In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.

Focused ultrasound excites action potentials in mammalian peripheral neurons in part through the mechanically gated ion channel PIEZO2.

Neurons of the peripheral nervous system (PNS) are tasked with diverse roles, from encoding touch, pain, and itch to interoceptive control of inflammation and organ physiology. Thus, technologies that allow precise control of peripheral nerve activity have the potential to regulate a wide range of biological processes. Noninvasive modulation of neuronal activity is an important translational application of focused ultrasound (FUS). Recent studies have identified effective strategies to modulate brain circuits; however, reliable parameters to control the activity of the PNS are lacking. To develop robust noninvasive technologies for peripheral nerve modulation, we employed targeted FUS stimulation and electrophysiology in mouse ex vivo skin-saphenous nerve preparations to record the activity of individual mechanosensory neurons. Parameter space exploration showed that stimulating neuronal receptive fields with high-intensity, millisecond FUS pulses reliably and repeatedly evoked one-to-one action potentials in all peripheral neurons recorded. Interestingly, when neurons were classified based on neurophysiological properties, we identified a discrete range of FUS parameters capable of exciting all neuronal classes, including myelinated A fibers and unmyelinated C fibers. Peripheral neurons were excited by FUS stimulation targeted to either cutaneous receptive fields or peripheral nerves, a key finding that increases the therapeutic range of FUS-based peripheral neuromodulation. FUS elicited action potentials with millisecond latencies compared with electrical stimulation, suggesting ion channel­mediated mechanisms. Indeed, FUS thresholds were elevated in neurons lacking the mechanically gated channel PIEZO2. Together, these results demonstrate that transcutaneous FUS drives peripheral nerve activity by engaging intrinsic mechanotransduction mechanisms in neurons [B. U. Hoffman, PhD thesis, (2019)].

Physiologic Targets and Modes of Action for CBL0137, a Lead for Human African Trypanosomiasis Drug Development.

CBL0137 is a lead drug for human African trypanosomiasis, caused by Trypanosoma brucei Herein, we use a four-step strategy to 1) identify physiologic targets and 2) determine modes of molecular action of CBL0137 in the trypanosome. First, we identified fourteen CBL0137-binding proteins using affinity chromatography. Second, we developed hypotheses of molecular modes of action, using predicted functions of CBL0137-binding proteins as guides. Third, we documented effects of CBL0137 on molecular pathways in the trypanosome. Fourth, we identified physiologic targets of the drug by knocking down genes encoding CBL0137-binding proteins and comparing their molecular effects to those obtained when trypanosomes were treated with CBL0137. CBL0137-binding proteins included glycolysis enzymes (aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, phosphoglycerate kinase) and DNA-binding proteins [universal minicircle sequence binding protein 2, replication protein A1 (RPA1), replication protein A2 (RPA2)]. In chemical biology studies, CBL0137 did not reduce ATP level in the trypanosome, ruling out glycolysis enzymes as crucial targets for the drug. Thus, many CBL0137-binding proteins are not physiologic targets of the drug. CBL0137 inhibited 1) nucleus mitosis, 2) nuclear DNA replication, and 3) polypeptide synthesis as the first carbazole inhibitor of eukaryote translation. RNA interference (RNAi) against RPA1 inhibited both DNA synthesis and mitosis, whereas RPA2 knockdown inhibited mitosis, consistent with both proteins being physiologic targets of CBL0137. Principles used here to distinguish drug-binding proteins from physiologic targets of CBL0137 can be deployed with different drugs in other biologic systems. SIGNIFICANCE STATEMENT: To distinguish drug-binding proteins from physiologic targets in the African trypanosome, we devised and executed a multidisciplinary approach involving biochemical, genetic, cell, and chemical biology experiments. The strategy we employed can be used for drugs in other biological systems.

Use, Yield, and Risk of Device-assisted Enteroscopy in the United States: Results From a Large Retrospective Multicenter Cohort.

