Pervasive mislocalization of pathogenic coding variants underlying human disorders.

Widespread sequencing has yielded thousands of missense variants predicted or confirmed as disease causing. This creates a new bottleneck: determining the functional impact of each variant-typically a painstaking, customized process undertaken one or a few genes and variants at a time. Here, we established a high-throughput imaging platform to assay the impact of coding variation on protein localization, evaluating 3,448 missense variants of over 1,000 genes and phenotypes. We discovered that mislocalization is a common consequence of coding variation, affecting about one-sixth of all pathogenic missense variants, all cellular compartments, and recessive and dominant disorders alike. Mislocalization is primarily driven by effects on protein stability and membrane insertion rather than disruptions of trafficking signals or specific interactions. Furthermore, mislocalization patterns help explain pleiotropy and disease severity and provide insights on variants of uncertain significance. Our publicly available resource extends our understanding of coding variation in human diseases.

Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms").

A proteome-scale map of the human interactome network.

Just as reference genome sequences revolutionized human genetics, reference maps of interactome networks will be critical to fully understand genotype-phenotype relationships. Here, we describe a systematic map of ?14,000 high-quality human binary protein-protein interactions. At equal quality, this map is ?30% larger than what is available from small-scale studies published in the literature in the last few decades. While currently available information is highly biased and only covers a relatively small portion of the proteome, our systematic map appears strikingly more homogeneous, revealing a "broader" human interactome network than currently appreciated. The map also uncovers significant interconnectivity between known and candidate cancer gene products, providing unbiased evidence for an expanded functional cancer landscape, while demonstrating how high-quality interactome models will help "connect the dots" of the genomic revolution.

Cytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL.

Allogeneic chimeric antigen receptor T-cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CARTs have been created using gene editing techniques that induce DNA double-stranded breaks (DSBs), resulting in unintended on-target editing outcomes with potentially unforeseen consequences. Cytosine base editors (CBEs) install C•G to T•A point mutations in T cells, with between 90% and 99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using 4 simultaneous base edits. We show that CBE, unlike CRISPR-Cas9, does not impact T-cell proliferation, lead to aberrant DNA damage response pathway activation, or result in karyotypic abnormalities following multiplexed editing. We demonstrate 7CAR8 to be highly efficacious against T-cell acute lymphoblastic leukemia (T-ALL) using multiple in vitro and in vivo models. Thus, CBE is a promising technology for applications requiring multiplexed gene editing and can be used to manufacture quadruple-edited 7CAR8 cells, with high potential for clinical translation for relapsed and refractory T-ALL.

Mixed emotions to social situations: An fMRI investigation.

BACKGROUND: Neuroscience research has generally studied emotions each taken in isolation. However, mixed emotional states (e.g., the co-occurrence of amusement and disgust, or sadness and pleasure) are common in everyday life. Psychophysiological and behavioral evidence suggests that mixed emotions may have response profiles that are distinguishable from their constituent emotions. Yet, the brain bases of mixed emotions remain unresolved. METHODS: We recruited 38 healthy adults who viewed short, validated film clips, eliciting either positive (amusing), negative (disgusting), neutral, or mixed (a mix of amusement and disgust) emotional states, while brain activity was assessed by functional magnetic resonance imaging (fMRI). We assessed mixed emotions in two ways: first by comparing neural reactivity to ambiguous (mixed) with that to unambiguous (positive and negative) film clips and second by conducting parametric analyses to measure neural reactivity with respect to individual emotional states. We thus obtained self-reports of amusement and disgust after each clip and computed a minimum feeling score (shared minimum of amusement and disgust) to quantify mixed emotional feelings. RESULTS: Both analyses revealed a network of the posterior cingulate (PCC), medial superior parietal lobe (SPL)/precuneus, and parieto-occipital sulcus to be involved in ambiguous contexts eliciting mixed emotions. CONCLUSION: Our results are the first to shed light on the dedicated neural processes involved in dynamic social ambiguity processing. They suggest both higher-order (SPL) and lower-order (PCC) processes may be needed to process emotionally complex social scenes.

