Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs.

Translating genetic findings for neurodevelopmental and psychiatric disorders (NPDs) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop codons (iSTOP) that lead to mRNA nonsense-mediated decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 genes. Using RNA sequencing (RNA-seq), we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Despite high editing efficiency, three schizophrenia risk genes (SETD1A, TRIO, and CUL1) only had heterozygous LoF alleles, suggesting their essential roles for cell growth. We found that CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.

Scaled and Efficient Derivation of Loss of Function Alleles in Risk Genes for Neurodevelopmental and Psychiatric Disorders in Human iPSC.

Translating genetic findings for neurodevelopmental and psychiatric disorders (NPD) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, here we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop-codons (iSTOP) that lead to mRNA nonsense-mediated-decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 NPD genes. Using RNAseq, we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Interestingly, for three schizophrenia risk genes (SETD1A, TRIO, CUL1), despite the high efficiency of base editing, we only obtained heterozygous LoF alleles, suggesting their essential roles for cell growth. We replicated the reported neural phenotypes of SHANK3-haploinsufficiency and found CUL1-LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.

Methionyl-tRNA synthetase synthetic and proofreading activities are determinants of antibiotic persistence.

Bacterial antibiotic persistence is a phenomenon where bacteria are exposed to an antibiotic and the majority of the population dies while a small subset enters a low metabolic, persistent, state and are able to survive. Once the antibiotic is removed the persistent population can resuscitate and continue growing. Several different molecular mechanisms and pathways have been implicated in this phenomenon. A common mechanism that may underly bacterial antibiotic persistence is perturbations in protein synthesis. To investigate this mechanism, we characterized four distinct metG mutants for their ability to increase antibiotic persistence. Two metG mutants encode changes near the catalytic site of MetRS and the other two mutants changes near the anticodon binding domain. Mutations in metG are of particular interest because MetRS is responsible for aminoacylation both initiator tRNAMet and elongator tRNAMet indicating that these mutants could impact translation initiation and/or translation elongation. We observed that all the metG mutants increased the level of antibiotic persistence as did reduced transcription levels of wild type metG. Although, the MetRS variants did not have an impact on MetRS activity itself, they did reduce translation rates. It was also observed that the MetRS variants affected the proofreading mechanism for homocysteine and that these mutants' growth is hypersensitive to homocysteine. Taken together with previous findings, our data indicate that both reductions in cellular Met-tRNAMet synthetic capacity and reduced proofreading of homocysteine by MetRS variants are positive determinants for bacterial antibiotic persistence.

Using the Woodcock-Johnson IV tests of cognitive abilities to detect feigned ADHD.

Research has suggested that many young adults can successfully feign ADHD, reporting clinically significant symptom levels and displaying deficits on cognitive tasks when asked to do so. Standalone performance validity tests (PVTs) have shown some success in identifying feigned ADHD, but these tests are rarely used in typical ADHD evaluation batteries. The present study attempted to develop embedded PVT indices from the Woodcock-Johnson IV Tests of Cognitive Abilities (WJ-IV). College students (N = 150) completed a battery including tasks from the WJ-IV, as well as an established standalone PVT and a rating scale measuring ADHD and related symptoms. Thirty of the students had been professionally diagnosed with ADHD; of the remaining 120 students, half were asked to perform honestly and to the best of their ability on the battery, whereas the other half were asked to try to simulate ADHD. Several processing speed and working memory scores from the WJ-IV effectively identified students feigning ADHD, detecting at least 50% of those students at score cutoffs that also maintained specificity of 90% or more, close to the efficiency of the standalone PVT. In addition, students with ADHD diagnoses generally did not show deficits on the WJ-IV. Implications for practice and future research are discussed.

Deacylated tRNA Accumulation Is a Trigger for Bacterial Antibiotic Persistence Independent of the Stringent Response.

