The CRTC1-SIK1 pathway regulates entrainment of the circadian clock.

Retinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.

Evaluation of BNT162b2 Covid-19 Vaccine in Children Younger than 5 Years of Age.

BACKGROUND: Safe and effective vaccines against coronavirus disease 2019 (Covid-19) are urgently needed in young children. METHODS: We conducted a phase 1 dose-finding study and are conducting an ongoing phase 2-3 safety, immunogenicity, and efficacy trial of the BNT162b2 vaccine in healthy children 6 months to 11 years of age. We present results for children 6 months to less than 2 years of age and those 2 to 4 years of age through the data-cutoff dates (April 29, 2022, for safety and immunogenicity and June 17, 2022, for efficacy). In the phase 2-3 trial, participants were randomly assigned (in a 2:1 ratio) to receive two 3-µg doses of BNT162b2 or placebo. On the basis of preliminary immunogenicity results, a third 3-µg dose (≥8 weeks after dose 2) was administered starting in January 2022, which coincided with the emergence of the B.1.1.529 (omicron) variant. Immune responses at 1 month after doses 2 and 3 in children 6 months to less than 2 years of age and those 2 to 4 years of age were immunologically bridged to responses after dose 2 in persons 16 to 25 years of age who received 30 µg of BNT162b2 in the pivotal trial. RESULTS: During the phase 1 dose-finding study, two doses of BNT162b2 were administered 21 days apart to 16 children 6 months to less than 2 years of age (3-µg dose) and 48 children 2 to 4 years of age (3-µg or 10-µg dose). The 3-µg dose level was selected for the phase 2-3 trial; 1178 children 6 months to less than 2 years of age and 1835 children 2 to 4 years of age received BNT162b2, and 598 and 915, respectively, received placebo. Immunobridging success criteria for the geometric mean ratio and seroresponse at 1 month after dose 3 were met in both age groups. BNT162b2 reactogenicity events were mostly mild to moderate, with no grade 4 events. Low, similar incidences of fever were reported after receipt of BNT162b2 (7% among children 6 months to <2 years of age and 5% among those 2 to 4 years of age) and placebo (6 to 7% among children 6 months to <2 years of age and 4 to 5% among those 2 to 4 years of age). The observed overall vaccine efficacy against symptomatic Covid-19 in children 6 months to 4 years of age was 73.2% (95% confidence interval, 43.8 to 87.6) from 7 days after dose 3 (on the basis of 34 cases). CONCLUSIONS: A three-dose primary series of 3-µg BNT162b2 was safe, immunogenic, and efficacious in children 6 months to 4 years of age. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04816643.).

Virtual discovery of melatonin receptor ligands to modulate circadian rhythms.

The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT1 and MT2. Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light-dark cycle1-4. The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep5,6 and depression1-4,7-9. Despite their importance, few in vivo active MT1-selective ligands have been reported2,8,10-12, hampering both the understanding of circadian biology and the development of targeted therapeutics. Here we docked more than 150 million virtual molecules to an MT1 crystal structure, prioritizing structural fit and chemical novelty. Of these compounds, 38 high-ranking molecules were synthesized and tested, revealing ligands with potencies ranging from 470 picomolar to 6 micromolar. Structure-based optimization led to two selective MT1 inverse agonists-which were topologically unrelated to previously explored chemotypes-that acted as inverse agonists in a mouse model of circadian re-entrainment. Notably, we found that these MT1-selective inverse agonists advanced the phase of the mouse circadian clock by 1.3-1.5 h when given at subjective dusk, an agonist-like effect that was eliminated in MT1- but not in MT2-knockout mice. This study illustrates the opportunities for modulating melatonin receptor biology through MT1-selective ligands and for the discovery of previously undescribed, in vivo active chemotypes from structure-based screens of diverse, ultralarge libraries.

Evaluation of the BNT162b2 Covid-19 Vaccine in Children 5 to 11 Years of Age.