Distribution of the reef manta ray Mobula alfredi and the oceanic manta ray Mobula birostris in the Philippines: a collaborative effort for conservation.

Little is known about manta ray population size, structure and connectivity in the Philippines. In collaboration with dive operators, non-governmental organizations and authorities, sightings of manta rays were collated into a single national database. Using in-water photographs and videos gathered through citizen science and dedicated research efforts, this study compiled sightings between 2004 and 2020, showing 22 separate sites throughout the archipelago with manta rays present. A total of 392 individual reef manta rays (Mobula alfredi) and 107 oceanic manta rays (Mobula birostris) were identified from the collected footage. Four specific sites in the provinces of Masbate and Palawan together hosted 89% of all identified individuals and accounted for 95% of sightings, highlighting these areas are key aggregation sites. This study also reports the movements of M. birostris within the Philippines, based on photo-identification of three individuals moving 150 km between Cebu and Masbate. Despite the growing number of recreational divers in Daanbantayan and San Jacinto, an 80% decline in M. birostris sightings was observed at these sites. To ensure effective future conservation, it is recommended that efforts focus on the identification and protection of manta ray hotspots and migratory corridors, the creation of a sustainable tourism framework and, most important, the implementation of mitigation strategies to reduce fisheries interactions.

Kidney-Related Research in the United States: A Position Statement From the National Kidney Foundation and the American Society of Nephrology.

Kidney disease is an important US public health problem because it affects over 37 million Americans, and Medicare expenditures for patients with chronic kidney disease now alone exceed $130 billion annually. Kidney disease is characterized by strong racial, ethnic, and socioeconomic disparities, and reducing kidney disease incidence will positively impact US health disparities. Due to the aging of the US population and an unabated obesity epidemic, the number of patients receiving treatment for kidney failure is anticipated to increase, which will escalate kidney disease health expenditures. The historical and current investment in kidney-related research via the National Institute of Diabetes and Digestive and Kidney Diseases has severely lagged behind ongoing expenditures for kidney disease care. Increasing research investment will identify, develop, and increase implementation of interventions to slow kidney disease progression, reduce incidence of kidney failure, enhance survival, and improve quality of life. This perspective states the urgent reasons why increasing investment in kidney-related research is important for US public health. The National Kidney Foundation and the American Society of Nephrology are working together to advocate for increased funding for the National Institute of Diabetes and Digestive and Kidney Diseases. The long-term goal is to reduce the burden of kidney disease in the US population and improve the quality of life of patients living with kidney disease.

Neutral competition in a deterministically changing environment: Revisiting continuum approaches.

Environmental variation can play an important role in ecological competition by influencing the relative advantage between competing species. Here, we consider such effects by extending a classical, competitive Moran model to incorporate an environment that fluctuates periodically in time. We adapt methods from work on these classical models to investigate the effects of the magnitude and frequency of environmental fluctuations on two important population statistics: the probability of fixation and the mean time to fixation. In particular, we find that for small frequencies, the system behaves similar to a system with a constant fitness difference between the two species, and for large frequencies, the system behaves similar to a neutrally competitive model. Most interestingly, the system exhibits nontrivial behavior for intermediate frequencies. We conclude by showing that our results agree quite well with recent theoretical work on competitive models with a stochastically changing environment, and discuss how the methods we develop ease the mathematical analysis required to study such models.

Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies.

N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or ß-1,2-xylose residues was produced. When compared with the innovator product Rituxan®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics' glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies.

An inter-species protein-protein interaction network across vast evolutionary distance.

In cellular systems, biophysical interactions between macromolecules underlie a complex web of functional interactions. How biophysical and functional networks are coordinated, whether all biophysical interactions correspond to functional interactions, and how such biophysical-versus-functional network coordination is shaped by evolutionary forces are all largely unanswered questions. Here, we investigate these questions using an "inter-interactome" approach. We systematically probed the yeast and human proteomes for interactions between proteins from these two species and functionally characterized the resulting inter-interactome network. After a billion years of evolutionary divergence, the yeast and human proteomes are still capable of forming a biophysical network with properties that resemble those of intra-species networks. Although substantially reduced relative to intra-species networks, the levels of functional overlap in the yeast-human inter-interactome network uncover significant remnants of co-functionality widely preserved in the two proteomes beyond human-yeast homologs. Our data support evolutionary selection against biophysical interactions between proteins with little or no co-functionality. Such non-functional interactions, however, represent a reservoir from which nascent functional interactions may arise.