Bacterial antibiotic persistence occurs when bacteria are treated with an antibiotic and the majority of the population rapidly dies off, but a small subpopulation enters into a dormant, persistent state and evades death. Diverse pathways leading to nucleoside triphosphate (NTP) depletion and restricted translation have been implicated in persistence, suggesting alternative redundant routes may exist to initiate persister formation. To investigate the molecular mechanism of one such pathway, functional variants of an essential component of translation (phenylalanyl-tRNA synthetase [PheRS]) were used to study the effects of quality control on antibiotic persistence. Upon amino acid limitation, elevated PheRS quality control led to significant decreases in aminoacylated tRNAPhe accumulation and increased antibiotic persistence. This increase in antibiotic persistence was most pronounced (65-fold higher) when the relA-encoded tRNA-dependent stringent response was inactivated. The increase in persistence with elevated quality control correlated with ∼2-fold increases in the levels of the RNase MazF and the NTPase MazG and a 3-fold reduction in cellular NTP pools. These data reveal a mechanism for persister formation independent of the stringent response where reduced translation capacity, as indicated by reduced levels of aminoacylated tRNA, is accompanied by active reduction of cellular NTP pools which in turn triggers antibiotic persistence. IMPORTANCE Bacterial antibiotic persistence is a transient physiological state wherein cells become dormant and thereby evade being killed by antibiotics. Once the antibiotic is removed, bacterial persisters are able to resuscitate and repopulate. It is thought that antibiotic bacterial persisters may cause reoccurring infections in the clinical setting. The molecular triggers and pathways that cause bacteria to enter into the persister state are not fully understood. Our results suggest that accumulation of deacylated tRNA is a trigger for antibiotic persistence independent of the RelA-dependent stringent response, a pathway thought to be required for persistence in many organisms. Overall, this provides a mechanism where changes in translation quality control in response to physiological cues can directly modulate bacterial persistence.

Did Amino Acid Side Chain Reactivity Dictate the Composition and Timing of Aminoacyl-tRNA Synthetase Evolution?

The twenty amino acids in the standard genetic code were fixed prior to the last universal common ancestor (LUCA). Factors that guided this selection included establishment of pathways for their metabolic synthesis and the concomitant fixation of substrate specificities in the emerging aminoacyl-tRNA synthetases (aaRSs). In this conceptual paper, we propose that the chemical reactivity of some amino acid side chains (e.g., lysine, cysteine, homocysteine, ornithine, homoserine, and selenocysteine) delayed or prohibited the emergence of the corresponding aaRSs and helped define the amino acids in the standard genetic code. We also consider the possibility that amino acid chemistry delayed the emergence of the glutaminyl- and asparaginyl-tRNA synthetases, neither of which are ubiquitous in extant organisms. We argue that fundamental chemical principles played critical roles in fixation of some aspects of the genetic code pre- and post-LUCA.

Differential Diagnosis of Sluggish Cognitive Tempo Symptoms in College Students.

Objective: Sluggish cognitive tempo (SCT) refers to a set of symptoms that prior research has found to be related to several different psychological disorders, especially the predominantly inattentive presentation of ADHD. This study collected evidence relevant to the question of whether SCT is a distinct disorder. Method: College students (N = 910) completed measures of SCT, ADHD, depression, anxiety, sleep quality, and substance misuse. Results: Students reporting clinically high SCT (reporting at least five symptoms often or very often) had significantly higher levels and rates of other types of psychopathology. Moreover, when students reporting clinically significant levels of ADHD, depression, and anxiety symptoms, poor sleep quality, or hazardous levels of alcohol or cannabis use were removed, very few students reporting high SCT remained (only 4.8% of the original high-SCT group). Conclusion: SCT may be best thought of as a symptom set common to many types of psychopathology, and it may be caused by sleep problems or substance misuse as well.

Profiles of Diagnosed and Undiagnosed College Students Meeting ADHD Symptom Criteria.

Objective: This study compared the psychological profiles of college students who self-reported an ADHD diagnosis in contrast to students without a diagnosis who reported above-threshold ADHD symptoms. Method: A large sample of college students yielded four subgroups: students with a self-reported diagnosis who met symptom criteria (Diagnosed, n = 40), students with a self-reported diagnosis who did not meet symptom criteria (Low Symptoms, n = 47), students who had no diagnosis and did not meet symptom criteria (Controls, n = 87), and students who had no diagnosis yet met symptom criteria (Undiagnosed, n = 38). All participants completed a battery of self-report rating scales assessing a variety of symptom and impairment domains. Results: The Diagnosed and Undiagnosed groups differed significantly from Controls on all domains, while the Low Symptom group did not differ significantly from Controls in any domain. Conclusion: Implications for engaging with reported diagnosis of ADHD in the college population are discussed.

Arabidopsis PIRL6 Is Essential for Male and Female Gametogenesis and Is Regulated by Alternative Splicing.