BACKGROUND: Safe, effective vaccines against coronavirus disease 2019 (Covid-19) are urgently needed in children younger than 12 years of age. METHODS: A phase 1, dose-finding study and an ongoing phase 2-3 randomized trial are being conducted to investigate the safety, immunogenicity, and efficacy of two doses of the BNT162b2 vaccine administered 21 days apart in children 6 months to 11 years of age. We present results for 5-to-11-year-old children. In the phase 2-3 trial, participants were randomly assigned in a 2:1 ratio to receive two doses of either the BNT162b2 vaccine at the dose level identified during the open-label phase 1 study or placebo. Immune responses 1 month after the second dose of BNT162b2 were immunologically bridged to those in 16-to-25-year-olds from the pivotal trial of two 30-µg doses of BNT162b2. Vaccine efficacy against Covid-19 at 7 days or more after the second dose was assessed. RESULTS: During the phase 1 study, a total of 48 children 5 to 11 years of age received 10 µg, 20 µg, or 30 µg of the BNT162b2 vaccine (16 children at each dose level). On the basis of reactogenicity and immunogenicity, a dose level of 10 µg was selected for further study. In the phase 2-3 trial, a total of 2268 children were randomly assigned to receive the BNT162b2 vaccine (1517 children) or placebo (751 children). At data cutoff, the median follow-up was 2.3 months. In the 5-to-11-year-olds, as in other age groups, the BNT162b2 vaccine had a favorable safety profile. No vaccine-related serious adverse events were noted. One month after the second dose, the geometric mean ratio of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing titers in 5-to-11-year-olds to those in 16-to-25-year-olds was 1.04 (95% confidence interval [CI], 0.93 to 1.18), a ratio meeting the prespecified immunogenicity success criterion (lower bound of two-sided 95% CI, >0.67; geometric mean ratio point estimate, ≥0.8). Covid-19 with onset 7 days or more after the second dose was reported in three recipients of the BNT162b2 vaccine and in 16 placebo recipients (vaccine efficacy, 90.7%; 95% CI, 67.7 to 98.3). CONCLUSIONS: A Covid-19 vaccination regimen consisting of two 10-µg doses of BNT162b2 administered 21 days apart was found to be safe, immunogenic, and efficacious in children 5 to 11 years of age. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04816643.).

Immunopeptidome profiling of human coronavirus OC43-infected cells identifies CD4 T-cell epitopes specific to seasonal coronaviruses or cross-reactive with SARS-CoV-2.

Seasonal "common-cold" human coronaviruses are widely spread throughout the world and are mainly associated with mild upper respiratory tract infections. The emergence of highly pathogenic coronaviruses MERS-CoV, SARS-CoV, and most recently SARS-CoV-2 has prompted increased attention to coronavirus biology and immunopathology, but the T-cell response to seasonal coronaviruses remains largely uncharacterized. Here we report the repertoire of viral peptides that are naturally processed and presented upon infection of a model cell line with seasonal coronavirus OC43. We identified MHC-bound peptides derived from each of the viral structural proteins (spike, nucleoprotein, hemagglutinin-esterase, membrane, and envelope) as well as non-structural proteins nsp3, nsp5, nsp6, and nsp12. Eighty MHC-II bound peptides corresponding to 14 distinct OC43-derived epitopes were identified, including many at very high abundance within the overall MHC-II peptidome. Fewer and less abundant MHC-I bound OC43-derived peptides were observed, possibly due to MHC-I downregulation induced by OC43 infection. The MHC-II peptides elicited low-abundance recall T-cell responses in most donors tested. In vitro assays confirmed that the peptides were recognized by CD4+ T cells and identified the presenting HLA alleles. T-cell responses cross-reactive between OC43, SARS-CoV-2, and the other seasonal coronaviruses were confirmed in samples of peripheral blood and peptide-expanded T-cell lines. Among the validated epitopes, spike protein S903-917 presented by DPA1*01:03/DPB1*04:01 and S1085-1099 presented by DRB1*15:01 shared substantial homology to other human coronaviruses, including SARS-CoV-2, and were targeted by cross-reactive CD4 T cells. Nucleoprotein N54-68 and hemagglutinin-esterase HE128-142 presented by DRB1*15:01 and HE259-273 presented by DPA1*01:03/DPB1*04:01 are immunodominant epitopes with low coronavirus homology that are not cross-reactive with SARS-CoV-2. Overall, the set of naturally processed and presented OC43 epitopes comprise both OC43-specific and human coronavirus cross-reactive epitopes, which can be used to follow CD4 T-cell cross-reactivity after infection or vaccination, and to guide selection of epitopes for inclusion in pan-coronavirus vaccines.

Unique pharmacodynamic properties and low abuse liability of the µ-opioid receptor ligand (S)-methadone.

