An activin receptor IIA ligand trap promotes erythropoiesis resulting in a rapid induction of red blood cells and haemoglobin.

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -ß (TGF-ß) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-ß family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

Measuring cereblon as a biomarker of response or resistance to lenalidomide and pomalidomide requires use of standardized reagents and understanding of gene complexity.

Cereblon, a member of the cullin 4 ring ligase complex (CRL4), is the molecular target of the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide and is required for the antiproliferative activity of these agents in multiple myeloma (MM) and immunomodulatory activity in T cells. Cereblon's central role as a target of lenalidomide and pomalidomide suggests potential utility as a predictive biomarker of response or resistance to IMiD therapy. Our studies characterized a cereblon monoclonal antibody CRBN65, with high sensitivity and specificity in Western analysis and immunohistochemistry that is superior to commercially available antibodies. We identified multiple cereblon splice variants in both MM cell lines and primary cells, highlighting challenges with conventional gene expression assays given this gene complexity. Using CRBN65 antibody and TaqMan quantitative reverse transcription polymerase chain reaction assays, we showed lack of correlation between cereblon protein and mRNA levels. Furthermore, lack of correlation between cereblon expression in MM cell lines and sensitivity to lenalidomide was shown. In cell lines made resistant to lenalidomide and pomalidomide, cereblon protein is greatly reduced. These studies show limitations to the current approaches of cereblon measurement that rely on commercial reagents and assays. Standardized reagents and validated assays are needed to accurately assess the role of cereblon as a predictive biomarker.

Optimized clinical performance of growth hormone with an expanded genetic code.

The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.

Lenalidomide downregulates the cell survival factor, interferon regulatory factor-4, providing a potential mechanistic link for predicting response.

Overexpression of the transcription factor interferon regulatory factor-4 (IRF4), which is common in multiple myeloma (MM), is associated with poor prognosis. Patients with higher IRF4 expression have significantly poorer overall survival than those with low IRF4 expression. Lenalidomide is an IMiD immunomodulatory compound that has both tumouricidal and immunomodulatory activity in MM. This study showed that lenalidomide downregulated IRF4 levels in MM cell lines and bone marrow samples within 8 h of drug exposure. This was associated with a decrease in MYC levels, as well as an initial G1 cell cycle arrest, decreased cell proliferation, and cell death by day 5 of treatment. In eight MM cell lines, high IRF4 levels correlated with increased lenalidomide sensitivity. The clinical significance of this observation was investigated in 154 patients with MM. Among MM patients with high levels of IRF4 expression, treatment with lenalidomide led to a significantly longer overall survival than other therapies in a retrospective analysis. These data confirm the central role of IRF4 in MM pathogenesis; indicate that this is an important mechanism by which lenalidomide exerts its antitumour effects; and may provide a mechanistic biomarker to predict response to lenalidomide.

Temperature gradients in the flight muscles of Manduca sexta imply a spatial gradient in muscle force and energy output.

There is a significant dorso-ventral temperature gradient in the dominant flight muscles [dorsolongitudinal muscles (DLM(1))] of the hawkmoth Manduca sexta during tethered flight. The mean temperature difference was 5.6°C (range=3.8-6.9°C) between the warmer, ventral-most subunits and the cooler, dorsal-most subunits. As force generation in muscle depends on temperature, the mechanical energy output of more dorsal subunits will differ from that of deeper and warmer muscle subunits. To test this hypothesis, we isolated the dorsal subunits and the ventral subunits and recorded both single and 25 Hz (wingbeat frequency) isometric contractions at a range of temperatures. Our data show that the contractile dynamics of the various regions of the DLM(1) are similarly affected by temperature, with higher temperatures leading to reduced contraction times. Furthermore, using standard electromyography, we showed that the different regions are activated nearly simultaneously (mean time difference=0.22 ms). These observations suggest that the existence of a temperature gradient will necessarily produce a mechanical energy gradient in the DLM(1) in M. sexta.

CCR2 receptor antagonists: optimization of biaryl sulfonamides to increase activity in whole blood.

A series of biarylsulfonamides was identified as hCCR2 receptor antagonist but suffered from high plasma protein binding resulting in a >100 fold shift in activity in a functional GTPγS assay run in tandem in the presence and absence of human serum albumin. Introduction of an aryl amide with ethylenediamine linker led to compounds with reduced shifts and improved activity in whole blood.

Waymo simulated driving behavior in reconstructed fatal crashes within an autonomous vehicle operating domain.

