Experimental demonstration of the mechanism of steady-state microbunching.

The use of particle accelerators as photon sources has enabled advances in science and technology1. Currently the workhorses of such sources are storage-ring-based synchrotron radiation facilities2-4 and linear-accelerator-based free-electron lasers5-14. Synchrotron radiation facilities deliver photons with high repetition rates but relatively low power, owing to their temporally incoherent nature. Free-electron lasers produce radiation with high peak brightness, but their repetition rate is limited by the driving sources. The steady-state microbunching15-22 (SSMB) mechanism has been proposed to generate high-repetition, high-power radiation at wavelengths ranging from the terahertz scale to the extreme ultraviolet. This is accomplished by using microbunching-enabled multiparticle coherent enhancement of the radiation in an electron storage ring on a steady-state turn-by-turn basis. A crucial step in unveiling the potential of SSMB as a future photon source is the demonstration of its mechanism in a real machine. Here we report an experimental demonstration of the SSMB mechanism. We show that electron bunches stored in a quasi-isochronous ring can yield sub-micrometre microbunching and coherent radiation, one complete revolution after energy modulation induced by a 1,064-nanometre-wavelength laser. Our results verify that the optical phases of electrons can be correlated turn by turn at a precision of sub-laser wavelengths. On the basis of this phase correlation, we expect that SSMB will be realized by applying a phase-locked laser that interacts with the electrons turn by turn. This demonstration represents a milestone towards the implementation of an SSMB-based high-repetition, high-power photon source.

A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis.

Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.

Cryptosporidium PI(4)K inhibitor EDI048 is a gut-restricted parasiticidal agent to treat paediatric enteric cryptosporidiosis.

Diarrhoeal disease caused by Cryptosporidium is a major cause of morbidity and mortality in young and malnourished children from low- and middle-income countries, with no vaccine or effective treatment. Here we describe the discovery of EDI048, a Cryptosporidium PI(4)K inhibitor, designed to be active at the infection site in the gastrointestinal tract and undergo rapid metabolism in the liver. By using mutational analysis and crystal structure, we show that EDI048 binds to highly conserved amino acid residues in the ATP-binding site. EDI048 is orally efficacious in an immunocompromised mouse model despite negligible circulating concentrations, thus demonstrating that gastrointestinal exposure is necessary and sufficient for efficacy. In neonatal calves, a clinical model of cryptosporidiosis, EDI048 treatment resulted in rapid resolution of diarrhoea and significant reduction in faecal oocyst shedding. Safety and pharmacological studies demonstrated predictable metabolism and low systemic exposure of EDI048, providing a substantial safety margin required for a paediatric indication. EDI048 is a promising clinical candidate for the treatment of life-threatening paediatric cryptosporidiosis.

Ligand-bound structures of the dengue virus protease reveal the active conformation.

Dengue is a mosquito-borne viral hemorrhagic disease that is a major threat to human health in tropical and subtropical regions. Here we report crystal structures of a peptide covalently bound to dengue virus serotype 3 (DENV-3) protease as well as the serine-protease inhibitor aprotinin bound to the same enzyme. These structures reveal, for the first time, a catalytically active, closed conformation of the DENV protease. In the presence of the peptide, the DENV-3 protease forms the closed conformation in which the hydrophilic ß-hairpin region of NS2B wraps around the NS3 protease core, in a manner analogous to the structure of West Nile virus (WNV) protease. Our results confirm that flavivirus proteases form the closed conformation during proteolysis, as previously proposed for WNV. The current DENV-3 protease structures reveal the detailed interactions at the P4' to P3 sites of the substrate. The new structural information explains the sequence preference, particularly for long basic residues in the nonprime side, as well as the difference in substrate specificity between the WNV and DENV proteases at the prime side. Structural analysis of the DENV-3 protease-peptide complex revealed a pocket that is formed by residues from NS2B and NS3; this pocket also exists in the WNV NS2B/NS3 protease structure and could be targeted for potential antivirus development. The structural information presented in the current study is invaluable for the design of specific inhibitors of DENV protease.

