Improved temporal resolution in ultrafast electron diffraction measurements through THz compression and time-stamping.

We present an experimental demonstration of ultrafast electron diffraction (UED) with THz-driven electron bunch compression and time-stamping that enables UED probes with improved temporal resolution. Through THz-driven longitudinal bunch compression, a compression factor of approximately four is achieved. Moreover, the time-of-arrival jitter between the compressed electron bunch and a pump laser pulse is suppressed by a factor of three. Simultaneously, the THz interaction imparts a transverse spatiotemporal correlation on the electron distribution, which we utilize to further enhance the precision of time-resolved UED measurements. We use this technique to probe single-crystal gold nanofilms and reveal transient oscillations in the THz near fields with a temporal resolution down to 50 fs. These oscillations were previously beyond reach in the absence of THz compression and time-stamping.

Recovering from the stress of the COVID-19 pandemic.

For our special issue on stress, we asked scientists about recovering from the stress of the pandemic, including some who shared insights with us in mid-2020. They discuss the importance of teamwork, reassessing priorities, and the added stresses of the cost-of-living crisis, funding cuts, and retaining scientists in academia.

Juneteenth in STEMM and the barriers to equitable science.

We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists.

Metatranscriptomic analysis uncovers prevalent viral ORFs compatible with mitochondrial translation.

RNA viruses are ubiquitous components of the global virosphere, yet relatively little is known about their genetic diversity or the cellular mechanisms by which they exploit the biology of their diverse eukaryotic hosts. A hallmark of (+)ssRNA (positive single-stranded RNA) viruses is the ability to remodel host endomembranes for their own replication. However, the subcellular interplay between RNA viruses and host organelles that harbor gene expression systems, such as mitochondria, is complex and poorly understood. Here we report the discovery of 763 new virus sequences belonging to the family Mitoviridae by metatranscriptomic analysis, the identification of previously uncharacterized mitovirus clades, and a putative new viral class. With this expanded understanding of the diversity of mitovirus and encoded RNA-dependent RNA polymerases (RdRps), we annotate mitovirus-specific protein motifs and identify hallmarks of mitochondrial translation, including mitochondrion-specific codons. This study expands the known diversity of mitochondrial viruses and provides additional evidence that they co-opt mitochondrial biology for their survival. IMPORTANCE Metatranscriptomic studies have rapidly expanded the cadre of known RNA viruses, yet our understanding of how these viruses navigate the cytoplasmic milieu of their hosts to survive remains poorly characterized. In this study, we identify and assemble 763 new viral sequences belonging to the Mitoviridae, a family of (+)ssRNA viruses thought to interact with and remodel host mitochondria. We exploit this genetic diversity to identify new clades of Mitoviridae, annotate clade-specific sequence motifs that distinguish the mitoviral RdRp, and reveal patterns of RdRp codon usage consistent with translation on host cell mitoribosomes. These results serve as a foundation for understanding how mitoviruses co-opt mitochondrial biology for their proliferation.

In Situ Analysis of Mitochondrial DNA Synthesis Using Metabolic Labeling Coupled to Fluorescence Microscopy.

Metabolic labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) enables the selective labeling of DNA synthesis in live cells. Newly synthesized EdU-containing DNA can be covalently modified after extraction or in fixed cells using copper-catalyzed azide-alkyne cycloaddition "click chemistry" reactions, enabling bioconjugation to various substrates including fluorophores for imaging studies. While often used to study nuclear DNA replication, EdU labeling can also be leveraged to detect the synthesis of organellar DNA in the cytoplasm of Eukaryotic cells. In this chapter, we outline methods for the application of EdU labeling to the study of mitochondrial genome synthesis in fixed cultured human cells, using fluorescent labeling and superresolution light microscopy.

The impact of COVID-19 on sleep for autistic children: A systematic review.

Background: Up to 80% of children with autism experience behavioural sleep problems, predominantly relating to bedtime resistance, sleep anxiety, sleep dysregulation, and shorter duration, which are associated with increased autistic symptom expression and emotional and behavioural difficulties. Researchers predicted the COVID-19 pandemic would worsen sleep and behavioural difficulties for autistic children, due to their need for routine and certainty. This systematic review is the first to focus on delineating the role of sleep disturbance in exacerbating autistic symptoms and internalising and externalising behaviours during the pandemic. Method: In this PROSPERO registered systematic review, we aggregated and synthesised findings from empirical studies from 2020 onwards that included children with autism and examined sleep outcomes, using narrative and framework synthesis due to the variety of methods and designs employed. We identified additional relevant themes through inductive thematic analysis. Results: Seventy-one studies met the search criteria, and we selected seventeen for review following screening and quality assessment. These studies reported mixed findings; with strongest support for worsening of sleep problems typically experienced by autistic children, including difficulties with sleep regulation and shorter sleep duration. Further, sleep problems were associated with increased expression of autistic characteristics. Conclusions: Preliminary findings of worsening sleep and increased autistic characteristics for autistic children throughout the COVID-19 pandemic highlight the need for ongoing, accessible and flexible service provision during exposure to environmental stressors. We propose that behavioural sleep interventions are well suited to telehealth adaptation and play an important role in supporting families when in-person treatment for sleep problems is not possible.

