Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform.

Reverse genetic systems enable the engineering of RNA virus genomes and are instrumental in studying RNA virus biology. With the recent outbreak of the coronavirus disease 2019 pandemic, already established methods were challenged by the large genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein we present an elaborated strategy for the rapid and straightforward rescue of recombinant plus-stranded RNA viruses with high sequence fidelity using the example of SARS-CoV-2. The strategy called CLEVER (CLoning-free and Exchangeable system for Virus Engineering and Rescue) is based on the intracellular recombination of transfected overlapping DNA fragments allowing the direct mutagenesis within the initial PCR-amplification step. Furthermore, by introducing a linker fragment - harboring all heterologous sequences - viral RNA can directly serve as a template for manipulating and rescuing recombinant mutant virus, without any cloning step. Overall, this strategy will facilitate recombinant SARS-CoV-2 rescue and accelerate its manipulation. Using our protocol, newly emerging variants can quickly be engineered to further elucidate their biology. To demonstrate its potential as a reverse genetics platform for plus-stranded RNA viruses, the protocol has been successfully applied for the cloning-free rescue of recombinant Chikungunya and Dengue virus.

The Improvement Default: People Presume Improvement When Lacking Information.

People erroneously think that things they know little about improve over time. We propose that, due to salient cultural narratives, improvement is a highly accessible expectation that leads people to presume improvement in the absence of diagnostic information. Five studies investigated an improvement default: a general tendency to presume improvement even in self-irrelevant domains. Participants erroneously presumed improvement over esoteric historical time periods associated with decline (Study 1). Participants arranged a stranger's experiences to produce trends of improvement (Study 2). Participants presumed improvement for a fictional city when given no diagnostic information about it (Study 3). Finally, participants who perceived more past improvement were less supportive of policies that may precipitate further improvement (Study 4). Implications for consequences, such as complacency toward improving inequality, are discussed.

Central retinal artery occlusion as a presenting symptom in Eales' disease: a case report.

BACKGROUND: Eales' disease is an idiopathic peripheral retinal vasculopathy characterized by retinal phlebitis, ischemia, retinal neovascularization, and recurrent vitreous hemorrhages. But CRAO is an unusual presentation. CASE PRESENTATION: A 27-year-old healthy female nurse of Indian descent presented with sudden vision loss in her right eye upon awakening. Central retinal artery occlusion (CRAO), combined with mild central retinal vein occlusion (CRVO), was diagnosed. During the second of three consecutive sessions of hyperbaric oxygen treatments, her vision rapidly improved. One week later, she developed peripheral phlebitis in the same eye. Infectious, inflammatory, and hematologic etiologies were excluded. The systemic evaluation was normal except for a positive Mantoux tuberculin skin test. Following systemic steroidal treatment, she experienced gradual improvement of her vasculitis. Two weeks later, mild retinal phlebitis appeared in her left eye. Eales' disease was diagnosed after the exclusion of other diseases. CONCLUSION: This is an unusual Eales' disease case, which presented as combined CRAO with mild CRVO. The association of CRAO and Eales' disease is reported here for the first time, to our best knowledge.

Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform.

Reverse genetic systems enable the engineering of RNA virus genomes and are instrumental in studying RNA virus biology. With the recent outbreak of the COVID-19 pandemic, already established methods were challenged by the large genome of SARS-CoV-2. Herein we present an elaborated strategy for the rapid and straightforward rescue of recombinant plus-stranded RNA viruses with high sequence fidelity, using the example of SARS-CoV-2. The strategy called CLEVER (CLoning-free and Exchangeable system for Virus Engineering and Rescue) is based on the intracellular recombination of transfected overlapping DNA fragments allowing the direct mutagenesis within the initial PCR-amplification step. Furthermore, by introducing a linker fragment - harboring all heterologous sequences - viral RNA can directly serve as a template for manipulating and rescuing recombinant mutant virus, without any cloning step. Overall, this strategy will facilitate recombinant SARS-CoV-2 rescue and accelerate its manipulation. Using our protocol, newly emerging variants can quickly be engineered to further elucidate their biology. To demonstrate its potential as a reverse genetics platform for plus-stranded RNA viruses, the protocol has been successfully applied for the cloning-free rescue of recombinant Chikungunya and Dengue virus.

Evaluating CloudResearch's Approved Group as a solution for problematic data quality on MTurk.

