A platform for oncogenomic reporting and interpretation.

Manual interpretation of variants remains rate limiting in precision oncology. The increasing scale and complexity of molecular data generated from comprehensive sequencing of cancer samples requires advanced interpretative platforms as precision oncology expands beyond individual patients to entire populations. To address this unmet need, we introduce a Platform for Oncogenomic Reporting and Interpretation (PORI), comprising an analytic framework that facilitates the interpretation and reporting of somatic variants in cancer. PORI integrates reporting and graph knowledge base tools combined with support for manual curation at the reporting stage. PORI represents an open-source platform alternative to commercial reporting solutions suitable for comprehensive genomic data sets in precision oncology. We demonstrate the utility of PORI by matching 9,961 pan-cancer genome atlas tumours to the graph knowledge base, calculating therapeutically informative alterations, and making available reports describing select individual samples.

Cell-to-cell communication mediates glioblastoma progression in Drosophila.

Glioblastoma (GB) is the most aggressive and lethal tumour of the central nervous system (CNS). GB cells grow rapidly and display a network of projections, ultra-long tumour microtubes (TMs), that mediate cell to cell communication. GB-TMs infiltrate throughout the brain, enwrap neurons and facilitate the depletion of the signalling molecule wingless (Wg)/WNT from the neighbouring healthy neurons. GB cells establish a positive feedback loop including Wg signalling upregulation that activates cJun N-terminal kinase (JNK) pathway and matrix metalloproteases (MMPs) production, which in turn promote further TMs infiltration, GB progression and neurodegeneration. Thus, cellular and molecular signals other than primary mutations emerge as central players of GB. Using a Drosophila model of GB, we describe the temporal organisation of the main cellular events that occur in GB, including cell-to-cell interactions, neurodegeneration and TM expansion. We define the progressive activation of JNK pathway signalling in GB mediated by the receptor Grindelwald (Grnd) and activated by the ligand Eiger (Egr)/TNFα produced by surrounding healthy brain tissue. We propose that cellular interactions of GB with the healthy brain tissue precede TM expansion and conclude that non-autonomous signals facilitate GB progression. These results contribute to deciphering the complexity and versatility of these incurable tumours.

A quality improvement initiative to implement the eat, sleep, console neonatal opioid withdrawal syndrome care tool in Massachusetts' PNQIN collaborative.

OBJECTIVE: To support hospitals in the Massachusetts PNQIN collaborative with adoption of the ESC Neonatal Opioid Withdrawal Syndrome (NOWS) Care Tool© and assess NOWS hospitalization outcomes. STUDY DESIGN: Statewide QI study where 11 hospitals adopted the ESC NOWS Care Tool©. Outcomes of pharmacotherapy and length of hospital stay (LOS) and were compared in Pre- and Post-ESC implementation cohorts. Statistical Process Control (SPC) charts were used to examine changes over time. RESULTS: The Post-ESC group had lower rates of pharmacotherapy (OR 0.35, 95% CI 0.26, 0.46) with shorter LOS (RR 0.79, 95% CI 0.76, 0.82). The 30-day NOWS readmission rate was 1.2% in the Pre- and 0.4% in the Post-ESC cohort. SPC charts indicate a shift in pharmacotherapy from 54.8 to 35.0% and LOS from 14.2 to 10.9 days Post-ESC. CONCLUSIONS: The ESC NOWS Care Tool was successfully implemented across a state collaborative with improvement in NOWS outcomes without short-term adverse effects.

Patient selection for a developmental therapeutics program using whole genome and Transcriptome analysis.

Introduction Given the high level of uncertainty surrounding the outcomes of early phase clinical trials, whole genome and transcriptome analysis (WGTA) can be used to optimize patient selection and study assignment. In this retrospective analysis, we reviewed the impact of this approach on one such program. Methods Patients with advanced malignancies underwent fresh tumor biopsies as part of our personalized medicine program (NCT02155621). Tumour molecular data were reviewed for potentially clinically actionable findings and patients were referred to the developmental therapeutics program. Outcomes were reviewed in all patients, including those where trial selection was driven by molecular data (matched) and those where there was no clear molecular rationale (unmatched). Results From January 2014 to January 2018, 28 patients underwent WGTA and enrolled in clinical trials, including 2 patients enrolled in two trials. Fifteen patients were matched to a treatment based on a molecular target. Five patients were matched to a trial based upon single-gene DNA changes, all supported by RNA data. Ten cases were matched on the basis of genome-wide data (n = 4) or RNA gene expression only (n = 6). With a median follow-up of 6.7 months, the median time on treatment was 8.2 weeks. Discussion When compared to single-gene DNA-based data alone, WGTA led to a 3-fold increase in treatment matching. In a setting where there is a high level of uncertainty around both the investigational agents and the biomarkers, more data are needed to fully evaluate the impact of routine use of WGTA.