(R,S)-methadone ((R,S)-MTD) is a µ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers. (S)-MTD is being developed as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. We compared the pharmacology of (R)-MTD and (S)-MTD and found they bind to MORs, but not NMDARs, and induce full analgesia. Unlike (R)-MTD, (S)-MTD was a weak reinforcer that failed to affect extracellular dopamine or induce locomotor stimulation. Furthermore, (S)-MTD antagonized motor and dopamine releasing effects of (R)-MTD. (S)-MTD acted as a partial agonist at MOR, with complete loss of efficacy at the MOR-galanin Gal1 receptor (Gal1R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use. One-sentence summary: (S)-MTD, like (R)-MTD, binds to and activates MORs in vitro, but (S)-MTD antagonizes the MOR-Gal1R heteromer, decreasing its abuse liability.

ToxoNet: A high confidence map of protein-protein interactions in Toxoplasma gondii.

The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen that is highly prevalent in the global population. The majority of the T. gondii proteome remains uncharacterized and the organization of proteins into complexes is unclear. To overcome this knowledge gap, we used a biochemical fractionation strategy to predict interactions by correlation profiling. To overcome the deficit of high-quality training data in non-model organisms, we complemented a supervised machine learning strategy, with an unsupervised approach, based on similarity network fusion. The resulting combined high confidence network, ToxoNet, comprises 2,063 interactions connecting 652 proteins. Clustering identifies 93 protein complexes. We identified clusters enriched in mitochondrial machinery that include previously uncharacterized proteins that likely represent novel adaptations to oxidative phosphorylation. Furthermore, complexes enriched in proteins localized to secretory organelles and the inner membrane complex, predict additional novel components representing novel targets for detailed functional characterization. We present ToxoNet as a publicly available resource with the expectation that it will help drive future hypotheses within the research community.

CD4 Effector TCR Avidity for Peptide on APC Determines the Level of Memory Generated.

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity in vivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory.

Safety and Immunogenicity of an Andes Virus DNA Vaccine by Needle-Free Injection: A Randomized, Controlled Phase 1 Study.

BACKGROUND: Andes virus (ANDV), a rodent-borne hantavirus, causes hantavirus pulmonary syndrome (HPS). The safety and immunogenicity of a novel ANDV DNA vaccine was evaluated. METHODS: Phase 1, double-blind, dose-escalation trial randomly assigned 48 healthy adults to placebo or ANDV DNA vaccine delivered via needle-free jet injection. Cohorts 1 and 2 received 2 mg of DNA or placebo in a 3-dose (days 1, 29, 169) or 4-dose (days 1, 29, 57, 169) schedule, respectively. Cohorts 3 and 4 received 4 mg of DNA or placebo in the 3-dose and 4-dose schedule, respectively. Subjects were monitored for safety and neutralizing antibodies by pseudovirion neutralization assay (PsVNA50) and plaque reduction neutralization test (PRNT50). RESULTS: While 98% and 65% of subjects had at least 1 local or systemic solicited adverse event (AE), respectively, most AEs were mild or moderate; no related serious AEs were detected. Cohorts 2, 3, and 4 had higher seroconversion rates than cohort 1 and seropositivity of at least 80% by day 197, sustained through day 337. PsVNA50 geometric mean titers were highest for cohort 4 on and after day 197. CONCLUSIONS: This first-in-human candidate HPS vaccine trial demonstrated that an ANDV DNA vaccine was safe and induced a robust, durable immune response. Clinical Trials Registration. NCT03682107.

Groundwater is a hidden global keystone ecosystem.

Groundwater is a vital ecosystem of the global water cycle, hosting unique biodiversity and providing essential services to societies. Despite being the largest unfrozen freshwater resource, in a period of depletion by extraction and pollution, groundwater environments have been repeatedly overlooked in global biodiversity conservation agendas. Disregarding the importance of groundwater as an ecosystem ignores its critical role in preserving surface biomes. To foster timely global conservation of groundwater, we propose elevating the concept of keystone species into the realm of ecosystems, claiming groundwater as a keystone ecosystem that influences the integrity of many dependent ecosystems. Our global analysis shows that over half of land surface areas (52.6%) has a medium-to-high interaction with groundwater, reaching up to 74.9% when deserts and high mountains are excluded. We postulate that the intrinsic transboundary features of groundwater are critical for shifting perspectives towards more holistic approaches in aquatic ecology and beyond. Furthermore, we propose eight key themes to develop a science-policy integrated groundwater conservation agenda. Given ecosystems above and below the ground intersect at many levels, considering groundwater as an essential component of planetary health is pivotal to reduce biodiversity loss and buffer against climate change.

Insights into the Mechanism of Neptunium Oxidation to the Heptavalent State.