INTRODUCTION: Since 2001, device-assisted enteroscopy (DAE) has revolutionized the diagnostic and therapeutic capabilities for managing small bowel pathology. Though commonly performed, there have been no recent large studies to assess the use, yield, and risks of DAE and none that include all 3 DAE modalities. We hypothesized that DAE is safe with high diagnostic and therapeutic yields achieved within reasonable procedure duration and here we present a large retrospective multicenter US study evaluating the use, yield, and complications of DAE. METHODS: After obtaining institutional review board approval, electronic records were used to identify all DAE's performed for luminal small bowel evaluation in adult patients at 4 US referral centers (Duke University Medical Center, New York University Langone Medical Center, Louisiana State University Health Sciences Center, and University of Massachusetts Medical Center) from January 1, 2014 to January 1, 2019. Electronic medical records were reviewed to collect and analyze a variety of procedure-related outcomes. Using the data pooled across centers, descriptive statistics were generated for the patient and procedure-related characteristics and outcomes; relationships between characteristics and outcomes were explored. RESULTS: A total of 1787 DAE's were performed over this 5-year period (392 at Duke University Medical Center, 887 at Louisiana State University Health Sciences Center, 312 at New York University Langone Medical Center, and 195 at University of Massachusetts Medical Center). Of these, there were 1017 (57%) double-balloon, 391 (29%) single-balloon, and 378 (21%) spiral enteroscopies. The mean age of patients undergoing DAE was 66 years and 53% of examinations were performed on women; 18% of patients in the cohort underwent >1 DAE over this time span. A total of 53% of examinations were performed for suspected small bowel bleeding, 31% were directly guided by video capsule endoscopy findings and 8% were performed for abnormal imaging. A total of 85% of examinations used an antegrade approach and DAE took a mean of 45 minutes to complete; 76% of examinations revealed abnormal findings, with vascular, inflammatory, and neoplastic findings seen in 49%, 17%, and 15% of the cohort, respectively. Older age was significantly associated with any abnormal finding, including arteriovenous malformations (P<0.0001); 50% of examinations included a therapeutic maneuver, most commonly argon plasma coagulation/cautery (43%). There were complications in 16 examinations (0.9%) including 2 perforations (0.1%), 6 cases with bleeding (0.3%) and 1 episode of pancreatitis (0.1%). CONCLUSIONS: DAE is most commonly performed to evaluate suspected small bowel bleeding and is commonly directed by video capsule findings. DAE is safe, has a high diagnostic yield, with 76% of examinations showing abnormal findings, and frequently features therapeutic maneuvers. Advancing age is associated with abnormal findings on DAE.

Interobserver Variability in the Recognition of Hypoxic-Ischemic Brain Injury on Computed Tomography Soon After Out-of-Hospital Cardiac Arrest.

BACKGROUND: Cerebral edema and loss of gray-white matter differentiation on head computed tomography (CT) after cardiac arrest generally portend a poor prognosis. The interobserver variability in physician recognition of hypoxic-ischemic brain injury (HIBI) on early CT after out-of-hospital cardiac arrest has not been studied. METHODS: In this survey study, participating physicians and a neuroradiologist reviewed 20 randomly selected head CTs obtained within 2 h of out-of-hospital cardiac arrest and decided if HIBI was present. All participants were blinded to clinical details. Interobserver agreement on the presence of HIBI (primary outcome) and pairwise agreement between participants and the neuroradiologist (secondary outcome) were determined using multi- and dual-rater kappa statistics with 95% confidence intervals (CIs). RESULTS: Agreement among physicians regarding the presence of HIBI on head CT was fair (kappa 0.34; 95% CI 0.19-0.49). Individual physician agreement with the neuroradiologist varied from poor to moderate (kappa 0.0-0.48), with 8 of 10 physicians having no more than fair agreement. Regarding the perceived severity of HIBI on head CT, physician agreement was moderate (ICC = 0.56; 95% CI 0.38-0.77). CONCLUSION: Physicians, including radiologists, demonstrated substantial interobserver variability when identifying HIBI on head CT soon after out-of-hospital cardiac arrest.

Computation predicts rapidly adapting mechanotransduction currents cannot account for tactile encoding in Merkel cell-neurite complexes.