Structured coaching as a means to improve sonographic visualization of the appendix: a quality improvement initiative.

PURPOSE: Ultrasound of the right lower quadrant for appendicitis requires specific training and practice. Improved visualization of the appendix can result in decreased utilization of computed tomography. Increasing the sensitivity of ultrasound for appendicitis thus decreases radiation exposure, reduces cost, and improves the patient healthcare experience in accordance with the three principles of the triple aim designated by the Institute for Healthcare Improvement. The purpose of this study was to examine the effect of a structured training program supplemented by technologist feedback on sonographic visualization of the appendix in a large pediatric health system. METHODS: The baseline ultrasound visualization frequency was computed for 20 ultrasound technologists in a large pediatric health system. Following this, technologists were informed of their individual and relative performance. Those with visualization frequencies less than 75% were provided structured training by a designated sonographer with an appendix visualization frequency above 75% while those with greater than 75% visualization continued to be monitored and informed of their monthly frequency. Following this structured training, appendix visualization frequencies were monitored over the next 5 months. RESULTS: There was no significant effect of sonographer career experience on the baseline appendix visualization frequency. Fourteen of 20 technologists demonstrated improved visualization frequencies post-intervention. The composite visualization frequency among these 20 technologists improved from 66.55 to 69.14%. This resulted in a potential savings of 38 CT scans during the post-intervention period. CONCLUSION: An appendix visualization rate monitoring program coupled with structured training was successful in improving the appendix visualization rate throughout a large pediatric health system. This program demonstrates the positive effect that monitoring and targeted intervention can have on a quality improvement program. The program continues to be carried out as part of a plan-study-do-act cycle.

Understanding the Benefits of Brief Classroom-Based Physical Activity Interventions on Primary School-Aged Children's Enjoyment and Subjective Wellbeing: A Systematic Review.

BACKGROUND: Engaging in classroom-based physical activity (PA) breaks may help increase children's subjective wellbeing and enjoyment of PA, important precursors for children to remain engaged in PA. We conducted a systematic review to investigate the effect of brief classroom-based PA interventions on primary school children's subjective wellbeing and enjoyment of PA. METHODS: Databases were reviewed between the earliest date of records and April 2020. Eligibility criteria included peer-reviewed empirical quantitative or mixed methods studies reporting enjoyment of PA or wellbeing outcomes of participating in a brief classroom-based PA program. Fifteen papers were included. Interventions supported 7022 child participants enrolled at baseline and 6286 children analyzed in interventions across 148 schools. RESULTS: Thirteen studies examined the effect of brief classroom-based PA interventions on subjective wellbeing. Four found a significant positive effect for children in the intervention group (specifically for children's self-efficacy in learning with video exercises, quality of life, and self-confidence). Eight studies examined the effect of brief classroom-based PA interventions on enjoyment of PA. Five studies reported a positive effect for children in the intervention group and only 1 study found a negative effect for 1 grade level of children in the intervention group. The remaining studies with wellbeing and/or enjoyment outcomes reported no significant effect. CONCLUSIONS: Findings from these studies indicate that the integration of brief PA breaks may support PA enjoyment and specific self-beliefs and quality of life perceptions that contribute to children's subjective wellbeing. However, more research is needed to confirm this effect.

Broadband Solenoidal Haloscope for Terahertz Axion Detection.

We introduce the Broadband Reflector Experiment for Axion Detection (BREAD) conceptual design and science program. This haloscope plans to search for bosonic dark matter across the [10^{-3},1] eV ([0.24, 240] THz) mass range. BREAD proposes a cylindrical metal barrel to convert dark matter into photons, which a novel parabolic reflector design focuses onto a photosensor. This unique geometry enables enclosure in standard cryostats and high-field solenoids, overcoming limitations of current dish antennas. A pilot 0.7 m^{2} barrel experiment planned at Fermilab is projected to surpass existing dark photon coupling constraints by over a decade with one-day runtime. Axion sensitivity requires <10^{-20} W/sqrt[Hz] sensor noise equivalent power with a 10 T solenoid and 10 m^{2} barrel. We project BREAD sensitivity for various sensor technologies and discuss future prospects.

Autism Spectrum Disorder and Intellectual Disability: A Pilot Study Exploring Associations between Child Sleep Problems, Child Factors and Parent Factors.