Maintaining data quality on Amazon Mechanical Turk (MTurk) has always been a concern for researchers. These concerns have grown recently due to the bot crisis of 2018 and observations that past safeguards of data quality (e.g., approval ratings of 95%) no longer work. To address data quality concerns, CloudResearch, a third-party website that interfaces with MTurk, has assessed ~165,000 MTurkers and categorized them into those that provide high- (~100,000, Approved) and low- (~65,000, Blocked) quality data. Here, we examined the predictive validity of CloudResearch's vetting. In a pre-registered study, participants (N = 900) from the Approved and Blocked groups, along with a Standard MTurk sample (95% HIT acceptance ratio, 100+ completed HITs), completed an array of data-quality measures. Across several indices, Approved participants (i) identified the content of images more accurately, (ii) answered more reading comprehension questions correctly, (iii) responded to reversed coded items more consistently, (iv) passed a greater number of attention checks, (v) self-reported less cheating and actually left the survey window less often on easily Googleable questions, (vi) replicated classic psychology experimental effects more reliably, and (vii) answered AI-stumping questions more accurately than Blocked participants, who performed at chance on multiple outcomes. Data quality of the Standard sample was generally in between the Approved and Blocked groups. We discuss how MTurk's Approval Rating system is no longer an effective data-quality control, and we discuss the advantages afforded by using the Approved group for scientific studies on MTurk.

Implicit Bias Reflects the Company That Words Keep.

In everyday language, concepts appear alongside (i.e., collocate with) related concepts. Societal biases often emerge in these collocations; e.g., female (vs. male) names collocate with art- (vs. science-) related concepts, and African American (vs. White American) names collocate with negative (vs. positive) concepts. It is unknown whether such collocations merely reflect societal biases or contribute to them. Concepts that are themselves neutral in valence but nevertheless collocate with valenced concepts provide a unique opportunity to address this question. For example, when asked, most people evaluate the concept "cause" as neutral, but "cause" is frequently followed by negative concepts (e.g., death, pain, and trouble). We use such semantically prosodic concepts to test the influence of collocation on the emergence of implicit bias: do neutral concepts that frequently collocate with valenced concepts have corresponding implicit bias? In evaluative priming tasks, participants evaluated positive/negative nouns (Study 1) or pictures (Study 2) after seeing verb primes that were (a) strongly valenced (e.g., hate and comfort), (b) neutral in valence but collocated with valenced concepts in corpora (e.g., ease and gain), or (c) neutral in valence and not collocated with valenced concepts in corpora (e.g., reply and describe). Throughout, neutral primes with positive (negative) collocates facilitated the evaluation of positive (negative) targets much like strongly valenced primes, whereas neutral primes without valenced collocates did not. That neutral concepts with valenced collocates parallel the influence of valenced concepts suggests that their collocations in natural language may be sufficient for fostering implicit bias. Societal implications of the causal embedding hypothesis are discussed.

Targeted proteoform mapping uncovers specific Neurexin-3 variants required for dendritic inhibition.

The diversification of cell adhesion molecules by alternative splicing is proposed to underlie molecular codes for neuronal wiring. Transcriptomic approaches mapped detailed cell-type-specific mRNA splicing programs. However, it has been hard to probe the synapse-specific localization and function of the resulting protein splice isoforms, or "proteoforms," in vivo. We here apply a proteoform-centric workflow in mice to test the synapse-specific functions of the splice isoforms of the synaptic adhesion molecule Neurexin-3 (NRXN3). We uncover a major proteoform, NRXN3 AS5, that is highly expressed in GABAergic interneurons and at dendrite-targeting GABAergic terminals. NRXN3 AS5 abundance significantly diverges from Nrxn3 mRNA distribution and is gated by translation-repressive elements. Nrxn3 AS5 isoform deletion results in a selective impairment of dendrite-targeting interneuron synapses in the dentate gyrus without affecting somatic inhibition or glutamatergic perforant-path synapses. This work establishes cell- and synapse-specific functions of a specific neurexin proteoform and highlights the importance of alternative splicing regulation for synapse specification.

Mother Nature's Fury: Antagonist Metaphors for Natural Disasters Increase Forecasts of Their Severity and Encourage Evacuation.

Natural disasters are often described as having antagonistic qualities (e.g., wildfires ravage). The information deficit model presumes that when people assess the risk of weather hazards, they ignore irrelevant metaphoric descriptors. However, metaphoric frames affect reasoning. The current research assessed whether antagonist metaphors for natural disasters affect perceptions of the risk they pose. Three studies (N = 1,936) demonstrated that participants forecasted an antagonist-framed natural hazard as being more severe, and intended to evacuate more often, than a literal-framed natural hazard. Thus, the metaphorical language used to discuss natural disasters deserves consideration in the development of effective risk communication.