Distinct firing properties among touch receptors are influenced by multiple, interworking anatomical structures. Our understanding of the functions and crosstalk of Merkel cells and their associated neurites-the end organs of slowly adapting type I (SAI) afferents-remains incomplete. Piezo2 mechanically activated channels are required both in Merkel cells and in sensory neurons for canonical SAI responses in rodents; however, a central unanswered question is how rapidly inactivating currents give rise to sustained action potential volleys in SAI afferents. The computational model herein synthesizes mechanotransduction currents originating from Merkel cells and neurites, in context of skin mechanics and neural dynamics. Its goal is to mimic distinct spike firing patterns from wildtype animals, as well as Atoh1 knockout animals that completely lack Merkel cells. The developed generator function includes a Merkel cell mechanism that represents its mechanotransduction currents and downstream voltage-activated conductances (slower decay of current) and a neurite mechanism that represents its mechanotransduction currents (faster decay of current). To mimic sustained firing in wildtype animals, a longer time constant was needed than the 200 ms observed for mechanically activated membrane depolarizations in rodent Merkel cells. One mechanism that suffices is to introduce an ultra-slowly inactivating current, with a time constant on the order of 1.7 s. This mechanism may drive the slow adaptation of the sustained response, for which the skin's viscoelastic relaxation cannot account. Positioned within the sensory neuron, this source of current reconciles the physiology and anatomical characteristics of Atoh1 knockout animals.

Comparison of the effectiveness of hands-on versus online education in child passenger safety.

BACKGROUND: Community paediatricians' knowledge of appropriate child safety seat (CSS) use in vehicles may be inadequate. We compared the effectiveness of hands-on and online education in improving and retaining child passenger safety (CPS) knowledge and skills among paediatric trainees. METHODS: Paediatric trainees were randomised to receive hands-on skills training versus a 1-hour online module in CPS. CSS knowledge and installation skills were assessed using a validated 10-item/point questionnaire and an assessment tool respectively at baseline and after 6 months. Preintervention and postintervention knowledge improvement and CSS installation skills between groups were assessed using paired t-tests and effect size (d). RESULTS: Forty-eight students agreed to participate and were randomised. Thirty-nine completed training (hands-on: 23 and online: 15). At entry, no significant differences in learners' demographics and prior CPS education existed. Baseline CPS knowledge scores did not differ significantly between groups (p=0.26). Postintervention, both groups demonstrated a significant increase in knowledge scores (hands-on=3.1 (95% CI 2.4 to 3.7), p<0.0001; online=2.6 (95% CI 1.9 to 3.3), p<0.0001), though the pre-post gain in knowledge scores were not significantly different between groups (p=0.35). At follow-up, both groups demonstrated a significant increase in knowledge scores (hands-on=1.8 (95% CI 1.2 to 2.4), p<0.0001; online=1.1 (95% CI 0.7 to 1.6), p<0.0001) with the hands-on group scores significantly better than the online group (p<0.02). The long-term gain in knowledge scores was not significantly different between groups (p=0.12).Baseline CSS installation skill scores did not significantly differ between groups for forward-facing seats (p=0.16) and rear-facing seats (p=0.51). At follow-up, mean CSS installation skill scores significantly increased for the hands-on group (forward-facing seat: 0.8 (95% CI 0.16 to 1.44), p<0.02; rear-facing seat: 1.2 (95% CI 0.6 to 1.7), p<0.001) but not for the online group (forward-facing seat: 0.9 (95% CI -0.08 to 1.9), p=0.07); rear-facing seat: -0.2 (95% CI -1.1 to 0.7), p=0.6). CONCLUSIONS: Among paediatric trainees, hands-on and online CPS education are both effective in improving long-term CPS knowledge. Long-term installation skills for forward-facing and rear-facing CSS persist for hands-on education but are inconclusive for online education.

Monitoring Charge Separation Processes in Quasi-One-Dimensional Organic Crystalline Structures.