Tetrazolone as an acid bioisostere: application to marketed drugs containing a carboxylic acid.

Matched molecular pair analysis was used to evaluate the ability of a tetrazolone group to act as a bioisostere of a carboxylic acid. Compound 7, a tetrazolone of the anti-hypertensive drug, telmisartan 6, was shown to be a potent AT1 antagonist (Kb = 0.14 nM), with activity comparable to telmisartan itself (Kb = 0.44 nM). Additionally, compound 9, a tetrazolone congener of the marketed anti-cancer agent, bexarotene 8, was shown to be an agonist at the retinoid X receptor alpha (EC50 = 64 nM). Compounds containing a tetrazolone group showed similar microsomal stability and plasma protein binding to marketed acid counterparts, while also reducing the value for clog P. Furthermore, compound 7 displayed an improved rat pharmacokinetic profile cf. telmisartan 6. Taken together, the results demonstrate that a tetrazolone group may serve as a bioisostere for a carboxylic acid.

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Functional connectivity mapping of regions associated with self- and other-processing.

Neuroscience literature increasingly suggests a conceptual self composed of interacting neural regions, rather than independent local activations, yet such claims have yet to be investigated. We, thus, combined task-dependent meta-analytic connectivity modeling (MACM) with task-independent resting-state (RS) connectivity analysis to delineate the neural network of the self, across both states. Given psychological evidence implicating the self's interdependence on social information, we also delineated the neural network underlying conceptual other-processing. To elucidate the relation between the self-/other-networks and their function, we mined the MACM metadata to generate a cognitive-behavioral profile for an empirically identified region specific to conceptual self, the pregenual anterior cingulate (pACC), and conceptual other, posterior cingulate/precuneus (PCC/PC). Mining of 7,200 published, task-dependent, neuroimaging studies, using healthy human subjects, yielded 193 studies activating the self-related seed and were conjoined with RS connectivity analysis to delineate a differentiated self-network composed of the pACC (seed) and anterior insula, relative to other functional connectivity. Additionally, 106 studies activating the other-related seed were conjoined with RS connectivity analysis to delineate a differentiated other-network of PCC/PC (seed) and angular gyrus/temporoparietal junction, relative to self-functional connectivity. The self-network seed related to emotional conflict resolution and motivational processing, whereas the other-network seed related to socially oriented processing and contextual information integration. Notably, our findings revealed shared RS connectivity between ensuing self-/other-networks within the ventromedial prefrontal cortex and medial orbitofrontal cortex, suggesting self-updating via integration of self-relevant social information. We, therefore, present initial neurobiological evidence corroborating the increasing claims of an intricate self-network, the architecture of which may promote social value processing.

A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1ß, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1ß, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.

When at rest: "Event-free" active inference may give rise to implicit self-models of coping potential.

Kalisch and colleagues highlight coping potential (CP) as a principle resilience mechanism during event engagement. We complement this discussion by exploring generative implicit CP self-models, arguably emerging during "resting-state," subsequent and prior to events. Resting-state affords a propitious environment for Bayesian learning, wherein appraisals/reappraisals may update active inferential CP self-models, which then mediate appraisal style organization and resilience factor valuation.

A public genome-scale lentiviral expression library of human ORFs.

Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.

Should infants presenting with an apparent life-threatening event undergo evaluation for serious bacterial infections and respiratory pathogens?

We sought to identify which patients with an apparent life-threatening event require infectious evaluation through an analysis of infants aged ≤12 months brought to an emergency department with an apparent life-threatening event. Among the 533 children evaluated, there were no cases of meningitis, 1 case of bacteremia, 17 cases of urinary tract infection, 22 cases of bacterial pneumonia, 22 cases of respiratory syncytial virus, and 2 cases of influenza virus identified in respiratory specimens.

