Successfully navigating the valley of death: the importance of accelerators to support academic drug discovery and development.

INTRODUCTION: The drug discovery and development 'valley of death' remains a challenge for promising new therapies originating from academic research laboratories. Drug discovery support centers and accelerators have been established to provide monetary and scientific support, but limited available funding along with cultural and expertise gaps remain obstacles for many promising technologies. AREAS COVERED: In this meta-opinion article, the authors summarize the literature around obstacles that academic drug discovery projects face, along with potential solutions and best practices. Topics covered include funding challenges, regulatory education, reproducibility, along with cultural and organizational considerations. It describes one accelerator in particular-Critical Path Institute's Translational Therapeutics Accelerator (TRxA)-that aims to overcome several of the mentioned challenges. EXPERT OPINION: The 'valley of death' remains a stubborn but not insurmountable part of the academic drug discovery and development landscape. Purposely designed accelerators can help, complementing more traditional intra- and extramural funding support.

SURVEILLANCE FOR AN EMERGENT HOOF DISEASE IN ELK (CERVUS ELAPHUS) IN THE US PACIFIC WEST SUPPLEMENTED BY 16S RRNA GENE AMPLICON SEQUENCING.

A novel hoof disease of elk (Cervus elaphus) was described in southwestern Washington, US, in 2008 and was subsequently diagnosed in an adjacent area in northwestern Oregon in 2014. The disease, currently referred to as treponeme-associated hoof disease (TAHD), is characterized by lesions ranging from mild erosions, to severe ulcers with underrunning of the hoof capsule and heel-sole junction, to overgrown and avulsed hoof capsules. Histologically, lesions exhibit epithelial erosion or ulceration, suppurative inflammation, and the presence of argyrophilic spirochetes. We used data collected by the Washington Department of Fish and Wildlife and Oregon Department of Fish and Wildlife from 2008 to 2017 as reference for disease distribution. We then conducted enhanced surveillance in 2018-20 by obtaining 164 submissions from four US Pacific West states. We detected TAHD for the first time in Idaho and northern California, as well as in multiple counties in Washington and Oregon where it had not been previously reported. Given the unexpectedly broad disease distribution, continued surveillance is warranted to determine the full geographic extent of TAHD. From samples of 22 elk, we investigated 16S rRNA gene amplicon sequencing as a technique that could be used to supplement TAHD surveillance. Operational taxonomic units of the family Spirochaetaceae were identified in 10 of 12 histologically diagnosed TAHD-positive cases and two of 10 TAHD-negative cases. Phyla Spirochaetae (P<0.008), Fusobacteria (P<0.006), and Tenericutes (P<0.01) were overrepresented in samples from TAHD-positive feet when compared with TAHD-negative elk. A unique spirochete, PT19, was detected in hooves of 11 elk and from at least one elk in each state. Results support the use of 16S rRNA gene amplicon sequencing as a reliable and informative tool to supplement investigations into distribution and etiology of this presumed polybacterial disease.

Traceback of the Psoroptes outbreak in British Columbian bighorn sheep (Ovis Canadensis).

Psoroptes are a non-burrowing, ectoparasitic, mange-causing mite that has been documented in American bighorn sheep populations throughout the 19th and 20th centuries; however, it was not seen on Canadian bighorn sheep until 2006. The aim of this study was to determine the potential source of the Psoroptes outbreak in Canadian bighorn sheep. Morphological and molecular analyses were used to compare mites recovered from outbreak-associated bighorn sheep, pet rabbits in Canada, and on historically infested bighorn sheep in the USA. The results revealed that Psoroptes acquired from the Canadian and outbreak-associated American bighorn sheep were morphologically more similar to those collected from rabbits than mites on historically infested bighorn sheep. Outer opisthosomal setae lengths measured an average of 81.7 µm (±7.7 µm) in outbreak associated bighorn mites, 88.9 µm (±12.0 µm) in rabbit mites and 151.2 µm (±16.6 µm) in historically infested bighorn mites. The opisthosomal lobe morphology of bighorn mites in the outbreak herds was also more similar to that of rabbit mites, previously described as P. cuniculi, than historically infested bighorn mites, which match previous descriptions of P. ovis. This finding was supported by DNA sequence data of the mitochondrial cytochrome B gene. This is the first report of Psoroptes of the rabbit ecotype on bighorn sheep. The morphological and molecular data therefore support the hypothesis that the source of Psoroptes outbreak in Canadian bighorn sheep represented a disease spillover event from rabbits rather than transmission from infested American bighorn sheep populations.