CYSTOID MACULAR EDEMA AFTER FOUR-POINT SCLERAL FIXATION OF INTRAOCULAR LENS.

PURPOSE: To assess the incidence of cystoid macular edema (CME) associated with 4-point Gore-Tex suture intraocular lens (IOL) scleral fixation, before and after institution of routine intravitreal triamcinolone acetonide prophylaxis and long-term topical nonsteroidal anti-inflammatory drug usage. METHODS: Consecutive patients were included after IOL implantation with concurrent pars plana vitrectomy for spontaneous IOL dislocation due to pseudoexfoliation syndrome. We compared short-term prophylactic nonsteroidal anti-inflammatory drugs only (Group A) to prophylactic intravitreal triamcinolone acetonide and long-term nonsteroidal anti-inflammatory drugs (Group B). RESULTS: Twenty-six eyes of 26 patients with pseudoexfoliation syndrome and spontaneous IOL dislocation were studied. Mean logMAR visual acuity improved from 1.27 ± 0.80 (20/375 Snellen equivalent) preoperatively to 0.46 ± 0.39 (Snellen 20/43) postoperatively (P < 0.001). Visual outcomes were similar for Groups A and B. In Group A, 10/16 eyes had CME, 4/16 had chronic CME longer than 6 months, and 1 longer than 12 months. In Group B, 1/10 had CME (which was both chronic and refractory). CONCLUSION: In eyes with pseudoexfoliation syndrome and spontaneous IOL dislocation, 4-point Gore-Tex suture IOL ab externo fixation yielded good visual outcomes, although CME was observed more than reported elsewhere. Prophylactic intravitreal triamcinolone acetonide and long-term nonsteroidal anti-inflammatory drugs seem to reduce the risk of postoperative CME.

Generation of iPSC line from a Parkinson patient with PARK7 mutation and CRISPR-edited Gibco human episomal iPSC line to mimic PARK7 mutation.

Mutations in the oncogene PARK7, which codes for DJ-1, have been associated with early-onset autosomal recessive Parkinson's disease (PD); however, the exact role of DJ-1 in PD remains elusive. Fibroblasts from a PD patient with a uniparental disomy, 1 bp deletion in PARK7 were reprogrammed into the induced pluripotent stem cell (iPSC) line: NIHTVBi015-A. For control purposes, CRISPR-Cas9 editing was used to mimic the mutation in the Gibco Human Episomal iPSC line: TMOi001-A is the control line (A18945) and TMOi001-A-3 is the control-edited line (2B10). All 3 lines exhibit normal karyotyping and expression of pluripotent markers: OCT4, SOX2, and NANOG. These lines provide a translational environment to study DJ-1-related function in PD.

Longitudinal Characterization of Transcriptomic, Functional, and Morphological Features in Human iPSC-Derived Neurons and Their Application to Investigate Translational Progranulin Disease Biology.

The disease biology of frontotemporal lobe dementia (FTD) is complex and not fully understood, with limited translational value appreciated from animal models to date. Human cellular systems that can recapitulate phenotypic features of disease offer promise as translational tools to not only increase our understanding of disease processes but also increase the probability of success of translating novel treatment options to patients. However not all researchers may necessarily have access to well-characterized induced pluripotent stem cell (iPSC)-derived human neurons. As an example, we therefore comprehensively profiled phenotypic features over time in one commercially-available IPSC-derived human neuron cell line. This included systems-level assessments of neurite outgrowth dynamics, neuronal network function, and genome-wide gene expression. By investigating progranulin biology as an example we then demonstrated the utility of these cells as a tool to investigate human disease biology. For example, by using the siRNA-mediated knockdown of the progranulin (GRN) gene, we demonstrated the establishment of an isogenic human cellular model to facilitate translational FTD research. We reproduced findings from rodent neurons by demonstrating that recombinant progranulin (rPGRN) mediated neuroprotection. Contrary to previous rodent data, in our human cellular models, growth factor treatment showed no consistent sensitivity to modulate neurite outgrowth dynamics. Our study further provides the first evidence that rRPGRN modulated neuronal firing and synchrony in human neurons. Taken together, our datasets are a valuable systems-level resource demonstrating the utility of the tested commercially-available human iPSC neurons for investigating basic human neurobiology, translational neuroscience, and drug discovery applications in neurodegenerative and other CNS diseases.

The road ahead for health and lifespan interventions.