Opioid Use Disorder Curricular Content in US-Based Doctor of Pharmacy Programs.

OBJECTIVES: To characterize the instructional settings, delivery methods, and assessment methods of opioid use disorder (OUD) content in Doctor of Pharmacy (PharmD) programs; assess faculty perceptions of OUD content; and assess faculty perceptions of a shared OUD curriculum. METHODS: This national, cross-sectional, descriptive survey study was designed to characterize OUD content, faculty perceptions, and faculty and institutional demographics. A contact list was developed for accredited, US-based PharmD programs with publicly-accessible online faculty directories (n = 137). Recruitment and telephone survey administration occurred between August and December 2021. Descriptive statistics were computed for all items. Open-ended items were reviewed to identify common themes. RESULTS: A faculty member from 67 (48.9%) of 137 institutions contacted completed the survey. All programs incorporated OUD content into required coursework. Didactic lectures were the most common delivery method (98.5%). Programs delivered a median of 7.0 h (range, 1.5-33.0) of OUD content in required coursework, with 85.1% achieving the 4-hour minimum for substance use disorder-related content recommended by the American Association of Colleges of Pharmacy. Just over half (56.8%) of faculty agreed or strongly agreed that their students were adequately prepared to provide opioid interventions; however, 50.0% or fewer perceived topics such as prescription interventions, screening and assessment interventions, resource referral interventions, and stigma to be covered adequately. Almost all (97.0%) indicated moderate, high, or extremely high interest in a shared OUD curriculum. CONCLUSION: Enhanced OUD education is needed in PharmD programs. A shared OUD curriculum was of interest to faculty and should be explored as a potentially viable solution for addressing this need.

Small molecule inhibitors that selectively block dengue virus methyltransferase.

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome.

Steady-state microbunching in a storage ring for generating coherent radiation.

Synchrotrons and storage rings deliver radiation across the electromagnetic spectrum at high repetition rates, and free electron lasers produce radiation pulses with high peak brightness. However, at present few light sources can generate both high repetition rates and high brightness outside the optical range. We propose to create steady-state microbunching (SSMB) in a storage ring to produce coherent radiation at a high repetition rate or in continuous wave mode. In this Letter we describe a general mechanism for producing SSMB and give sample parameters for extreme ultraviolet lithography and submillimeter sources. We also describe a similar arrangement to produce two pulses with variable spacing for pump-probe experiments. With technological advances, SSMB could reach the soft x-ray range (<10 nm).

Conversation with Chen-Ning Yang: reminiscence and reflection.

Chen-Ning Yang ( ) is the most distinguished Chinese theoretical physicist. In 1954, together with Robert Mills, he formulated the Yang-Mills Gauge Theory, which led to the development of the Standard Model, the leading framework for understanding particle physics. In 1956, Yang and Tsung-Dao Lee ( ) proposed the possibility of parity non-conservation in weak interaction, which won them the Nobel Prize in Physics in 1957. Besides these two major achievements, Yang made many other seminal contributions to particle physics, statistical physics and condensed matter physics. At the end of 2003, Yang returned to China from the US and established the Institute for Advanced Study at Tsinghua University in Beijing. NSR's Executive Editor-in-Chief Mu-ming Poo ( ), a neurobiologist, and Alexander Wu Chao ( ), an accelerator physicist at Stanford University, talked with Professor Yang on a variety of topics, ranging from his retrospective view on Yang-Mills theory, on his contemporary physicists, on tastes in scientific research, and on the current and future developments of Chinese science. The following is an excerpt from this conversation that took place on 21 March 2019 at Tsinghua University, Beijing.

Precision cancer monitoring using a novel, fully integrated, microfluidic array partitioning digital PCR platform.