Plasmodium vivax and human hexokinases share similar active sites but display distinct quaternary architectures.

Malaria is a devastating disease caused by a protozoan parasite. It affects over 300 million individuals and results in over 400 000 deaths annually, most of whom are young children under the age of five. Hexokinase, the first enzyme in glucose metabolism, plays an important role in the infection process and represents a promising target for therapeutic intervention. Here, cryo-EM structures of two conformational states of Plasmodium vivax hexokinase (PvHK) are reported at resolutions of ∼3 Å. It is shown that unlike other known hexokinase structures, PvHK displays a unique tetrameric organization (∼220 kDa) that can exist in either open or closed quaternary conformational states. Despite the resemblance of the active site of PvHK to its mammalian counterparts, this tetrameric organization is distinct from that of human hexokinases, providing a foundation for the structure-guided design of parasite-selective antimalarial drugs.

Providing a Single Ground-Truth for Illuminant Estimation for the ColorChecker Dataset.

The ColorChecker dataset is one of the most widely used image sets for evaluating and ranking illuminant estimation algorithms. However, this single set of images has at least 3 different sets of ground-truth (i.e., correct answers) associated with it. In the literature it is often asserted that one algorithm is better than another when the algorithms in question have been tuned and tested with the different ground-truths. In this short correspondence we present some of the background as to why the 3 existing ground-truths are different and go on to make a new single and recommended set of correct answers. Experiments reinforce the importance of this work in that we show that the total ordering of a set of algorithms may be reversed depending on whether we use the new or legacy ground-truth data.

Methylomic correlates of autophagy activity in cystic fibrosis.

Autophagy is a highly regulated, biological process that provides energy during periods of stress and starvation. This conserved process also acts as a defense mechanism and clears microbes from the host cell. Autophagy is impaired in Cystic Fibrosis (CF) patients and CF mice, as their cells exhibit low expression levels of essential autophagy molecules. The genetic disorder in CF is due to mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene that encodes for a chloride channel. CF patients are particularly prone to infection by pathogens that are otherwise cleared by autophagy in healthy immune cells including Burkholderia cenocepacia (B. cenocepacia). The objective of this study is to determine the mechanism underlying weak autophagic activity in CF macrophages and find therapeutic targets to correct it. Using reduced representation bisulfite sequencing (RRBS) to determine DNA methylation profile, we found that the promoter regions of Atg12 in CF macrophages are significantly more methylated than in the wild-type (WT) immune cells, accompanied by low protein expression. The natural product epigallocatechin-3-gallate (EGCG) significantly reduced the methylation of Atg12 promoter improving its expression. Accordingly, EGCG restricted B. cenocepacia replication within CF mice and their derived macrophages by improving autophagy and preventing dissemination. In addition, EGCG improved the function of CFTR protein. Altogether, utilizing RRBS for the first time in the CF field revealed a previously unrecognized mechanism for reduced autophagic activity in CF. Our data also offers a mechanism by which EGCG exerts its positive effects in CF.

Learning to Detect Blue-White Structures in Dermoscopy Images With Weak Supervision.

