in combination.

Oncogenic GNAS drives a gastric pylorus program in intraductal papillary mucinous neoplasms of the pancreas.

OBJECTIVE: Intraductal Papillary Mucinous Neoplasms (IPMNs) are cystic lesions and bona fide precursors for pancreatic ductal adenocarcinoma (PDAC). Recently, we showed that acinar to ductal metaplasia, an injury repair program, is characterized by a transcriptomic program similar to gastric spasmolytic polypeptide expressing metaplasia (SPEM), suggesting common mechanisms of reprogramming between the stomach and pancreas. The aims of this study were to assay IPMN for pyloric markers and to identify molecular drivers of this program. DESIGN: We analyzed RNA-seq studies of IPMN for pyloric markers, which were validated by immunostaining in patient samples. Cell lines expressing Kras G12D +/- GNAS R201C were manipulated to identify distinct and overlapping transcriptomic programs driven by each oncogene. A PyScenic-based regulon analysis was performed to identify molecular drivers in the pancreas. Expression of candidate drivers was evaluated by RNA-seq and immunostaining. RESULTS: Pyloric markers were identified in human IPMN. GNAS R201C drove expression of these markers in cell lines and siRNA targeting of GNAS R201C or Kras G12D demonstrates that GNAS R201C amplifies a mucinous, pyloric phenotype. Regulon analysis identified a role for transcription factors SPDEF, CREB3L1, and CREB3L4, which are expressed in patient samples. siRNA-targeting of Spdef inhibited mucin production. CONCLUSION: De novo expression of a SPEM phenotype has been identified in pancreatitis and a pyloric phenotype in Kras G12D -driven PanIN and Kras G12D ;GNAS R201C -driven IPMN, suggesting common mechanisms of reprogramming between these lesions and the stomach. A transition from a SPEM to pyloric phenotype may reflect disease progression and/or oncogenic mutation. IPMN-specific GNAS R201C amplifies a mucinous phenotype, in part, through SPDEF.

Insect genomics: A lousy fly.

The bee louse (Braula coeca) is a honeybee inquiline and a long-standing taxonomic mystery. A new study unravels their genomic architecture and shows that these enigmatic flies have evolved from scale insect-exploiting drosophilid ancestors and share genetic similarities with their honeybee hosts.

The AAHKS Clinical Research Award: Extended Postoperative Oral Tranexamic Acid in Total Knee Arthroplasty: A Randomized Controlled Pilot Study.

BACKGROUND: Perioperative tranexamic acid (TXA) use with total knee arthroplasty (TKA) is widely accepted today. Recently, a few international groups have published on the safety and outcomes of extending TXA use in the postoperative period. Through a double-blinded, randomized control trial (RCT), we aimed to investigate the safety and clinical efficacy of extended postoperative oral TXA use in TKA performed in an American, free-standing ambulatory surgery center (ASC). METHODS: Based on a power analysis, 40 patients undergoing primary TKA were randomized into 2 groups: extended oral TXA versus placebo. Both groups received a standard 1g intravenous TXA dose prior to incision and at the time of closure. The extended TXA group received an additional 1.95 g oral TXA dose following ambulation the day of surgery, plus on postoperative days 1,2, and 3. Patients who had a history of venous thromboembolism (VTE) or cancer were excluded. All patients received 81 mg of aspirin twice daily for VTE prophylaxis. Patients were followed on postoperative day 3 and weeks 2 and 6. Paired t-tests determined statistical significance. RESULTS: Extended TXA patients showed significantly increased knee flexion at 6 weeks (116.05 versus 106.5, P = .0308), improved VAS at 2 (2.5 versus 3.85, P = .039) and 6 weeks (1.35 versus 2.8, P = .011), and superior KOOS JR at 2 (66.87 versus 60.63, P = .03) and 6 weeks (73.33 versus 62.47, P = .0019) compared to placebo patients. No significant differences were found for changes in hemoglobin levels at any time points. No significant differences were found at 12 weeks for any clinical endpoints. No adverse events were noted in either cohort. CONCLUSIONS: When compared to placebo, the extended use of oral TXA in the postoperative period may safely result in improved motion, pain, and functional scores. Further investigation into 1-to-2-year outcomes, as well as the duration and dose of postoperative TXA use is warranted.