Alpha band signatures of social synchrony.

Previous research has reported changes in mu rhythm, the central rhythm of the alpha frequency band, in both intentional and spontaneous interpersonal coordination. The current study was designed to extend existing findings on social synchrony to the pendulum swinging task and simultaneously measured time unfolding behavioral synchrony and EEG estimation of mu activity during spontaneous, intentional in-phase and intentional anti-phase interpersonal coordination. As expected, the behavioral measures of synchrony demonstrated the expected pattern of weak synchronization for spontaneous coordination, moderate synchronization for intentional anti-phase coordination, and strong synchronization for in-phase coordination. With respect to the EEG measures, we found evidence for mu enhancement for spontaneous coordination in contrast to mu suppression for intentional coordination (both in phase and anti-phase), with higher levels of synchronization associated with higher levels of mu suppression in the right hemisphere. The implications of the research findings and methodology for understanding the underlying mechanisms contributing to social problems in psychological disorders, leader-follower relationships, and inter-brain dynamics are discussed.

Relationship Between Theory of Mind, Emotion Recognition, and Social Synchrony in Adolescents With and Without Autism.

Difficulty in social communication and interaction is a primary diagnostic feature of ASD. Research has found that adolescents with ASD display various impairments in social behavior such as theory of mind (ToM), emotion recognition, and social synchrony. However, not much is known about the relationships among these dimensions of social behavior. Adolescents with and without ASD participated in the study. ToM ability was measured by viewing social animations of geometric shapes, recognition of facial emotions was measured by viewing pictures of faces, and synchrony ability was measured with a spontaneously arising interpersonal movement task completed with a caregiver and an intentional interpersonal task. Attention and social responsiveness were measured using parent reports. We then examined the relationship between ToM, emotion recognition, clinical measures of attention and social responsiveness, and social synchronization that arises either spontaneously or intentionally. Results indicate that spontaneous synchrony was related to ToM and intentional synchrony was related to clinical measures of attention and social responsiveness. Facial emotion recognition was not related to either ToM or social synchrony. Our findings highlight the importance of biological motion perception and production and attention for more fully understanding the social behavior characteristic of ASD. The findings suggest that the processes underlying difficulties in spontaneous synchrony in ASD are different than the processes underlying difficulties in intentional synchronization.

Category selectivity of the N170 and the role of expertise in deaf signers.

Deafness is known to affect processing of visual motion and information in the visual periphery, as well as the neural substrates for these domains. This study was designed to characterize the effects of early deafness and lifelong sign language use on visual category sensitivity of the N170 event-related potential. Images from nine categories of visual forms including upright faces, inverted faces, and hands were presented to twelve typically hearing adults and twelve adult congenitally deaf signers. Classic N170 category sensitivity was observed in both participant groups, whereby faces elicited larger amplitudes than all other visual categories, and inverted faces elicited larger amplitudes and slower latencies than upright faces. In hearing adults, hands elicited a right hemispheric asymmetry while in deaf signers this category elicited a left hemispheric asymmetry. Pilot data from five hearing native signers suggests that this effect is due to lifelong use of American Sign Language rather than auditory deprivation itself.

Impairments of Social Motor Synchrony Evident in Autism Spectrum Disorder.

Social interactions typically involve movements of the body that become synchronized over time and both intentional and spontaneous interactional synchrony have been found to be an essential part of successful human interaction. However, our understanding of the importance of temporal dimensions of social motor synchrony in social dysfunction is limited. Here, we used a pendulum coordination paradigm to assess dynamic, process-oriented measures of social motor synchrony in adolescents with and without autism spectrum disorder (ASD). Our data indicate that adolescents with ASD demonstrate less synchronization in both spontaneous and intentional interpersonal coordination. Coupled oscillator modeling suggests that ASD participants assembled a synchronization dynamic with a weaker coupling strength, which corresponds to a lower sensitivity and decreased attention to the movements of the other person, but do not demonstrate evidence of a delay in information transmission. The implication of these findings for isolating an ASD-specific social synchronization deficit that could serve as an objective, bio-behavioral marker is discussed.

"Look who's talking!" Gaze Patterns for Implicit and Explicit Audio-Visual Speech Synchrony Detection in Children With High-Functioning Autism.