Neptunium can exist in multiple oxidation states, including the rare and poorly understood heptavalent form. In this work, we monitored the formation of heptavalent neptunium [Np(VII)O4(OH)2]3- during ozonolysis of aqueous MOH (M=Li, Na, K) solutions using a combined experimental and theoretical approach. All experimental reactions were closely monitored via absorption and vibrational spectroscopy to follow both the oxidation state and the speciation of neptunium guided by the calculated vibrational frequencies for various neptunium species. The mechanism of the reaction partly involves oxidative dissolution of transient Np(VI) oxide/hydroxide solid phases, the identity of which are dependent on the co-precipitating counter-cation Li+/Na+/K+. Additional calculations suggest that the most favorable energetic pathway occurs through the reaction of a [Np(V)O2(OH)4]3- with the hydroxide radical to form [Np(VI)O2(OH)4]2-, followed by an additional oxidation with HO⋅ to create [Np(VII)O4(OH)2]3-.

How Social Connectedness Helps Patients Stay Home After Hospital at Home Enrollment: A Mixed Methods Study.

BACKGROUND: While enrolled in Hospital at Home (HaH) programs, patients rely on their social network to provide supportive behaviors that are routinely provided by hospital staff in the inpatient setting. OBJECTIVE: This study investigated how social connectedness is associated with patient outcomes in a HaH program. DESIGN: The explanatory iterative sequential mixed methods design included an electronic health record review to collect quantitative measures to describe the severity of patient illness and healthcare utilization and then qualitative interviews to explain quantitative findings. PARTICIPANTS: The quantitative phase included 100 patients (18 years or older) admitted to the hospital who were subsequently enrolled in the HaH program. In the qualitative phase, 33 of the 100 patients participated in semi-structured interviews. ANALYSIS: Qualitative data was analyzed using the Sort & Sift, Think & Shift method. Integrated analysis included merged data displays of healthcare utilization data and patient descriptions of their care and genogram-type illustrations to enable variable-oriented analysis of structural support. We then examined patient narratives by two variables: life course and care elevation, to understand differences in the trajectories of six subsets of patients as identified by the quantitative data. KEY RESULTS: Three factors prompted patients to enroll in HaH: low attention from hospital staff during hospital stay; loneliness and isolation during hospital stay; and family encouragement to enroll. After discharge, social support within the home structure facilitated recovery during HaH. Conversely, HaH patients with limited support within the home were more likely to be readmitted. CONCLUSIONS: Structural social connectedness facilitates patient recovery in HaH. Before enrolling patients in HaH, clinicians should take an in-depth social history, including questions about social/familial roles, household responsibilities, and technology acceptance. Clinicians should engage formal and informal caregivers in these conversations early and communicate a clear picture of what caregivers should do to support the patient through recovery.

Chemotherapy-induced gut microbiome disruption, inflammation, and cognitive decline in female patients with breast cancer.

Chemotherapy is notorious for causing behavioral side effects (e.g., cognitive decline). Notably, the gut microbiome has recently been reported to communicate with the brain to affect behavior, including cognition. Thus, the aim of this clinical longitudinal observational study was to determine whether chemotherapy-induced disruption of the gut microbial community structure relates to cognitive decline and circulating inflammatory signals. Fecal samples, blood, and cognitive measures were collected from 77 patients with breast cancer before, during, and after chemotherapy. Chemotherapy altered the gut microbiome community structure and increased circulating TNF-α. Both the chemotherapy-induced changes in microbial relative abundance and decreased microbial diversity were related to elevated circulating pro-inflammatory cytokines TNF-α and IL-6. Participants reported subjective cognitive decline during chemotherapy, which was not related to changes in the gut microbiome or inflammatory markers. In contrast, a decrease in overall objective cognition was related to a decrease in microbial diversity, independent of circulating cytokines. Stratification of subjects, via a reliable change index based on 4 objective cognitive tests, identified objective cognitive decline in 35% of the subjects. Based on a differential microbial abundance analysis, those characterized by cognitive decline had unique taxonomic shifts (Faecalibacterium, Bacteroides, Fusicatenibacter, Erysipelotrichaceae UCG-003, and Subdoligranulum) over chemotherapy treatment compared to those without cognitive decline. Taken together, gut microbiome change was associated with cognitive decline during chemotherapy, independent of chemotherapy-induced inflammation. These results suggest that microbiome-related strategies may be useful for predicting and preventing behavioral side effects of chemotherapy.