We propose a novel approach to identify one of the most significant dermoscopic criteria in the diagnosis of cutaneous Melanoma: the blue-white structure (BWS). In this paper, we achieve this goal in a multiple instance learning (MIL) framework using only image-level labels indicating whether the feature is present or not. To this aim, each image is represented as a bag of (nonoverlapping) regions, where each region may or may not be identified as an instance of BWS. A probabilistic graphical model is trained (in MIL fashion) to predict the bag (image) labels. As output, we predict the classification label for the image (i.e., the presence or absence of BWS in each image) and we also localize the feature in the image. Experiments are conducted on a challenging dataset with results outperforming state-of-the-art techniques, with BWS detection besting competing methods in terms of performance. This study provides an improvement on the scope of modeling for computerized image analysis of skin lesions. In particular, it propounds a framework for identification of dermoscopic local features from weakly labeled data.

Potential disease agents in domestic goats and relevance to bighorn sheep (Ovis canadensis) management.

Domestic goats are raised for meat, milk and hair production, in herds for rangeland weed control, and as pack animals. Domestic sheep, goats and wild bighorn sheep are all susceptible to a multifactorial pneumonia. We sampled 43 herd goats from 7 herds and 48 pack goats from 11 herds for viral and bacterial serology, parasitology, and Pasteurellaceae microbiology. The goats in this study were in generally good health, although most goats did harbor various pathogens and parasites including several bacteria, specifically Pasteurellaceae, which have been associated with pneumonia in free-ranging bighorn sheep. It is not known if domestic goats can transmit the Pasteurellaceae or other pathogens found in this study readily to wild bighorn sheep. However, due the possibility of transmission, domestic goats in areas in or near bighorn sheep habitat should be managed to minimize the risk of spreading disease agents to bighorn sheep.

Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 µM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.

Genomics reveals historic and contemporary transmission dynamics of a bacterial disease among wildlife and livestock.

Whole-genome sequencing has provided fundamental insights into infectious disease epidemiology, but has rarely been used for examining transmission dynamics of a bacterial pathogen in wildlife. In the Greater Yellowstone Ecosystem (GYE), outbreaks of brucellosis have increased in cattle along with rising seroprevalence in elk. Here we use a genomic approach to examine Brucella abortus evolution, cross-species transmission and spatial spread in the GYE. We find that brucellosis was introduced into wildlife in this region at least five times. The diffusion rate varies among Brucella lineages (∼3 to 8 km per year) and over time. We also estimate 12 host transitions from bison to elk, and 5 from elk to bison. Our results support the notion that free-ranging elk are currently a self-sustaining brucellosis reservoir and the source of livestock infections, and that control measures in bison are unlikely to affect the dynamics of unrelated strains circulating in nearby elk populations.

Incorporating Colour Information for Computer-Aided Diagnosis of Melanoma from Dermoscopy Images: A Retrospective Survey and Critical Analysis.

Cutaneous melanoma is the most life-threatening form of skin cancer. Although advanced melanoma is often considered as incurable, if detected and excised early, the prognosis is promising. Today, clinicians use computer vision in an increasing number of applications to aid early detection of melanoma through dermatological image analysis (dermoscopy images, in particular). Colour assessment is essential for the clinical diagnosis of skin cancers. Due to this diagnostic importance, many studies have either focused on or employed colour features as a constituent part of their skin lesion analysis systems. These studies range from using low-level colour features, such as simple statistical measures of colours occurring in the lesion, to availing themselves of high-level semantic features such as the presence of blue-white veil, globules, or colour variegation in the lesion. This paper provides a retrospective survey and critical analysis of contributions in this research direction.

Spectral edge: gradient-preserving spectral mapping for image fusion.

This paper describes a novel approach to image fusion for color display. Our goal is to generate an output image whose gradient matches that of the input as closely as possible. We achieve this using a constrained contrast mapping paradigm in the gradient domain, where the structure tensor of a high-dimensional gradient representation is mapped exactly to that of a low-dimensional gradient field which is then reintegrated to form an output. Constraints on output colors are provided by an initial RGB rendering. Initially, we motivate our solution with a simple "ansatz" (educated guess) for projecting higher-D contrast onto color gradients, which we expand to a more rigorous theorem to incorporate color constraints. The solution to these constrained optimizations is closed-form, allowing for simple and hence fast and efficient algorithms. The approach can map any N-D image data to any M-D output and can be used in a variety of applications using the same basic algorithm. In this paper, we focus on the problem of mapping N-D inputs to 3D color outputs. We present results in five applications: hyperspectral remote sensing, fusion of color and near-infrared or clear-filter images, multilighting imaging, dark flash, and color visualization of magnetic resonance imaging diffusion-tensor imaging.