Aging is a modifiable risk factor for most chronic diseases and an inevitable process in humans. The development of pharmacological interventions aimed at delaying or preventing the onset of chronic conditions and other age-related diseases has been at the forefront of the aging field. Preclinical findings have demonstrated that species, sex and strain confer significant heterogeneity on reaching the desired health- and lifespan-promoting pharmacological responses in model organisms. Translating the safety and efficacy of these interventions to humans and the lack of reliable biomarkers that serve as predictors of health outcomes remain a challenge. Here, we will survey current pharmacological interventions that promote lifespan extension and/or increased healthspan in animals and humans, and review the various anti-aging interventions selected for inclusion in the NIA's Interventions Testing Program as well as the ClinicalTrials.gov database that target aging or age-related diseases in humans.

The War on Prevention II: Battle Metaphors Undermine Cancer Treatment and Prevention and Do Not Increase Vigilance.

Bellicose metaphors for cancer are ubiquitous. But are they good metaphors for health communicators to use? Because metaphors can guide reasoning about abstract concepts, framing cancer with metaphors of battle, war, and enemies leads people to apply attributes of these concepts to cancer. The current research investigates how this affects inferences about cancer treatment, prevention, and monitoring. Battles and war are usually seen as being difficult. Indeed, reading about a person's "battle" or "fight" against cancer makes cancer treatment seem more difficult (studies 1-4). One way to approach a battle is to surrender and give up control. Consistent with this implication, battle metaphors increase fatalistic beliefs about cancer prevention (e.g. believing that there is little one can do to prevent getting cancer; study 3). Finally, even though battles invoke vigilance and action, Study 4 failed to find that such metaphors motivate people to immediately see their doctor when imagining a cancer scare. These findings suggest that bellicose metaphors for cancer can influence the health beliefs of nonpatients in ways that may make them less willing to enact healthy behaviors.

SAM68-Specific Splicing Is Required for Proper Selection of Alternative 3' UTR Isoforms in the Nervous System.

Neuronal alternative splicing is a core mechanism for functional diversification. We previously found that STAR family proteins (SAM68, SLM1, SLM2) regulate spatiotemporal alternative splicing in the nervous system. However, the whole aspect of alternative splicing programs by STARs remains unclear. Here, we performed a transcriptomic analysis using SAM68 knockout and SAM68/SLM1 double-knockout midbrains. We revealed different alternative splicing activity between SAM68 and SLM1; SAM68 preferentially targets alternative 3' UTR exons. SAM68 knockout causes a long-to-short isoform switch of a number of neuronal targets through the alteration in alternative last exon (ALE) selection or alternative polyadenylation. The altered ALE usage of a novel target, interleukin 1 receptor accessory protein (Il1rap), results in remarkable conversion from a membrane-bound type to a secreted type in Sam68KO brains. Proper ALE selection is necessary for IL1RAP neuronal function. Thus the SAM68-specific splicing program provides a mechanism for neuronal selection of alternative 3' UTR isoforms.

Social Exclusion Shifts Personal Network Scope.

Social exclusion has the potential to alter subsequent social interactions with the members of personal networks, especially given their online availability in contemporary life. Nonetheless, there is minimal research examining how social challenges such as exclusion alter ensuing interactions with personal ties. Here, we tested whether being excluded during a social interaction changed which relationships are most salient in an ostensibly unrelated, online news sharing task. Across three operationalizations of tie strength, exclusion (vs. inclusion) increased sharing to close friends, but (unexpectedly) decreased sharing to close family members. The findings provide preliminary evidence that negative encounters may shift attention toward certain types of network ties and away from others. Future work is needed to examine how social experiences influence personal network scope - i.e., who comes to mind - in the background of daily life.

Dopamine: Functions, Signaling, and Association with Neurological Diseases.

The dopaminergic system plays important roles in neuromodulation, such as motor control, motivation, reward, cognitive function, maternal, and reproductive behaviors. Dopamine is a neurotransmitter, synthesized in both central nervous system and the periphery, that exerts its actions upon binding to G protein-coupled receptors. Dopamine receptors are widely expressed in the body and function in both the peripheral and the central nervous systems. Dopaminergic signaling pathways are crucial to the maintenance of physiological processes and an unbalanced activity may lead to dysfunctions that are related to neurodegenerative diseases. Unveiling the neurobiology and the molecular mechanisms that underlie these illnesses may contribute to the development of new therapies that could promote a better quality of life for patients worldwide. In this review, we summarize the aspects of dopamine as a catecholaminergic neurotransmitter and discuss dopamine signaling pathways elicited through dopamine receptor activation in normal brain function. Furthermore, we describe the potential involvement of these signaling pathways in evoking the onset and progression of some diseases in the nervous system, such as Parkinson's, Schizophrenia, Huntington's, Attention Deficit and Hyperactivity Disorder, and Addiction. A brief description of new dopaminergic drugs recently approved and under development treatments for these ailments is also provided.