Tranexamic acid for angiotensin converting enzyme inhibitor induced angioedema: A retrospective multicenter study.

BACKGROUND: Angiotensin converting enzyme inhibitors (ACE-Is) prevent the breakdown of bradykinin and can lead to life threatening angioedema. Tranexamic acid is an antifibrinolytic that inhibits formation of precursors involved in bradykinin synthesis and, in case reports, has been described as a potential treatment for ACE-I angioedema. METHODS: This retrospective study included patients who presented to the emergency department (ED) from January 2018 to August 2021 with angioedema while taking an ACE-I. Patients who received tranexamic acid (treatment group) were compared with patients who did not receive tranexamic acid (control group). Primary outcome was length of stay (LOS). Secondary outcomes evaluated included ICU admissions, intubations, and safety events. RESULTS: A total of 262 patients were included in this study (73 treatment; 189 control). Overall, the median ED LOS was longer in the treatment group than controls (20.9 h vs 4.8 h, p < 0.001). ICU admission rates were higher in the treatment group (45% vs 16%, p < 0.001). More patients were intubated in the treatment group (12% vs 3%, p = 0.018). No difference was seen between the treatment group and the controls for return within 7 days, complications related to thrombosis, and death. In patients presenting with severe angioedema symptoms who were admitted to the hospital, median LOS was not different between the two groups (58.7 h vs 55.7 h, p = 0.61). CONCLUSIONS: Patients who received tranexamic acid had increased ED LOS, rates of ICU admission, and need for intubation. This finding may be related to the severity of presentation. Administration of tranexamic acid appears safe to use in ACE-I angioedema. Prospective randomized controlled studies should be considered to determine whether tranexamic acid is an effective treatment for ACE-I angioedema.

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.

Networks of gut bacteria relate to cardiovascular disease in a multi-ethnic population: the HELIUS study.

AIMS: Gut microbiota have been linked to blood lipid levels and cardiovascular diseases (CVDs). The composition and abundance of gut microbiota trophic networks differ between ethnicities. We aim to evaluate the relationship between gut microbiotal trophic networks and CVD phenotypes. METHODS AND RESULTS: We included cross-sectional data from 3860 individuals without CVD history from 6 ethnicities living in the Amsterdam region participating in the prospective Healthy Life in Urban Setting (HELIUS) study. Genetic variants were genotyped, faecal gut microbiota were profiled, and blood and anthropometric parameters were measured. A machine learning approach was used to assess the relationship between CVD risk (Framingham score) and gut microbiota stratified by ethnicity. Potential causal relationships between gut microbiota composition and CVD were inferred by performing two-sample Mendelian randomization with hard CVD events from the Pan-UK Biobank and microbiome genome-wide association studies summary data from a subset of the HELIUS cohort (n = 4117). Microbial taxa identified to be associated with CVD by machine learning and Mendelian randomization were often ethnic-specific, but some concordance across ethnicities was found. The microbes Akkermansia muciniphila and Ruminococcaceae UCG-002 were protective against ischaemic heart disease in African-Surinamese and Moroccans, respectively. We identified a strong inverse association between blood lipids, CVD risk, and the combined abundance of the correlated microbes Christensenellaceae-Methanobrevibacter-Ruminococcaceae (CMR). The CMR cluster was also identified in two independent cohorts and the association with triglycerides was replicated. CONCLUSION: Certain gut microbes can have a potentially causal relationship with CVD events, with possible ethnic-specific effects. We identified a trophic network centred around Christensenellaceae, Methanobrevibacter, and various Ruminococcaceae, frequently lacking in South-Asian Surinamese, to be protective against CVD risk and associated with low triglyceride levels.

Oncogenic Fatty Acid Metabolism Rewires Energy Supply Chain in Gastric Carcinogenesis.