Patient-clinician concordance, face-time and access.

PURPOSE: People in socially disadvantageous positions may receive less time with their clinicians and consequently reduced access to healthcare resources, potentially magnifying health disparities. Socio-cultural characteristics of clinicians and patients may influence the time spent together. The purpose of this paper is to explore the relationship between clinician/patient time and clinician and patient characteristics using real-time location systems (RTLS). DESIGN/METHODOLOGY/APPROACH: In the MGH/MGPO Outpatient RFID (radio-frequency identification) project clinicians and patients wore RTLS tags during the workday to measure face-time (FT), the duration patients and clinicians are co-located, wait time (WT); i.e. from registration to clinical encounter and flow time (FLT) from registration to discharge. Demographic data were derived from the health system's electronic medical record (EMR). The RTLS and EMR data were synthesized and analyzed using standard structured-query language and statistical analytic methods. FINDINGS: From January 1, 2009 to January 1, 2011, 1,593 clinical encounters were associated with RTLS measured FTs, which differed with socioeconomic status and gender: women and lower income people received greater FT. WT was significantly longer for lower socioeconomic patients and for patients seeing trainee clinicians, women or majority ethnic group clinicians (Caucasian). FLT was shortest for men, higher socioeconomic status and for attending physician patients. Demographic concordance between patient and clinician did not significantly affect process times. RESEARCH LIMITATIONS/IMPLICATIONS: The study demonstrates the feasibility of using RTLS to capture clinically relevant process measures and suggests that the clinical delivery system surrounding a clinical encounter may more significantly influence access to clinician time than individual patient and clinician characteristics. ORIGINALITY/VALUE: Applying RTLS to healthcare is coming. We can now successfully install and run these systems in healthcare settings and extract useful information from them. Interactions with the clinical delivery system are at least as important as interactions with clinicians for providing access to care: measure FT, WT and FLT with RTLS; link clinical behavior, e.g. FT, with patient characteristics; explore how individual characteristics interact with system behavior.

A probabilistic approach to quantification of melanin and hemoglobin content in dermoscopy images.

We describe a technique that employs the stochastic Latent Topic Models framework to allow quantification of melanin and hemoglobin content in dermoscopy images. Such information bears useful implications for analysis of skin hyperpigmentation, and for classification of skin diseases. The proposed method outperforms existing approaches while allowing for more stringent and probabilistic modeling than previously.

Exemplar-Based Color Constancy and Multiple Illumination.

Exemplar-based learning or, equally, nearest neighbor methods have recently gained interest from researchers in a variety of computer science domains because of the prevalence of large amounts of accessible data and storage capacity. In computer vision, these types of technique have been successful in several problems such as scene recognition, shape matching, image parsing, character recognition, and object detection. Applying the concept of exemplar-based learning to the problem of color constancy seems odd at first glance since, in the first place, similar nearest neighbor images are not usually affected by precisely similar illuminants and, in the second place, gathering a dataset consisting of all possible real-world images, including indoor and outdoor scenes and for all possible illuminant colors and intensities, is indeed impossible. In this paper, we instead focus on surfaces in the image and address the color constancy problem by unsupervised learning of an appropriate model for each training surface in training images. We find nearest neighbor models for each surface in a test image and estimate its illumination based on comparing the statistics of pixels belonging to nearest neighbor surfaces and the target surface. The final illumination estimation results from combining these estimated illuminants over surfaces to generate a unique estimate. We show that it performs very well, for standard datasets, compared to current color constancy algorithms, including when learning based on one image dataset is applied to tests from a different dataset. The proposed method has the advantage of overcoming multi-illuminant situations, which is not possible for most current methods since they assume the color of the illuminant is constant all over the image. We show a technique to overcome the multiple illuminant situation using the proposed method and test our technique on images with two distinct sources of illumination using a multiple-illuminant color constancy dataset. The concept proposed here is a completely new approach to the color constancy problem and provides a simple learning-based framework.