Differences in Stability, Activity and Mutation Effects Between Human and Mouse Leucine-Rich Repeat Kinase 2.

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene have been implicated in the pathogenesis of Parkinson's disease (PD). Identification of PD-associated LRRK2 mutations has led to the development of novel animal models, primarily in mice. However, the characteristics of human LRRK2 and mouse Lrrk2 protein have not previously been directly compared. Here we show that proteins from different species have different biochemical properties, with the mouse protein being more stable but having significantly lower kinase activity compared to the human orthologue. In examining the effects of PD-associated mutations and risk factors on protein function, we found that conserved substitutions such as G2019S affect human and mouse LRRK2 proteins similarly, but variation around position 2385, which is not fully conserved between humans and mice, induces divergent in vitro behavior. Overall our results indicate that structural differences between human and mouse LRRK2 are likely responsible for the different properties we have observed for these two species of LRRK2 protein. These results have implications for disease modelling of LRRK2 mutations in mice and on the testing of pharmacological therapies in animals.

Are Manipulation Checks Necessary?

Researchers are concerned about whether manipulations have the intended effects. Many journals and reviewers view manipulation checks favorably, and they are widely reported in prestigious journals. However, the prototypical manipulation check is a verbal (rather than behavioral) measure that always appears at the same point in the procedure (rather than its order being varied to assess order effects). Embedding such manipulation checks within an experiment comes with problems. While we conceptualize manipulation checks as measures, they can also act as interventions which initiate new processes that would otherwise not occur. The default assumption that manipulation checks do not affect experimental conclusions is unwarranted. They may amplify, undo, or interact with the effects of a manipulation. Further, the use of manipulation checks in mediational analyses does not rule out confounding variables, as any unmeasured variables that correlate with the manipulation check may still drive the relationship. Alternatives such as non-verbal and behavioral measures as manipulation checks and pilot testing are less problematic. Reviewers should view manipulation checks more critically, and authors should explore alternative methods to ensure the effectiveness of manipulations.

Proteomic analysis reveals co-ordinated alterations in protein synthesis and degradation pathways in LRRK2 knockout mice.

Mutations in leucine-rich repeat kinase 2 (LRRK2) segregate with familial Parkinson's disease (PD) and genetic variation around LRRK2 contributes to risk of sporadic disease. Although knockout (KO) of Lrrk2 or knock-in of pathogenic mutations into the mouse germline does not result in a PD phenotype, several defects have been reported in the kidneys of Lrrk2 KO mice. To understand LRRK2 function in vivo, we used an unbiased approach to determine which protein pathways are affected in LRRK2 KO kidneys. We nominated changes in cytoskeletal-associated proteins, lysosomal proteases, proteins involved in vesicular trafficking and in control of protein translation. Changes were not seen in mice expressing the pathogenic G2019S LRRK2 mutation. Using cultured epithelial kidney cells, we replicated the accumulation of lysosomal proteases and demonstrated changes in subcellular distribution of the cation-independent mannose-6-phosphate receptor. These results show that loss of LRRK2 leads to co-ordinated responses in protein translation and trafficking and argue against a dominant negative role for the G2019S mutation.

Hexokinases link DJ-1 to the PINK1/parkin pathway.

BACKGROUND: Early onset Parkinson's disease is caused by variants in PINK1, parkin, and DJ-1. PINK1 and parkin operate in pathways that preserve mitochondrial integrity, but the function of DJ-1 and how it relates to PINK1 and parkin is poorly understood. METHODS: A series of unbiased high-content screens were used to analyze changes at the protein, RNA, and metabolite level in rodent brains lacking DJ-1. Results were validated using targeted approaches, and cellular assays were performed to probe the mechanisms involved. RESULTS: We find that in both rat and mouse brains, DJ-1 knockout results in an age-dependent accumulation of hexokinase 1 in the cytosol, away from its usual location at the mitochondria, with subsequent activation of the polyol pathway of glucose metabolism in vivo. Both in the brain and in cultured cells, DJ-1 deficiency is associated with accumulation of the phosphatase PTEN that antagonizes the kinase AKT. In cells, addition of an inhibitor of AKT (MK2206) or addition of a peptide to dissociate association of hexokinases from mitochondria both inhibit the PINK1/parkin pathway, which works to maintain mitochondrial integrity. CONCLUSION: Hexokinases are an important link between three major genetic causes of early onset Parkinson's disease. Because aging is associated with deregulated nutrient sensing, these results help explain why DJ-1 is associated with age-dependent disease.