BACKGROUND & AIMS: Gastric carcinogenesis develops within a sequential carcinogenic cascade from precancerous metaplasia to dysplasia and adenocarcinoma, and oncogenic gene activation can drive the process. Metabolic reprogramming is considered a key mechanism for cancer cell growth and proliferation. However, how metabolic changes contribute to the progression of metaplasia to dysplasia remains unclear. We have examined metabolic dynamics during gastric carcinogenesis using a novel mouse model that induces Kras activation in zymogen-secreting chief cells. METHODS: We generated a Gif-rtTA;TetO-Cre;KrasG12D (GCK) mouse model that continuously induces active Kras expression in chief cells after doxycycline treatment. Histologic examination and imaging mass spectrometry were performed in the GCK mouse stomachs at 2 to 14 weeks after doxycycline treatment. Mouse and human gastric organoids were used for metabolic enzyme inhibitor treatment. The GCK mice were treated with a stearoyl- coenzyme A desaturase (SCD) inhibitor to inhibit the fatty acid desaturation. Tissue microarrays were used to assess the SCD expression in human gastrointestinal cancers. RESULTS: The GCK mice developed metaplasia and high-grade dysplasia within 4 months. Metabolic reprogramming from glycolysis to fatty acid metabolism occurred during metaplasia progression to dysplasia. Altered fatty acid desaturation through SCD produces a novel eicosenoic acid, which fuels dysplastic cell hyperproliferation and survival. The SCD inhibitor killed both mouse and human dysplastic organoids and selectively targeted dysplastic cells in vivo. SCD was up-regulated during carcinogenesis in human gastrointestinal cancers. CONCLUSIONS: Active Kras expression only in gastric chief cells drives the full spectrum of gastric carcinogenesis. Also, oncogenic metabolic rewiring is an essential adaptation for high-energy demand in dysplastic cells.

Lasso Peptides: Exploring the Folding Landscape of Nature's Smallest Interlocked Motifs.

Lasso peptides make up a class of natural products characterized by a threaded structure. Given their small size and stability, chemical synthesis would offer tremendous potential for the development of novel therapeutics. However, the accessibility of the pre-folded lasso architecture has limited this advance. To better understand the folding process de novo, simulations are used herein to characterize the folding propensity of microcin J25 (MccJ25), a lasso peptide known for its antimicrobial properties. New algorithms are developed to unambiguously distinguish threaded from nonthreaded precursors and determine handedness, a key feature in natural lasso peptides. We find that MccJ25 indeed forms right-handed pre-lassos, in contrast to past predictions but consistent with all natural lasso peptides. Additionally, the native pre-lasso structure is shown to be metastable prior to ring formation but to readily transition to entropically favored unfolded and nonthreaded structures, suggesting that de novo lasso folding is rare. However, by altering the ring forming residues and appending thiol and thioester functionalities, we are able to increase the stability of pre-lasso conformations. Furthermore, conditions leading to protonation of a histidine imidazole side chain further stabilize the modified pre-lasso ensemble. This work highlights the use of computational methods to characterize lasso folding and demonstrates that de novo access to lasso structures can be facilitated by optimizing sequence, unnatural modifications, and reaction conditions like pH.

Residual Seizure Rate of Intermittent Inpatient EEG Compared to a Continuous EEG Model.

BACKGROUND: Subclinical seizures are common in hospitalized patients and require electroencephalography (EEG) for detection and intervention. At our institution, continuous EEG (cEEG) is not available, but intermittent EEGs are subject to constant live interpretation. As part of quality improvement (QI), we sought to estimate the residual missed seizure rate at a typical quaternary Canadian health care center without cEEG. METHODS: We calculated residual risk percentages using the clinically validated 2HELPS2B score to risk-stratify EEGs before deriving a risk percentage using a MATLAB calculator which modeled the risk decay curve for each recording. We generated a range of estimated residual seizure rates depending on whether a pre-cEEG screening EEG was simulated, EEGs showing seizures were included, or repeat EEGs on the same patient were excluded. RESULTS: Over a 4-month QI period, 499 inpatient EEGs were scored as low (n = 125), medium (n = 123), and high (n = 251) seizure risk according to 2HELPS2B criteria. Median recording duration was 1:00:06 (interquartile range, IQR 30:40-2:21:10). The model with highest residual seizure rate included recordings with confirmed electrographic seizures (median 20.83%, IQR 20.6-26.6%), while the model with lowest residual seizure rate was in seizure-free recordings (median 10.59%, IQR 4%-20.6%). These rates were significantly higher than the benchmark 5% miss-rate threshold set by 2HELPS2B (p<0.0001). CONCLUSIONS: We estimate that intermittent inpatient EEG misses 2-4 times more subclinical seizures than the 2HELPS2B-determined acceptable 5% seizure miss-rate threshold for cEEG. Future research is needed to determine the impact of potentially missed seizures on clinical care.