Preventing and mitigating high severity collisions is one of the main opportunities for Automated Driving Systems (ADS) to improve road safety. This study evaluated the Waymo Driver's performance within real-world fatal collision scenarios that occurred in a specific operational design domain (ODD). To address the rare nature of high-severity collisions, this paper describes the addition of novel techniques to established safety impact assessment methodologies. A census of fatal, human-involved collisions was examined for years 2008 through 2017 for Chandler, AZ, which overlaps the current geographic ODD of the Waymo One fully automated ride-hailing service. Crash reconstructions were performed on all available fatal collisions that involved a passenger vehicle as one of the first collision partners and an available map in this ODD to determine the pre-impact kinematics of the vehicles involved in the original crashes. The final dataset consisted of a total of 72 crashes and 91 vehicle actors (52 initiators and 39 responders) for simulations. Next, a novel counterfactual "what-if'' simulation method was developed to synthetically replace human-driven crash participants one at a time with the Waymo Driver. This study focused on the Waymo Driver's performance when replacing one of the first two collision partners. The results of these simulations showed that the Waymo Driver was successful in avoiding all collisions when replacing the crash initiator, that is, the road user who made the initial, unexpected maneuver leading to a collision. Replacing the driver reacting (the responder) to the actions of the crash initiator with the Waymo Driver resulted in an estimated 82% of simulations where a collision was prevented and an additional 10% of simulations where the collision severity was mitigated (reduction in crash-level serious injury risk). The remaining 8% of simulations with the Waymo Driver in the responder role had a similar outcome to the original collision. All of these "unchanged" collisions involved both the original vehicle and the Waymo Driver being struck in the rear in a front-to-rear configuration. These results demonstrate the potential of fully automated driving systems to improve traffic safety compared to the performance of the humans originally involved in the collisions. The findings also highlight the major importance of driving behaviors that prevent entering a conflict situation (e.g. maintaining safe time gaps and not surprising other road users). However, methodological challenges in performing single instance counterfactual simulations based solely on police report data and uncertainty in ADS performance may result in variable performance, requiring additional analysis and supplemental methodologies. This study's methods provide insights on rare, severe events that would otherwise only be experienced after operating in extreme real-world driving distances (many billions of driving miles).

An omni-directional model of injury risk in planar crashes with application for autonomous vehicles.

Objective: Automated driving systems (ADS) are actively being deployed within the driving fleet. ADS are designed to safely navigate roadways, which entails an expectation of encountering varying degrees of potential conflict with other road users. The ADS design and evaluation process benefits from estimating injury severity probabilities for collisions that may occur. Current regression models in the literature are typically bespoke analyses involving targeted principal directions of force (PDOFs) and occupant positions. It is preferable to rely on injury severity models derived from a single source to provide a continuous function of risk for all planar collisions, while also accounting for specific vehicle and occupant characteristics. The novel feature of the proposed models is continuous, parametric injury risk surfaces that encompass the full spectrum of available United States field data.Methods: We used years 2001-2015 of the National Automotive Sampling System, Crashworthiness Data System (NASS-CDS) and years 2017-2019 of the Crash Investigation Sampling System (CISS) to estimate injury risk at the maximum abbreviated injury scale (MAIS) 3 and higher (3+) and 5 and higher (5+) levels for all adult occupants traveling in 2002 or newer passenger vehicles which were less than 10 years old at the time of the crash. The models account for occupant, vehicle, and crash characteristics. Interactions with vulnerable road users (e.g., pedestrian, bicyclist) were not considered.Results: We present statistical models suitable to predict injury in all non-rollover crashes at the maximum MAIS3+ and 5+ levels, and show that these models can be comparable to similar single scenario (e.g., frontal) crash models. We discuss challenges with imputing missing field data, and discuss handling of covariates that may not be known at the time of the crash.Conclusions: Collision severity assessment is a vital component of the ADS design process. We developed a novel injury risk function that can assess occupant injury risks across the spectrum of foreseeable planar collisions. These models can provide insight on potential outcomes of counterfactual simulations, injury risk and crashworthiness considerations for human driven vehicles, and provide an evaluation tool that can be applied in ADS safety impact evaluation.

Monte carlo method for estimating whole-body injury metrics from pedestrian impact simulation results.

The goal of the current study was to develop a method to estimate whole-body injury metrics (WBIMs), which measure the overall impact of injuries, using stochastic injury prediction results from a computational human surrogate. First, hospitalized pedestrian data was queried to identify injuries sustained by pedestrians and their frequencies. Second, with consideration for an understanding of injury mechanisms and the capability of the computational human surrogate, the whole-body was divided into 17 body regions. Then, an injury pattern database was constructed for each body region for various maximum abbreviated injury scale (MAIS) levels. Third, a two-step Monte Carlo sampling process was employed to generate N virtual pedestrians with an assigned list of injuries in AIS codes. Then, the expected values of WBIMs such as injury severity score (ISS), probability of death, whole-body functional capacity index (WBFCI), and lost years of life (LYL), were estimated. Lastly, the proposed method was verified using injury information from the inpatient pedestrian database. Also, the proposed method was applied to pedestrian impact simulations with various impact speeds to estimate the probability of death with respect to the impact speed. The probability of death from the proposed method was compared with those from epidemiological studies. The proposed method accurately estimated WBIMs such as ISS and WBFCI using either for a given distribution of injury risk or MAIS levels. The predicted probability of death with respect to the impact speed showed a good correlation with those from the epidemiological study. These results imply that if we have a human surrogate that can predict the risk of injury accurately, we can accurately estimate WBIMs using the proposed method. The proposed method can simplify a vehicle design optimization process by transforming the multi-objective optimization problem into the single-objective one. Lastly, the proposed method can be applied to other human surrogates such as occupant models.