A novel digital PCR (dPCR) platform combining off-the-shelf reagents, a micro-molded plastic microfluidic consumable with a fully integrated single dPCR instrument was developed to address the needs for routine clinical diagnostics. This new platform offers a simplified workflow that enables: rapid time-to-answer; low potential for cross contamination; minimal sample waste; all within a single integrated instrument. Here we showcase the capability of this fully integrated platform to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards. Next, we validated the platform with an established chronic myeloid leukemia (CML) fusion gene (BCR-ABL1) assay down to 0.01% mutant allele frequency to highlight the platform's utility for precision cancer monitoring. Thirdly, using a juvenile myelomonocytic leukemia (JMML) patient-specific assay we demonstrate the ability to precisely track an individual cancer patient's response to therapy and show the patient's achievement of complete molecular remission. These three applications highlight the flexibility and utility of this novel fully integrated dPCR platform that has the potential to transform personalized medicine for cancer recurrence monitoring.

Development of a Cytopathic Effect-Based Phenotypic Screening Assay against Cryptosporidium.

Cryptosporidiosis is a diarrheal disease predominantly caused by Cryptosporidium parvum ( Cp) and Cryptosporidium hominis ( Ch), apicomplexan parasites which infect the intestinal epithelial cells of their human hosts. The only approved drug for cryptosporidiosis is nitazoxanide, which shows limited efficacy in immunocompromised children, the most vulnerable patient population. Thus, new therapeutics and in vitro infection models are urgently needed to address the current unmet medical need. Toward this aim, we have developed novel cytopathic effect (CPE)-based Cp and Ch assays in human colonic tumor (HCT-8) cells and compared them to traditional imaging formats. Further model validation was achieved through screening a collection of FDA-approved drugs and confirming many previously known anti- Cryptosporidium hits as well as identifying a few novel candidates. Collectively, our data reveals this model to be a simple, functional, and homogeneous gain of signal format amenable to high throughput screening, opening new avenues for the discovery of novel anticryptosporidials.

Change in practice patterns of an academic division of vascular surgery.

HYPOTHESIS: Technological advances have required that faculty of academic divisions of vascular surgery acquire new technical skills and significantly alter their past clinical practice patterns. DESIGN: Retrospective medical record review. SETTING: An academic tertiary referral center and a community teaching hospital. PATIENTS: All patients undergoing 10 specific vascular procedures during a 5-year period. MAIN OUTCOME MEASURES: We analyzed volumes for 10 specific open and endovascular index procedures performed by 5 vascular surgeons during a 60-month period. Procedures reviewed included open abdominal aortic aneurysm repair, endovascular abdominal aortic aneurysm repair, carotid endarterectomy, carotid artery stent, suprainguinal arterial reconstruction, suprainguinal percutaneous transluminal angioplasty/stent (PTA/S), infrainguinal arterial reconstruction, infrainguinal PTA/S, renal and visceral arterial reconstruction, and renal and visceral PTA/S. In-hospital length of stay was compared between open procedures and their endovascular counterparts. RESULTS: In 2000, 453 open and 44 endovascular index procedures were performed. In contrast, by 2005, open index cases had decreased by 47.0% (239) and endovascular index cases had increased by 679.5% (299). Open abdominal aortic aneurysm repairs had decreased by 54.5% (68 vs 31), carotid endarterectomies by 28.8% (139 vs 99), suprainguinal arterial reconstructions by 47.5% (40 vs 21), infrainguinal arterial reconstructions by 56.5% (186 vs 81), and renal/visceral arterial reconstructions by 65.0% (20 vs 7). In 2005, 62 endovascular abdominal aortic aneurysm repairs and 45 carotid stents were performed, whereas none were performed in 2000. In addition, infrainguinal PTA/S had increased by 675.0% (12 vs 81) and suprainguinal PTA/S by 20.0% (20 vs 24). CONCLUSIONS: Although the total number of procedures performed has remained relatively constant, there has been a dramatic increase in the number of endovascular procedures as well as an associated decline in the number of open procedures. This change in practice pattern has allowed the members of our division to maintain a significant role in the care of patients undergoing vascular surgery, as evidenced by stable overall procedural volume. This will provide a platform for future outcome-related analyses of open vs endovascular procedures performed within a single specialty group.