Plant intracellular Ras-group leucine-rich repeat (LRR) proteins (PIRLs) are related to Ras-interacting animal LRR proteins that participate in developmental cell signaling. Systematic knockout analysis has implicated some members of the Arabidopsis (Arabidopsis thaliana) PIRL family in pollen development. However, for PIRL6, no bona fide knockout alleles have been recovered, suggesting that it may have an essential function in both male and female gametophytes. To test this hypothesis, we investigated PIRL6 expression and induced knockdown by RNA interference. Knockdown triggered defects in gametogenesis, resulting in abnormal pollen and early developmental arrest in the embryo sac. Consistent with this, PIRL6 was expressed in gametophytes: functional transcripts were detected in wild-type flowers but not in sporocyteless (spl) mutant flowers, which do not produce gametophytes. A genomic PIRL6-GFP fusion construct confirmed expression in both pollen and the embryo sac. Interestingly, PIRL6 is part of a convergent overlapping gene pair, a scenario associated with an increased likelihood of alternative splicing. We detected multiple alternative PIRL6 mRNAs in vegetative organs and spl mutant flowers, tissues that lacked the functionally spliced transcript. cDNA sequencing revealed that all contained intron sequences and premature termination codons. These alternative mRNAs accumulated in the nonsense-mediated decay mutant upf3, indicating that they are normally subjected to degradation. Together, these results demonstrate that PIRL6 is required in both male and female gametogenesis and suggest that sporophytic expression is negatively regulated by unproductive alternative splicing. This posttranscriptional mechanism may function to minimize PIRL6 protein expression in sporophyte tissues while allowing the overlapping adjacent gene to remain widely transcribed.

Shifting understandings of labour pain in Canadian medical history.

While pain in childbirth is a universal, cross-cultural, biological reality, individual experiences and perceptions of this pain are historically and culturally specific. At the turn of the 20th century-a key period in terms of both the medicalisation of birth and the professionalisation of obstetrics in the Canadian context-Canadian physicians understood and conceptualised 'birth pangs' in a number of varying (and at times competing) ways. Throughout the 19th century, doctors emphasised the broader utility of pain as a diagnostic tool and a physiologically necessary part of the birthing process. With the advent of anaesthetics, including chloroform and ether, however, a growing subset of the medical profession simultaneously lauded the professional, physiological, and humanitarian benefits of pain relief. By the first decades of the 20th century, shifting understandings of labour pain-and particularly growing distinctions between 'pain' and 'contraction' in mainstream medical discourses-underscored the increasing use of obstetric anaesthesia. Drawing on a broad range of medical texts and professional literature, and focusing on a key historical moment when the introduction and adoption of a new medical technology opened up possibilities for professional debate, this paper unpacks both the micropolitics and the macropolitics of shifting understandings of labour pain in modern Canadian medical history.

Indirect tRNA aminoacylation during accurate translation and phenotypic mistranslation.

The fact that most bacteria do not contain a full set of aminoacyl-tRNA synthetases (aaRS) is often underappreciated. In the absence of asparaginyl-tRNA and/or glutaminyl-tRNA synthetase (AsnRS and GlnRS), Asn-tRNAAsn and/or Gln-tRNAGln are produced by an indirect tRNA aminoacylation pathway that relies on misacylation of these two tRNAs by two different misacylating aaRSs, followed by transamidation by an amidotransferase (GatCAB in bacteria). This review highlights the central importance of indirect tRNA aminoacylation to accurate protein translation, mechanistic peculiarities that appear to be unique to this system, and the newly recognized connection between indirect tRNA aminoacylation and mistranslation as a strategy used by bacteria to respond to environmental stressors like antibiotics.

Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells.

Many plasmids used for gene cloning and heterologous protein expression in Escherichia coli cells are low copy number or single copy number plasmids. The extraction of these types of plasmids from small bacterial cell cultures produces low DNA yields. In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium. TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB. By contrast, increasing ampicillin concentration to enhance plasmid retention did not improve plasmid DNA recovery. These experiments demonstrate that yields of low and single copy number plasmid DNAs from minipreps can be strongly enhanced using simple and inexpensive media.

Executive Dysfunction and Functional Impairment Associated With Sluggish Cognitive Tempo in Emerging Adulthood.