Use of point-of-care lactate in the prehospital aeromedical environment.

INTRODUCTION: Lactate measurement has been used to identify critical medical illness and initiate early treatment strategies. The prehospital environment offers an opportunity for very early identification of critical illness and commencement of care. HYPOTHESIS: The investigators hypothesized that point-of-care lactate measurement in the prehospital aeromedical environment would: (1) identify medical patients with high mortality; (2) influence fluid, transfusion, and intubation; and (3) increase early central venous catheter (CVC) placement. METHODS: Critically ill, medical, nontrauma patients who were transported from September 2007 through February 2009 by University of Massachusetts (UMass) Memorial LifeFlight, a university-based emergency medical helicopter service, were eligible for enrollment. Patients were prospectively randomized to receive a fingerstick whole-blood lactate measurement on an alternate-day schedule. Flight crews were not blinded to results. Flight crews were asked to inform the receiving attending physician of the results. The primary endpoint was the ability of a high, prehospital lactate value [> 4 millimoles per liter (mmol/L)] to identify mortality. Secondary endpoints included differences in post-transport fluid, transfusion, and intubation, and decrease in time to central venous catheter (CVC) placement. Categorical variables were compared between groups by Fisher's Exact Test, and continuous variables were compared by t-test. RESULTS: Patients (N = 59) were well matched for age, gender, and acuity. In the lactate cohort (n = 20), mean lactate was 7 mmol/L [Standard error of the mean, SEM = 1]. Initial analysis revealed that prehospital lactate levels of ≥ 4 mmol/L did show a trend toward higher mortality with an odds ratio of 2.1 (95% CI, 0.3-13.8). Secondary endpoints did not show a statistically significant change in management between the lactate and non lactate groups. There was a trend toward decreased time to post-transport CVC in the non lactate faction. CONCLUSION: Prehospital aeromedical point-of-care lactate measurement levels ≥ 4 mmol/L may help stratify mortality. Further investigation is needed, as this is a small, limited study. The initial analysis did not find a significant change in post-transport management.

Use of the relationship between absolute lymphocyte count and CD4 count to improve earlier consideration of pneumocystis pneumonia in HIV-positive emergency department patients with pneumonia.

BACKGROUND: The ability to accurately assess the level of immunosuppression in HIV+ patients in the emergency department (ED) is often limited and can affect management of these patients. OBJECTIVE: To evaluate the relationship between the absolute lymphocyte count (ALC) and CD4 count in HIV patients admitted through the ED with pneumonia and how utilization of this relationship may affect early consideration and evaluation of Pneumocystis jiroveci pneumonia (PCP). METHODS: Retrospective multicenter 5-year study of HIV+ patients with an ICD-9 diagnosis of pneumonia. Included patients had an ALC measured on ED presentation and a CD4 count measured in < 24 h. A receiver operator curve (ROC), decision plot analysis, and McNemar test of proportions were used to characterize the relationship between study variables. RESULTS: Six hundred eighty six patients were enrolled, 23.2% (95% confidence interval [CI] 20.2-26.1) were diagnosed with PCP. The geometric mean CD4 count and ALC were 81 and 1089, respectively. The correlation between ALC and CD4 was r = 0.60 (95% CI 0.55-65, p < 0.01). The ROC was 0.78 (0.75-0.82). An ALC < 1700 cells/mm(3) had a sensitivity of 84% (95% CI 80-87) and specificity of 55% (95% CI 48-70) for a CD4 < 200 cells/mm(3). An ALC threshold of 1700 cells/mm(3) would have identified 86% of patients with PCP but falsely identified 2.5 patients without PCP for every one accurately identified. CONCLUSION: The ALC threshold of 1700 cells/mm(3) retains significant discriminatory value and would moderately improve identification of patients with a CD4 < 200 cells/mm(3) but is not likely to be reliable as the sole method of early recognition and evaluation of PCP.