Sleep problems are common in children with autism spectrum disorder (ASD). However, few studies have undertaken group comparisons of sleep profiles and factors associated with poorer sleep between children with ASD without intellectual disability (ID; hereafter referred to as ASD) and ASD with co-occurring ID (hereafter referred to as ASD + ID). This study aimed to (1) compare child (sleep problems and emotional and behavioural problems (EBPs)) and parent factors (parenting stress and mental health) for children with ASD compared to children with ASD + ID, and (2) examine the associations between sleep problems and child and parent factors in both groups. Parents of 56 children with ASD (22 ASD, 34 ASD + ID) aged 6-13 years took part in the study. No statistically significant differences in sleep problems were found between children with ASD compared to children with ASD + ID. However, total EBPs were independently associated with child sleep problems in both groups. Further, 'Self-Absorbed' and 'Communication Disturbance' EBPs were significantly greater in the ASD + ID compared to the ASD group. Overall treatment outcomes for children with ASD may be further improved if consideration is given to the specific types of EBPs being experienced by the child and their association with sleep problems.

Diverse inhibitory projections from the cerebellar interposed nucleus.

The cerebellum consists of parallel circuit modules that contribute to diverse behaviors, spanning motor to cognitive. Recent work employing cell-type-specific tracing has identified circumscribed output channels of the cerebellar nuclei (CbN) that could confer tight functional specificity. These studies have largely focused on excitatory projections of the CbN, however, leaving open the question of whether inhibitory neurons also constitute multiple output modules. We mapped output and input patterns to intersectionally restricted cell types of the interposed and adjacent interstitial nuclei in mice. In contrast to the widespread assumption of primarily excitatory outputs and restricted inferior olive-targeting inhibitory output, we found that inhibitory neurons from this region ramified widely within the brainstem, targeting both motor- and sensory-related nuclei, distinct from excitatory output targets. Despite differences in output targeting, monosynaptic rabies tracing revealed largely shared afferents to both cell classes. We discuss the potential novel functional roles for inhibitory outputs in the context of cerebellar theory.

Multimodal assessment of autophagy in mammalian cells with a novel, LC3-based tandem reporter.

Autophagy is an important intracellular pathway for the degradation of superfluous or harmful subcellular materials, thereby playing a critical role in the maintenance of cell health under normal and stress-related conditions. Researchers interrogating autophagic activity in mammalian cell lines often leverage complementary assay technologies to confirm observations. The Autophagy LC3 HiBiT Reporter assay system utilizes a tandem reporter module, HiBiT-HaloTag, fused to a key marker of autophagic activity, LC3B protein, to enable multiple, cell-based assay modalities. This novel autophagy reporter expressed in a single cell line supports (a) a bioluminescent, homogeneous, plate-reader assay for rapid and quantitative assessment of changes in the level of the LC3-based reporter, (b) a fluorescence-based imaging approach to monitor reporter subcellular distribution in live cells, and (c) an antibody-free, protein blotting method to detect the relative amounts of the LC3-I and LC-II forms of the reporter associated with modulation of autophagic flux. Here we detail protocols for all three assay modalities applied to a U2OS human osteosarcoma cell line stably expressing the novel autophagy reporter, enabling the identification of modulators of autophagic activity and subsequent confirmation of mechanism of action.

Responding and navigating racialized microaggressions in STEM.

While it is commonly thought that microaggressions are isolated incidents, microaggressions are ingrained throughout the academic research institution (Young, Anderson and Stewart 2015; Lee et al. 2020). Persons Excluded from science because of Ethnicity and Race (PEERs) frequently experience microaggressions from various academicians, including graduate students, postdocs and faculty (Asai 2020; Lee et al. 2020). Here, we elaborate on a rationale for concrete actions to cope with and diminish acts of microaggressions that may otherwise hinder the inclusion of PEERs. We encourage Science, Technology, Engineering and Mathematics (STEM) departments and leadership to affirm PEER scholar identities and promote allyship by infusing sensitivity, responsiveness and anti-bias awareness.

Patching the Leaks: Revitalizing and Reimagining the STEM Pipeline.

We identify problematic areas throughout the Science, Technology, Engineering and Mathematics (STEM) pipeline that perpetuate racial disparities in academia. Distinct ways to curtail these disparities include early exposure and access to resources, supportive mentoring networks and comprehensive training programs specifically for racially minoritized students and trainees at each career stage. These actions will revitalize the STEM pipeline.

Regional variations of jaw bone characteristics in an ovariectomized rat model.