OBJECTIVE: Research has identified a relationship between sluggish cognitive tempo (SCT) symptoms and symptoms of ADHD, anxiety, and depression; however, no study has controlled for symptoms of ADHD, anxiety, and depression when examining impairment related to SCT symptoms. This study aimed to examine (a) the extent to which functional impairment and executive function (EF) problems were accounted for by SCT symptoms when controlling for ADHD, anxiety, and depression symptoms, and (b) which type of symptoms were associated with the greatest amount of impairment. METHOD: College students ( N = 458) completed self-report scales of ADHD, SCT, anxiety, and depression symptoms, as well as functional impairment and EF problems. RESULTS: Thirteen percent of the sample was found to have high levels of SCT symptoms. SCT symptoms showed a moderate to strong correlation with the other symptom sets; however, high levels of SCT symptoms often occurred separate from high levels of ADHD, anxiety, or depression symptoms. SCT symptoms accounted for the most unique variance for both EF problems and functional impairment. Students with high levels of SCT symptoms, with or without high levels of ADHD symptoms, exhibited more impairment and EF problems than the controls. CONCLUSION: SCT is a clinical construct worthy of additional study, particularly among college students.

Sluggish Cognitive Tempo and Speed of Performance.

OBJECTIVE: This study examined whether college students who reported higher levels of sluggish cognitive tempo (SCT) symptoms were actually more "sluggish" in their performance while completing speeded cognitive and academic measures. METHOD: College students ( N = 253) completed self-reports of SCT and their reading and test-taking abilities as well as tests of processing speed, reading fluency, and reading comprehension. RESULTS: Across all variables, SCT symptoms were most significantly associated with self-reported difficulty on timed reading tasks. However, students with high SCT scores were not significantly slower than controls on any of the timed tasks. CONCLUSION: In college students, self-reports of high SCT levels do not suggest actual slow performance on cognitive and academic tasks.

Characterization of tunnel mutants reveals a catalytic step in ammonia delivery by an aminoacyl-tRNA amidotransferase.

The Helicobacter pylori Asp-tRNA(A) (sn) /Glu-tRNA(G) (ln) amidotransferase (GatCAB) utilizes an uncommonly hydrophilic, ~ 40 Å ammonia tunnel for ammonia/ammonium transport between isolated active sites. Hydrophilicity of this tunnel requires a distinct ammonia transport mechanism, which hypothetically occurs through a series of deprotonation and protonation steps. To explore the initiation of this relay mechanism, the highly conserved tunnel residue D185 (in the GatA subunit) was enzymatically and computationally investigated by comparing D185A, D185N, and D185E mutant enzymes to wild-type GatCAB. Our results indicate that D185 acts as an acid/base residue, participating directly in catalysis. To our knowledge, this is the first example of acid/base chemistry in a glutamine-dependent amidotransferase ammonia tunnel.

"The Luxurious Daughters of Artificial Life": Female "Delicacy" and Pain in Late-Victorian Advice Literature.

The second half of the 19th century marked the rise of obstetrics as a legitimate physician-dominated medical specialty. In this period of transition, distanced from traditional cultures of social childbirth but not yet embracing hospital deliveries, many middle-class North American women turned to prescriptive literature to fill a crucial gap. In the medical advice they directed at young wives and expectant mothers, physicians consistently emphasized the middle-class woman's heightened sensitivity to the pain of giving birth, relying on arguments that resonated with the class, gender, and racial tensions of the late-Victorian period while consistently reaffirming physicians' expanding authority.

Enhancement of plasmid DNA transformation efficiencies in early stationary-phase yeast cell cultures.

Chemical-based methods have been developed for transformation of DNA into log-phase cells of the budding yeast Saccharomyces cerevisiae with high efficiency. Transformation of early stationary-phase cells, e.g. cells grown in overnight liquid cultures or as colonies on plates, is less efficient than log-phase cells but is simpler and more adaptable to high-throughput projects. In this study we have tested different approaches for transformation of early stationary-phase cell cultures and identified a method utilizing polyethylene glycol (PEG), lithium acetate and dimethyl sulphoxide (DMSO) as the most efficient. Plasmid DNA transformations using this method could be improved modestly by allowing cells to recover from the chemical treatment in rich broth before plating to selective media. Strong increases in transformation efficiencies were observed when cells were treated briefly with dithiothreitol (DTT). Tests using several different yeast strain backgrounds indicated that DTT treatment could enhance transformation efficiencies by up to 40-fold. Evaluation of multiple parameters affecting the efficiency of the method led to development of an optimized protocol achieving > 50 000 transformants/µg DNA in most backgrounds tested.