Cryptosporidiosis Drug Discovery: Opportunities and Challenges.

The apicomplexan parasite Cryptosporidium is the second most important diarrheal pathogen causing life-threatening diarrhea in children, which is also associated with long-term growth faltering and cognitive deficiency. Cryptosporidiosis is a parasitic disease of public health concern caused by Cryptosporidium parvum and Cryptosporidium hominis. Currently, nitazoxanide is the only approved treatment for cryptosporidium infections. Unfortunately, it has limited efficacy in the most vulnerable patients, thus there is an urgent need for a safe and efficacious cryptosporidiosis drug. In this work, we present our current perspectives on the target product profile for novel cryptosporidiosis therapies and the perceived challenges and possible mitigation plans at different stages in the cryptosporidiosis drug discovery process.

Transcriptome Profiling of Patient-Specific Human iPSC-Cardiomyocytes Predicts Individual Drug Safety and Efficacy Responses In Vitro.

Understanding individual susceptibility to drug-induced cardiotoxicity is key to improving patient safety and preventing drug attrition. Human induced pluripotent stem cells (hiPSCs) enable the study of pharmacological and toxicological responses in patient-specific cardiomyocytes (CMs) and may serve as preclinical platforms for precision medicine. Transcriptome profiling in hiPSC-CMs from seven individuals lacking known cardiovascular disease-associated mutations and in three isogenic human heart tissue and hiPSC-CM pairs showed greater inter-patient variation than intra-patient variation, verifying that reprogramming and differentiation preserve patient-specific gene expression, particularly in metabolic and stress-response genes. Transcriptome-based toxicology analysis predicted and risk-stratified patient-specific susceptibility to cardiotoxicity, and functional assays in hiPSC-CMs using tacrolimus and rosiglitazone, drugs targeting pathways predicted to produce cardiotoxicity, validated inter-patient differential responses. CRISPR/Cas9-mediated pathway correction prevented drug-induced cardiotoxicity. Our data suggest that hiPSC-CMs can be used in vitro to predict and validate patient-specific drug safety and efficacy, potentially enabling future clinical approaches to precision medicine.

Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening.

Dengue virus (DENV) NS5 protein comprises an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain (RdRp). DENV RdRp is responsible for viral RNA synthesis via a de novo initiation mechanism and represents an attractive target for anti-viral therapy. Herein we describe the characterization of its de novo initiation activities by PAGE analyses and the knowledge gained was used to develop a fluorescent-based assay. A highly processive and robust assay was achieved by addition of cysteine in the assay buffer. This stabilized the apo-enzyme, and rendered optimal de novo initiation activity while balancing its intrinsic terminal transferase activity. Steady-state kinetic parameters of the NTP and RNA substrates under these optimal conditions were determined for DENV1-4 FL NS5. Heavy metal ions such as Zn(++) and Co(++) as well as high levels of monovalent salts, suppressed DENV polymerase de novo initiation activities. This assay was validated with nucleotide chain terminators and used to screen two diverse small library sets. The screen data obtained was further compared with concurrent screens performed with a DENV polymerase elongation fluorescent assay utilizing pre-complexed enzyme-RNA. A higher hit-rate was obtained for the de novo initiation assay compared to the elongation assay (∼2% versus ∼0.1%). All the hits from the latter assay are also identified in the de novo initiation assay, indicating that the de novo initiation assay performed with the stabilized apo-enzyme has the advantage of providing additional chemical starting entities for inhibiting this enzyme.

Time to failure of arterial shunts in a pig hemorrhagic shock model.