Conversation requires integration of information from faces and voices to fully understand the speaker's message. To detect auditory-visual asynchrony of speech, listeners must integrate visual movements of the face, particularly the mouth, with auditory speech information. Individuals with autism spectrum disorder may be less successful at such multisensory integration, despite their demonstrated preference for looking at the mouth region of a speaker. We showed participants (individuals with and without high-functioning autism (HFA) aged 8-19) a split-screen video of two identical individuals speaking side by side. Only one of the speakers was in synchrony with the corresponding audio track and synchrony switched between the two speakers every few seconds. Participants were asked to watch the video without further instructions (implicit condition) or to specifically watch the in-synch speaker (explicit condition). We recorded which part of the screen and face their eyes targeted. Both groups looked at the in-synch video significantly more with explicit instructions. However, participants with HFA looked at the in-synch video less than typically developing (TD) peers and did not increase their gaze time as much as TD participants in the explicit task. Importantly, the HFA group looked significantly less at the mouth than their TD peers, and significantly more at non-face regions of the image. There were no between-group differences for eye-directed gaze. Overall, individuals with HFA spend less time looking at the crucially important mouth region of the face during auditory-visual speech integration, which is maladaptive gaze behavior for this type of task.

Eye tracking research to answer questions about augmentative and alternative communication assessment and intervention.

Recently, eye tracking technologies (i.e., technologies that automatically track the point of an individual's gaze while that person views or interacts with a visual image) have become available for research purposes. Based on the sampling of the orientation of the individual's eyes, researchers can quantify which locations within the visual image were fixated (viewed), for how long, and how many times. These automated eye tracking research technologies open up a wealth of avenues for investigating how individuals with developmental or acquired communication disabilities may respond to aided augmentative and alternative communication (AAC) systems. In this paper, we introduce basic terminology and explore some of the special challenges of conducting eye tracking research with populations with disabilities who might use AAC, including challenges of inferring attention from the presence of fixation and challenges related to calibration that may result from participant characteristics, behavioral idiosyncracies, and/or the number of calibration points. We also examine how the technology can be applied to ask well-structured experimental questions that have direct clinical relevance, with a focus on the unique contributions that eye tracking research can provide by (a) allowing evaluation of skills in individuals who are difficult to assess via traditional methods, and (b) facilitating access to information on underlying visual cognitive processes that is not accessible via traditional behavioral measures.

Selected toxicities of targeted therapies: presentation and management.

The introduction of targeted therapy has changed the landscape of metastatic renal cell carcinoma (mRCC) but has also brought challenges. One such challenge of treating this chronic disease is long-term therapy, which produces ongoing toxicity. Knowledge of the spectrum of toxicity from targeted therapy and appropriate and timely intervention is required to maintain adequate dosing required for optimal outcome. This article addresses some of the major toxicities of newer agents in RCC and discusses management strategies.

Visual field bias in hearing and deaf adults during judgments of facial expression and identity.

The dominance of the right hemisphere during face perception is associated with more accurate judgments of faces presented in the left rather than the right visual field (RVF). Previous research suggests that the left visual field (LVF) bias typically observed during face perception tasks is reduced in deaf adults who use sign language, for whom facial expressions convey important linguistic information. The current study examined whether visual field biases were altered in deaf adults whenever they viewed expressive faces, or only when attention was explicitly directed to expression. Twelve hearing adults and 12 deaf signers were trained to recognize a set of novel faces posing various emotional expressions. They then judged the familiarity or emotion of faces presented in the left or RVF, or both visual fields simultaneously. The same familiar and unfamiliar faces posing neutral and happy expressions were presented in the two tasks. Both groups were most accurate when faces were presented in both visual fields. Across tasks, the hearing group demonstrated a bias toward the LVF. In contrast, the deaf group showed a bias toward the LVF during identity judgments that shifted marginally toward the RVF during emotion judgments. Two secondary conditions tested whether these effects generalized to angry faces and famous faces and similar effects were observed. These results suggest that attention to facial expression, not merely the presence of emotional expression, reduces a typical LVF bias for face processing in deaf signers.

Behavioral and neural evidence of increased attention to the bottom half of the face in deaf signers.

PURPOSE: This study examined the effects of deafness and sign language use on the distribution of attention across the top and bottom halves of faces. METHODS: In a composite face task, congenitally deaf signers and typically hearing controls made same/different judgments of the top or bottom halves of faces presented with the halves aligned or spatially misaligned, while event-related potentials (ERPs) were recorded. RESULTS: Both groups were more accurate when judging misaligned than aligned faces, which indicates holistic face processing. Misalignment affected all ERP components examined, with effects on the N170 resembling those of face inversion. Hearing adults were similarly accurate when judging the top and bottom halves of the faces, but deaf signers were more accurate when attending to the bottom than the top. Attending to the top elicited faster P1 and N170 latencies for both groups; within the deaf group, this effect was greatest for individuals who produced the highest accuracies when attending to the top. CONCLUSIONS: These findings dovetail with previous research by providing behavioral and neural evidence of increased attention to the bottom half of the face in deaf signers, and by documenting that these effects generalize to a speeded task, in the absence of gaze shifts, with neutral facial expressions.

Binding of DC-SIGN to glycoproteins expressed in glycoengineered Pichia pastoris.