Mechanisms of cellular invasion by intracellular parasites.

Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.

Plasmodium falciparum erythrocytic stage parasites require the putative autophagy protein PfAtg7 for normal growth.

Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite's erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth.

Interrogating a hexokinase-selected small-molecule library for inhibitors of Plasmodium falciparum hexokinase.

Parasites in the genus Plasmodium cause disease throughout the tropic and subtropical regions of the world. P. falciparum, one of the deadliest species of the parasite, relies on glycolysis for the generation of ATP while it inhabits the mammalian red blood cell. The first step in glycolysis is catalyzed by hexokinase (HK). While the 55.3-kDa P. falciparum HK (PfHK) shares several biochemical characteristics with mammalian HKs, including being inhibited by its products, it has limited amino acid identity (~26%) to the human HKs, suggesting that enzyme-specific therapeutics could be generated. To that end, interrogation of a selected small-molecule library of HK inhibitors has identified a class of PfHK inhibitors, isobenzothiazolinones, some of which have 50% inhibitory concentrations (IC50s) of <1 µM. Inhibition was reversible by dilution but not by treatment with a reducing agent, suggesting that the basis for enzyme inactivation was not covalent association with the inhibitor. Lastly, six of these compounds and the related molecule ebselen inhibited P. falciparum growth in vitro (50% effective concentration [EC50] of ≥ 0.6 and <6.8 µM). These findings suggest that the chemotypes identified here could represent leads for future development of therapeutics against P. falciparum.

Face time versus test ordering: is there a trade-off?

BACKGROUND: As clinician-patient face time comes under pressure, clinicians might consider substituting testing for time spent in diagnostic reasoning, history, and physical exam. OBJECTIVES: To explore the relationship between clinician-patient time and medical resource utilization. METHODS: In the Massachusetts General Hospital/Massachusetts General Physician Organization outpatient radio frequency identification project, clinicians and patients wore real-time location system (RTLS) tags. "Face time" was defined as the duration patients and clinicians were colocated. Radiology testing was used as a proxy for medical resource use. A radiology test was determined to be associated with a clinical encounter if it involved the same patient and clinician and occurred less than 3 months after the index encounter. Radiologic data were derived from the electronic health record and test appropriateness proxy score from the radiology order entry system. Data were synthesized and analyzed using standard structured query language and statistical analysis. RESULTS: From July 2008 to October 2010, 2086 clinical encounter medical records and RTLSmeasured face times could be associated: 1957 for primary care (PC) and 129 for urgent care (UC). Of these, 471 met study criteria. In PC, shorter face time was associated with more testing, but shorter wait times and flow times. In UC, testing was not associated with shorter face times, but was associated with shorter wait times and longer flow times. CONCLUSION: Our pilot suggests RTLS can capture face time and trade-offs between face time and testing. Ongoing studies will elucidate how these trade-offs affect patients, clinicians, and healthcare systems.

Automatic detection of blue-white veil by discrete colour matching in dermoscopy images.

Skin lesions are often comprised of various colours. The presence of multiple colours with an irregular distribution can signal malignancy. Among common colours under dermoscopy, blue-grey (blue-white veil) is a strong indicator of malignant melanoma. Since it is not always easy to visually identify and recognize this feature, a computerised automatic colour analysis method can provide the clinician with an objective second opinion. In this paper, we put forward an innovative method, through colour analysis and computer vision techniques, to automatically detect and segment blue-white veil areas in dermoscopy images. The proposed method is an attempt to mimic the human perception of lesion colours, and improves and outperforms the state-of-the-art as shown in our experiments.