Temporary vascular shunts (TVSs) are relied on frequently in the current military theater. Details of their physiology and outcome are of increasing interest and needed to further define their place in the management of trauma. This study was conducted using a porcine hemorrhagic shock model and aimed to 1) establish the time to failure of short, proximally placed vascular shunts; and 2) examine histological changes in the shunted artery. The external iliac artery of male pigs was transected and a straight shunt secured in place. Hemorrhagic shock was then induced by rapidly removing 40 per cent of blood volume and maintaining severe hypotension for 40 minutes before resuscitation. Animals were observed for up to 6 days for limb function as well as overall physiological status. At the conclusion of the experiment, status of the shunt and adjacent artery was evaluated by an independent pathologist. The presence of thrombi and inflammation within the proximal and distal artery was graded. Among the 10 animals tested, nine subjects survived the initial surgery. All shunts remained patent with mean time of 4.2±1.2 days of observation. Three-position histological analysis of the connected arterial tissues revealed minimal acute inflammation and minimal or thrombus. The results suggest that TVSs in proximal arteries remain functional for at least 48 to 72 hours in proximal arteries even without anticoagulation in the setting of brief hemorrhagic shock. This knowledge may aid combat evacuation patterns.

Higher catalytic efficiency of N-7-methylation is responsible for processive N-7 and 2'-O methyltransferase activity in dengue virus.

Methyltransferases (MTases) from the genus Flavivirus encode both N-7 and 2'-O activities needed for type 1 (m(7)GpppNm) cap structure formation. We performed kinetic studies to understand the mechanisms of its progressive N-7 and 2'-O methylations. Sequential N-7 to 2'-O methylation occurred via a random bi bi and processive mechanism that does not involve enzyme-RNA dissociation. Analyses of steady state kinetic parameters showed that N-7 precedes 2'-O methylation as it turnovers RNA faster (k(cat)) resulting in 2.4-fold higher catalytic efficiency. Michaelis constants for S-adenosyl-methionine (AdoMet) in both reactions were about 10-fold lower than for their respective RNA substrates, suggesting that the rate-limiting steps in methylase reactions were associated with RNA templates. In the context of long viral RNA sequences, and compared to S-adenosyl-homocysteine, sinefungin was about 60- and 12-folds more potent against dengue N-7 and 2'-O MTase activity, exhibiting IC(50) values of 30 and 41nM, respectively.

Preliminary retrograde visceral artery reconstruction for thoracoabdominal aortic aneurysms.

The recent availability of thoracic endografts has expanded the options for treatment of thoracoabdominal aortic pathology. However, disease that involves the visceral aortic segment presents a special challenge due to the need to preserve mesenteric perfusion. We present three patients in whom preliminary retrograde visceral artery reconstruction was used as an adjunct prior to endovascular repair.

Traumatic injuries to the portal vein: case study.

BACKGROUND: Injuries to the portal vein are rare but devastating. Contemporary studies have debated the most effective management for this injury. The purpose of this case study was to provide an update on portal vein injury and add information regarding its management. METHODS: A retrospective review investigated the 10-year experience with portal vein injury in a level 1 trauma center. RESULTS: Of the 18,900 trauma patients (0.08%) evaluated during a 10-year period, 15 sustained injuries to the portal vein. All the injuries resulted from penetrating trauma, and the overall survival rate was 60% (9 of the 15 patients). Four patients died of exsanguination and two patients died later as a result of multisystem organ failure. Postoperative complications were common. Sepsis and wound infection were the most common postoperative complications, occurring in seven (78%) of the nine survivors. All the patients had associated nonvascular injuries, whereas 9 (60%) of the 15 had associated vascular injuries. Associated injuries to the other structures in the portal triad occurred in 7 (47%) of the 15 patients, and 5 (71%) of these patients survived. Survival rates by procedure were 86% for venorrhaphy and 67% for ligation. CONCLUSIONS: Injuries to the portal vein are rare. In this study, exsanguination was the main cause of death. The key to a favorable outcome is prompt control of hemorrhage with an early decision to proceed with either venorrhaphy or ligation. Ligation can be effective for the management of hemodynamically unstable patients.