Generalizability of COBRA: A Parsimonious Perioperative Venous Thromboembolism Risk Assessment Model.

INTRODUCTION: Standardized use of venous thromboembolism (VTE) risk assessment models (RAMs) in surgical patients has been limited, in part due to the cumbersome workflow addition required to use available models. The COBRA score-capturing cancer diagnosis, (old) age, body mass index, race, and American Society of Anesthesiologists Physical Status score-has been reported as a potentially automatable VTE RAM that circumvents the cumbersome workflow addition that most RAMs represent. We aimed to test the ability of the COBRA model to effectively risk-stratify patients across various populations. METHODS: Patients were included from the 2014-2019 American College of Surgeons National Surgical Quality Improvement Program (NSQIP) Participant Use Data File for two hospitals, representing colorectal, endocrine, breast, transplant, plastic, and general surgery services. COBRA score was calculated for each patient using preoperative characteristics. We calculated negative predictive value (NPV) for VTE outcomes and compared the COBRA score to NSQIP's expected VTE rate for all patients, between the two hospitals, and between subspecialty service lines. RESULTS: Of the 10,711 patients included, those with COBRA <4 (31%) had projected median VTE rate of 0.21% (interquartile range, 0.09-0.68%; mean, 0.54%). Patients with higher scores (69%) had median rate of 0.88% (0.26-2.07%; 1.46%); relative rate 2.7. The median projected VTE rates for patients identified as low risk were 0.21% and 0.16% and as high risk were 0.87% and 0.89% at hospitals one and 2, respectively. The median projected VTE rates for patients identified as low risk were 0.17%, 0.61%, and 0.08% and as high risk were 0.52%, 1.43%, and 0.18% among general, colorectal, and endocrine surgery patients, respectively. COBRA had NPV of 0.995 and sensitivity of 0.871 as compared to NPV 0.997 and sensitivity 0.857 of the NSQIP model. CONCLUSIONS: The COBRA score is concordant with the traditional gold standard NSQIP VTE RAM and demonstrates interhospital and service-specific generalizability, although performance was limited in especially low-risk patients. The model adequately risk-stratifies surgical patients preoperatively, potentially providing clinical decision support for perioperative workflows.

Using sulfur stable isotope ratios (δ34 S) for animal geolocation: Estimating the delay mechanisms between diet ingestion and isotope incorporation in tail hair.

RATIONALE: Metabolism and diet quality play an important role in determining delay mechanisms between an animal ingesting an element and depositing the associated isotope signal in tissue. While many isotope mixing models assume instantaneous reflection of diet in an animal- tissue, this is rarely the case. Here we use data from wildebeest to measure the lag time between ingestion of 34 S and its detection in tail hair. METHODS: We use time-lagged regression analysis of δ34 S data from GPS-collared blue wildebeest from the Serengeti ecosystem in combination with δ34 S isoscape data to estimate the lag time between an animal ingesting and depositing 34 S in tail hair. RESULTS: The best fitting regression model of δ34 S in tail hair and an individual- position on the δ34 S isoscape is generated assuming an average time delay of 78 days between ingestion and detection in tail hair. This suggests that sulfur may undergo multiple metabolic transitions before being deposited in tissue. CONCLUSION: Our findings help to unravel the underlying complexities associated with sulfur metabolism and are broadly consistent with results from other species. These findings will help to inform research aiming to apply the variation of δ34 S in inert biological material for geolocation or understanding dietary changes, especially for fast moving migratory ungulates such as wildebeest.

Modular and divergent synthesis of 2,N3-disubstituted 4-quinazolinones facilitated by regioselective N-alkylation.

The synthesis of a biologically relevant 2-amino-N3-alkylamido 4-quinazolinone has been accomplished in four steps from commercially available materials using design principles from both modular and divergent synthesis. N3-Alkylation of 2-chloro-4(3H)-quinazolinone using methyl bromoacetate, followed by C2-amination produced a suitable scaffold for introducing molecular diversity. Optimization of alkylation conditions afforded full regioselectivity, enabling exclusive access to the N-alkylated isomer. Subsequent C2-amination using piperidine, pyrrolidine, or diethylamine, followed by amide bond formation using variously substituted phenethylamines, generated fifteen unique 4-quinazolinones bearing C2-amino and N3-alkylamido substituents. These efforts highlight the reciprocal influence of C2 and N3 substitution on functionalization at either position, establish an effective synthetic pathway toward 2,N3-disubstituted 4-quinazolinones, and enable preliminary bioactivity studies while providing an experiential learning opportunity for undergraduate student researchers.