We perform the transient absorption spectroscopy experiments to investigate the dynamics of the low-energy collective electron-hole excitations in α-copper phthalocyanine thin films. The results are interpreted in terms of the third-order nonlinear polarization response function. It is found that, initially excited in the molecular plane, the intramolecular Frenkel exciton polarization reorients with time to align along the molecular chain direction to form coupled Frenkel-charge-transfer exciton states, the eigenstates of the one-dimensional periodic molecular lattice. The process pinpoints the direction of the charge separation in α-copper phthalocyanine and similar organic molecular structures. Being able to observe and monitor such processes is important both for understanding the physical principles of organic thin film solar energy conversion device operation and for the development of organic optoelectronics in general.

Association of IL6ST (gp130) Polymorphism with Functional Outcome Following Spontaneous Intracerebral Hemorrhage.

BACKGROUND AND PURPOSE: Genes associated with the inflammatory response and cytostructural integrity may influence recovery following a brain injury. To examine this in the setting of spontaneous intracerebral hemorrhage (ICH), selected single nucleotide polymorphisms (SNPs) were assessed for associations with patient outcome. METHODS: A cohort of 54 patients with supratentorial ICH were enrolled. Based on known involvement with neuroinflammation and cytostructural integrity, 10 preselected SNPs from 6 candidate genes were tested for associations with 6-month functional outcome (modified Rankin Scale [mRS] ≥ 3), mortality, and in-hospital deterioration (Glasgow Coma Scale decrease by >2 within 7 days of admission) following ICH. Fisher's exact test and logistic regression with adjustment for race and ICH score were performed. RESULTS: SNP rs10940495 (gp130 G/A) within the gp130 gene was the only SNP significantly associated with lower odds of an unfavorable 6-month functional outcome (odds ratio = .16 for mRS ≥ 3; 95% confidence interval, .03-.87, P = .03). Compared with major allele (A) homozygotes, minor allele (G) carriers in the IL6 signal transducer gene (gp130) locus were 84% less likely to have a poor outcome (mRS ≥ 3) at 6 months following spontaneous ICH. The SNP rs10940495 (gp130 G/A) and SNP rs3219119 (PARP-1 A/T) were associated with 6-month mortality (P = .02 and .04, respectively) only on univariate analysis. None of the SNPs examined were associated with in-hospital deterioration. CONCLUSION: In this exploratory study, SNP rs10940495 in the gp130 locus was associated with functional outcome at 6 months following spontaneous ICH. These findings, which should be validated through a larger study, suggest that inflammation plays an important role in mediating outcomes after ICH.

Rear-Facing Car Safety Seat Use for Children 18 Months of Age: Prevalence and Determinants.

OBJECTIVE: To examine the prevalence and potential determinants of rear-facing car safety seat use among children approximately 18 months of age born at a university hospital. STUDY DESIGN: We administered a telephone survey to caregivers of children 17-19 months of age who were born between November 2013 and May 2014. The survey was designed to assess the prevalence of rear-facing car safety seat use and estimate the likelihood of rear-facing car safety seat use, compared with forward-facing car seat use, in reference to hypothesized determinants. aORs and 95% CIs were calculated using multivariable logistic regression. RESULTS: In total, 56% of potentially eligible caregivers (491/877) completed the survey; 62% of these reported rear-facing car safety seat use. Race, education, rurality, and household income were associated with rear-facing car safety seat use after controlling for potential confounders. Additionally, caregivers who reported having discussed car seats with their child's provider (aOR 1.7; 95% CI 1.1-2.6); receiving their child's primary care in pediatrics compared with family practice clinics (aOR 2.4; 95% CI 1.1-2.6); and being aware of the American Academy of Pediatrics rear-facing recommendation (aOR 2.8; 95% CI 1.8-4.1) were significantly more likely to report rear-facing car safety seat use. Conversely, caregivers who previously used a car seat with another child were less likely to have their child rear facing at 18 months of age (aOR 0.6; 95% CI 0.4-0.9). CONCLUSIONS: A large proportion of children were forward facing at 18 months of age. Future efforts focused on encouraging providers to discuss car seats during patient visits, increasing awareness of the American Academy of Pediatrics' rear-facing recommendation, and targeting high-risk populations may improve the prevalence of children who remain rear facing until 2 years of age.