Public knowledge, perception and source of information on ebola virus disease - lagos, Nigeria; september, 2014.

BACKGROUND: The first ever outbreak of Ebola virus disease (EVD) in Nigeria was declared in July, 2014. Level of public knowledge, perception and adequacy of information on EVD were unknown. We assessed the public preparedness level to adopt disease preventive behavior which is premised on appropriate knowledge, perception and adequate information. METHODS: We enrolled 5,322 respondents in a community-based cross-sectional study. We used interviewer-administered questionnaire to collect data on socio-demographic characteristics, EVD-related knowledge, perception and source of information. We performed univariate and bivariate data analysis using Epi-Info software setting p-value of 0.05 as cut-off for statistical significance. RESULTS: Mean age of respondents was 34 years (± 11.4 years), 52.3% were males. Forty one percent possessed satisfactory general knowledge; 44% and 43.1% possessed satisfactory knowledge on mode of spread and preventive measures, respectively. Residing in EVD cases districts, male respondents and possessing at least secondary education were positively associated with satisfactory general knowledge (p-value: 0.01, 0.001 and 0.000004, respectively). Seventy one percent perceived EVD as a public health problem while 61% believed they cannot contract the disease. Sixty two percent and 64% of respondents will not shake hands and hug a successfully treated EVD patient respectively. Only 2.2% of respondents practice good hand-washing practice. Television (68.8%) and radio (55.0%) are the most common sources of information on EVD. CONCLUSIONS: Gaps in EVD-related knowledge and perception exist. Targeted public health messages to raise knowledge level, correct misconception and discourage stigmatization should be widely disseminated, with television and radio as media of choice.

Insect-machine interface: a carbon nanotube-enhanced flexible neural probe.

We developed microfabricated flexible neural probes (FNPs) to provide a bi-directional electrical link to the moth Manduca sexta. These FNPs can deliver electrical stimuli to, and capture neural activity from, the insect's central nervous system. They are comprised of two layers of polyimide with gold sandwiched in between in a split-ring geometry that incorporates the bi-cylindrical anatomical structure of the insect's ventral nerve cord. The FNPs provide consistent left and right abdominal stimulation both across animals and within an individual animal. The features of the stimulation (direction, threshold charge) are aligned with anatomical features of the moth. We also have used these FNPs to record neuronal activity in the ventral nerve cord of the moth. Finally, by integrating carbon nanotube (CNT)-Au nanocomposites into the FNPs we have reduced the interfacial impedance between the probe and the neural tissue, thus reducing the magnitude of stimulation voltage. This in turn allows use of the FNPs with a wireless stimulator, enabling stimulation and flight biasing of freely flying moths. Together, these FNPs present a potent new platform for manipulating and measuring the neural circuitry of insects, and for other nerves in humans and other animals with similar dimensions as the ventral nerve cord of the moth.

Flexible split-ring electrode for insect flight biasing using multisite neural stimulation.

We describe a flexible multisite microelectrode for insect flight biasing using neural stimulation. The electrode is made of two layers of polyimide (PI) with gold sandwiched in between in a split-ring geometry. The split-ring design in conjunction with the flexibility of the PI allows for a simple insertion process and provides good attachment between the electrode and ventral nerve cord of the insect. Stimulation sites are located at the ends of protruding tips that are circularly distributed inside the split-ring structure. These protruding tips penetrate into the connective tissue surrounding the nerve cord. We have been able to insert the electrode into pupae of the giant sphinx moth Manduca sexta as early as seven days before the adult moth emerges, and we are able to use the multisite electrode to deliver electrical stimuli that evoke multidirectional, graded abdominal motions in both pupae and adult moths. Finally, in loosely tethered flight, we have used stimulation through the flexible microelectrodes to alter the abdominal angle, thus causing the flying moth to deviate to the left or right of its intended path.

Reverse endocytosis of transmembrane ephrin-B ligands via a clathrin-mediated pathway.

Eph/ephrin receptors and ligands mediate cell-cell interaction through reciprocal signaling upon juxtacrine contact, and play a critical role in embryonic patterning, neuronal targeting, and vascular assembly. To study transmembrane ephrin-B ligand trafficking, we determined the cellular localization of ephrin-B1-GFP upon engagement by EphB1. Under normal culture conditions ephrin-B1-GFP is localized to the plasma membrane, mostly at the lateral cell borders. Addition of soluble EphB1-Fc receptor induces ephrin-B1-GFP clustering on the cell surface and subsequent internalization, as judged by biochemical studies, electron microscopy, and co-localization with endosomal markers. A dominant-negative mutant of dynamin or potassium depletion blocks ephrin-B1 endocytosis. These results suggest that ephrin-B1 internalization is an active receptor-mediated process that utilizes the clathrin-mediated endocytic pathway.