Postmenopausal osteoporosis causes severe loss of bone quantity and quality in limb bone but has a lesser effect on jaw bone. Thus, the objective of this study was to examine whether ovariectomy (OVX) and mastication alter the regional variation of jaw bone characteristics. Sprague-Dawley female rats (6 months) were given a bilateral OVX or a sham operation (SHAM) (n = 10 for each group). After 2 months post-OVX, the hemi-mandible from each rat was dissected. A micro-computed tomography based mean, standard deviation (SD), the lower and upper 5th percentile (Low5 and High5) values of tissue mineral density (TMD) histograms were assessed for whole bone (WB), alveolar bone (AB), cortical bone (CB), and trabecular bone (TB) regions. Morphology of TB and periodontal ligament (PDL) was also obtained. Layers of AB were segmented up to 400 µm from the PDL. Mechanical properties at the tissue level were measured by nanoindentation at the same site by a single loading-unloading cycle of indentation in hydration. The AB and TB regions had significantly lower TMD Mean, Low5, and High5 but higher SD than the CB region for both sham and OVX groups (p < 0.01). TMD parameters of the OVX group rapidly increased up to 60 µm away from the PDL and were significantly higher than those of the sham group starting at 280 µm and farther in the CB region (p < 0.05). All values of morphological and nanoindentation parameters were not significantly different between sham and OVX groups (p > 0.06). Estrogen deficiency induced by OVX did not deteriorate bone characteristics including mineral density, morphology, and nanoindentation parameters in rat mandibles. Masticatory loading had an effect on the TMD parameters at the limited region of AB. These results provide insight into why osteoporosis-associated jaw bone fractures are extremely rare.

GLI3 resides at the intersection of hedgehog and androgen action to promote male sex differentiation.

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3XtJ mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3XtJ testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3XtJ mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3XtJ testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract.

Testing adults by questionnaire for social and communication disorders, including autism spectrum disorders, in an adult mental health service population.

OBJECTIVES: Autism is difficult to identify in adults due to lack of validated self-report questionnaires. We compared the effectiveness of the autism-spectrum quotient (AQ) and the Ritvo autism-Asperger's diagnostic scale-revised (RAADS-R) questionnaires in adult mental health services in two English counties. METHODS: A subsample of adults who completed the AQ and RAADS-R were invited to take part in an autism diagnostic observation schedule (ADOS Module 4) assessment with probability of selection weighted by scores on the questionnaires. RESULTS: There were 364 men and 374 women who consented to take part. Recorded diagnoses were most commonly mood disorders (44%) and mental and behavioural disorders due to alcohol/substance misuse (19%), and 4.8% (95% CI [2.9, 7.5]) were identified with autism (ADOS Module 4 10+). One had a pre-existing diagnosis of autism; five (26%) had borderline personality disorders (all female) and three (17%) had mood disorders. The AQ and RAADS-R had fair test accuracy (area under receiver operating characteristic [ROC] curve 0.77 and 0.79, respectively). AQ sensitivity was 0.79 (95% CI [0.54, 0.94]) and specificity was 0.77 (95% CI [0.65, 0.86]); RAADS-R sensitivity was 0.75 (95% CI [0.48, 0.93]) and specificity was 0.71 (95% CI [0.60, 0.81]). CONCLUSIONS: The AQ and RAADS-R can guide decisions to refer adults in mental health services to autism diagnostic services.

Characterization of the HAYSTAC axion dark matter search cavity using microwave measurement and simulation techniques.

Many searches for axion cold dark matter rely on the use of tunable electromagnetic resonators. Current detectors operate at or near microwave frequencies and use cylindrical cavities with cylindrical tuning rods. The cavity performance strongly impacts the signal power of the detector, which is expected to be very small even under optimal conditions. There is strong motivation to characterize these microwave cavities and improve their performance in order to maximize the achievable signal power. We present the results of a study characterizing the HAYSTAC (Haloscope At Yale Sensitive to Axion Cold dark matter) cavity using bead perturbation measurements and detailed 3D electromagnetic simulations. This is the first use of bead perturbation methods to characterize an axion haloscope cavity. In this study, we measured impacts of misalignments on the order of 0.001 in. and demonstrated that the same impacts can be predicted using electromagnetic simulations. We also performed a detailed study of mode crossings and hybridization between the TM010 mode used in operation and other cavity modes. This mixing limits the tuning range of the cavity that can be used during an axion search. By characterizing each mode crossing in detail, we show that some mode crossings are benign and are potentially still useful for data collection. The level of observed agreement between measurements and simulations demonstrates that finite element modeling can capture non-ideal cavity behavior and the impacts of very small imperfections. 3D electromagnetic simulations and bead perturbation measurements are standard tools in the microwave engineering community, but they have been underutilized in an axion cavity design. This work demonstrates their potential to improve understanding of existing cavities and to optimize future designs.

Correction: Comparative evaluation of a new magnetic bead-based DNA extraction method from fecal samples for downstream next-generation 16S rRNA gene sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0202858.].