Previous studies have shown that glycoproteins expressed in wild-type Pichia pastoris bind to Dendritic cell-SIGN (DC-Specific Intercellular adhesion molecule-3 Grabbing Nonintegrin), a mannose-binding receptor found on dendritic cells in peripheral tissues which is involved in antigen presentation and the initiation of an immune response. However, the binding of DC-SIGN to glycoproteins purified from P. pastoris strains engineered to express humanized N- and O-linked glycans has not been tested to date. In this study, the binding of glycoproteins with specific high-mannose or human N- and O-linked glycan structures to DC-SIGN was tested. Proteins with humanized N-glycans including Man5 structures and O-glycans (up to as many as 24) with single mannose chain length showed DC-SIGN binding that was comparable to that measured for a CHO-produced IgG1 which lacks O-linked mannose. Glycoproteins with wild-type N-glycans and mannotriose and higher O-glycans bound to DC-SIGN in a manner that was strongly inhibited by either the use of enzymatic N-deglycosylation or sodium meta-periodate oxidation. Mannan purified from humanized P. pastoris also showed lower ability to inhibit DC-SIGN binding to glycoproteins with wild type fungal glycosylation than mannan purified from wild type strains. This study shows that humanized P. pastoris can produce glycoproteins that do not bind to DC-SIGN.

Optimization of erythropoietin production with controlled glycosylation-PEGylated erythropoietin produced in glycoengineered Pichia pastoris.

Pichia pastoris is a methylotropic yeast that has gained great importance as an organism for protein expression in recent years. Here, we report the expression of recombinant human erythropoietin (rhEPO) in glycoengineered P. pastoris. We show that glycosylation fidelity is maintained in fermentation volumes spanning six orders of magnitude and that the protein can be purified to high homogeneity. In order to increase the half-life of rhEPO, the purified protein was coupled to polyethylene glycol (PEG) and then compared to the currently marketed erythropoiesis stimulating agent, Aranesp(®) (darbepoetin). In in vitro cell proliferation assays the PEGylated protein was slightly, and the non-PEGylated protein was significantly more active than comparator. Pharmacodynamics as well as pharmacokinetic activity of PEGylated rhEPO in animals was comparable to that of Aranesp(®). Taken together, our results show that glycoengineered P. pastoris is a suitable production host for rhEPO, yielding an active biologic that is comparable to those produced in current mammalian host systems.

Gaze patterns during identity and emotion judgments in hearing adults and deaf users of American Sign Language.

Deaf individuals rely on facial expressions for emotional, social, and linguistic cues. In order to test the hypothesis that specialized experience with faces can alter typically observed gaze patterns, twelve hearing adults and twelve deaf, early-users of American Sign Language judged the emotion and identity of expressive faces (including whole faces, and isolated top and bottom halves), while accuracy and fixations were recorded. Both groups recognized individuals more accurately from top than bottom halves, and emotional expressions from bottom than top halves. Hearing adults directed the majority of fixations to the top halves of faces in both tasks, but fixated the bottom half slightly more often when judging emotion than identity. In contrast, deaf adults often split fixations evenly between the top and bottom halves regardless of task demands. These results suggest that deaf adults have habitual fixation patterns that may maximize their ability to gather information from expressive faces.

Elimination of ß-mannose glycan structures in Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, such as those containing ß-mannose (Man) linkages, can elicit an immune response or bind to Man receptors, thus reducing their efficacy. Recent studies have confirmed that P. pastoris has four genes from the ß-mannosyl transferase (BMT) family and that Bmt2p is responsible for the majority of ß-Man linkages on glycans. While expressing recombinant human erythropoietin (rhEPO) in a developmental glycoengineered strain devoid of BMT2 gene expression, cross-reactivity was observed with an antibody raised against host cell antigens. Treatment of the rhEPO with protein N-glycosidase F eliminated cross-reactivity, indicating that the antigen was associated with the glycan. Thorough analysis of the glycan profile of rhEPO demonstrated the presence of low amounts of α-1,2-mannosidase resistant high-Man glycoforms. In an attempt to eliminate the α-mannosidase resistant glycoforms, we used a systemic approach to genetically knock-out the remaining members of the BMT family culminating in a quadruple bmt2,4,1,3 knock-out strain. Data presented here conclude that the additive elimination of Bmt2p, Bmt3p and Bmt1p activities are required for total abolition of ß-Man-associated glycans and their related antigenicity. Taken together, the elimination of ß-Man containing glycoforms represents an important step forward for the Pichia production platform as a suitable system for the production of therapeutic glycoproteins.

Structural elucidation of an α-1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris.

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-ß-Man-(1 → 2)-ß-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple ß-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study.

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependant cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.

A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris.

To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N-terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high-throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N-glycosylation.