A Hybrid Auricular Framework of Autologous Rib Cartilage and a Porous Polyethylene Implant for Reconstruction of Congenital Microtia: A Modification of Nagata's Technique.

Background: An implant (porous polyethylene) is an alternative to rib cartilage for microtia reconstruction but carries a risk of extrusion. Objective: To evaluate the outcome of a hybrid framework of implant with rib cartilage for microtia reconstruction. Methods: Patients who underwent Nagata's technique for microtia reconstruction were reviewed for complications and aesthetic score. In stage 1, a rib cartilage framework or a hybrid framework of implant with rib cartilage was used. In stage 2, the framework was elevated and supported by an implant for projection. Postoperative outcomes were reported for both groups. Results: Forty-four ears of 40 patients underwent surgery. Eleven ears received a rib auricular framework and 33 ears a hybrid auricular framework. The mean postoperative follow-up for the rib and hybrid groups was 76.3 and 43.1 months, respectively. No supporting postauricular implant extruded, whereas stainless-steel wires extruded in seven ears (15.9%). Five (15.2%) hybrid frameworks were removed due to infection or extrusion. Mean operating time was 2 h shorter in the hybrid group. Aesthetic outcomes were similar for both groups. Conclusion: A hybrid framework of rib and implant that requires less harvested cartilage is feasible for microtia reconstruction, but caution should be used due to its higher explantation rate.

Correlation of gene expression with magnetic resonance imaging features of retinoblastoma: a multi-center radiogenomics validation study.

OBJECTIVES: To validate associations between MRI features and gene expression profiles in retinoblastoma, thereby evaluating the repeatability of radiogenomics in retinoblastoma. METHODS: In this retrospective multicenter cohort study, retinoblastoma patients with gene expression data and MRI were included. MRI features (scored blinded for clinical data) and matched genome-wide gene expression data were used to perform radiogenomic analysis. Expression data from each center were first separately processed and analyzed. The end product normalized expression values from different sites were subsequently merged by their Z-score to permit cross-sites validation analysis. The MRI features were non-parametrically correlated with expression of photoreceptorness (radiogenomic analysis), a gene expression signature informing on disease progression. Outcomes were compared to outcomes in a previous described cohort. RESULTS: Thirty-six retinoblastoma patients were included, 15 were female (42%), and mean age was 24 (SD 18) months. Similar to the prior evaluation, this validation study showed that low photoreceptorness gene expression was associated with advanced stage imaging features. Validated imaging features associated with low photoreceptorness were multifocality, a tumor encompassing the entire retina or entire globe, and a diffuse growth pattern (all p < 0.05). There were a number of radiogenomic associations that were also not validated. CONCLUSIONS: A part of the radiogenomic associations could not be validated, underlining the importance of validation studies. Nevertheless, cross-center validation of imaging features associated with photoreceptorness gene expression highlighted the capability radiogenomics to non-invasively inform on molecular subtypes in retinoblastoma. CLINICAL RELEVANCE STATEMENT: Radiogenomics may serve as a surrogate for molecular subtyping based on histopathology material in an era of eye-sparing retinoblastoma treatment strategies. KEY POINTS: • Since retinoblastoma is increasingly treated using eye-sparing methods, MRI features informing on molecular subtypes that do not rely on histopathology material are important. • A part of the associations between retinoblastoma MRI features and gene expression profiles (radiogenomics) were validated. • Radiogenomics could be a non-invasive technique providing information on the molecular make-up of retinoblastoma.

The African Light Source: history, context and future.

The African Light Source (AfLS) project is now almost eight years old. This article assesses the history, current context and future of the project. There is by now considerable momentum in building the user community, including deep training, facilitating access to current facilities, growing the scientific output, scientific networks and growing the local laboratory-scale research infrastructure. The Conceptual Design Report for the AfLS is in its final editing stages. This document specifies the socio-economic and scientific rationales and the technical aspects amongst others. The AfLS is supported by many national and Pan-African scientific professional bodies and voluntary associates across many scientific disciplines, and there are stakeholders throughout the continent and beyond. The current roadmap phases have expanded to include national and Pan-African level conversations with policy makers through new Strategic Task Force groups. The document summarizes this progress and discusses the future of the project.