Emotional and behavioral outcomes among youths with mental disorders during the first Covid lockdown and school closures in England: a large clinical population study using health care record integrated surveys.

PURPOSE: Emotional and behavioral problems in children and young people (CYP) have increased over the pandemic. Those with pre-existing mental disorders are more vulnerable but have been understudied. We investigated emotional and behavioral outcomes in this population; differences across diagnostic groups; and social, educational, and clinical determinants. METHODS: We invited 5386 caregivers and CYP (aged 5-17) under child mental health services pre-pandemic to complete an online survey on CYP's emotional/behavioral symptoms and pandemic-related circumstances, and integrated responses with clinicodemographic information extracted from electronic health records. We compared four parent-rated outcomes (total emotional/behavioral scores and emotional/behavioral changes as compared to before the pandemic) across the three most common diagnostic groups in our population (Attention Deficit Hyperactivity Disorder (ADHD), Autism Spectrum Disorder (ASD) and emotional disorders (EmD)). We then estimated the association of clinicodemographic and pandemic-related characteristics with emotional/behavioral outcomes. RESULTS: A total of 1741 parents (32.3%) completed the survey. Parents of CYP with ADHD or ASD reported more behavioral difficulties (t(591) = 5.618 (0.001); t(663) = 6.527 (0.001)); greater emotional deterioration (t(591) = 2.592 (0.009); t(664) = 4.670 (< 0.001); and greater behavioral deterioration (t(594) = 4.529 (< 0.001); t(664) = 5.082 (< 0.001)) as compared to the EmD group. Those with ASD and EmD showed more emotional difficulties than ADHD (t(891) = - 4.431 (< 0.001); t(590) = - 3.254 (0.001)). Across diagnoses, poor parental mental health and challenges with education were most strongly associated with worse outcomes. CONCLUSIONS: Within our clinical population, CYP with ADHD/ASD were the most adversely affected during lockdown. Enhancing clinical service provision that tackles parental stress and supports education may help mitigate the impact of future restrictions.

Donor white blood cell differential is the single largest determinant of whole blood gene expression patterns.

It has become widely accepted that sample cellular composition is a significant determinant of the gene expression patterns observed in any transcriptomic experiment performed with bulk tissue. Despite this, many investigations currently performed with whole blood do not experimentally account for possible inter-specimen differences in cellularity, and often assume that any observed gene expression differences are a result of true differences in nuclear transcription. In order to determine how confounding of an assumption this may be, in this study, we recruited a large cohort of human donors (n = 138) and used a combination of next generation sequencing and flow cytometry to quantify and compare the underlying contributions of variance in leukocyte counts versus variance in other biological factors to overall variance in whole blood transcript levels. Our results suggest that the combination of donor neutrophil and lymphocyte counts alone are the primary determinants of whole blood transcript levels for up to 75% of the protein-coding genes expressed in peripheral circulation, whereas the other factors such as age, sex, race, ethnicity, and common disease states have comparatively minimal influence. Broadly, this infers that a majority of gene expression differences observed in experiments performed with whole blood are driven by latent differences in leukocyte counts, and that cell count heterogeneity must be accounted for to meaningfully biologically interpret the results.

MCU (mitochondrial Ca2+ uniporter) makes the calcium go round.

Store-operated Ca2+ entry (SOCE) is a major mechanism controlling Ca2+ signaling and Ca2+-dependent functions and has been implicated in immunity, cancer, and organ development. SOCE-dependent cytosolic Ca2+ signals are affected by mitochondrial Ca2+ transport through several competing mechanisms. However, how these mechanisms interact in shaping Ca2+ dynamics and regulating Ca2+-dependent functions remains unclear. In a recent issue, Yoast et al. shed light on these questions by defining multiple roles of the mitochondrial Ca2+ uniporter in regulating SOCE, Ca2+ dynamics, transcription, and lymphocyte activation.

The role of microglia in 67NR mammary tumor-induced suppression of brain responses to immune challenges in female mice.

It is poorly understood how solid peripheral tumors affect brain neuroimmune responses despite the various brain-mediated side effects and higher rates of infection reported in cancer patients. We hypothesized that chronic low-grade peripheral tumor-induced inflammation conditions microglia to drive suppression of neuroinflammatory responses to a subsequent peripheral immune challenge. Here, Balb/c murine mammary tumors attenuated the microglial inflammatory gene expression responses to lipopolysaccharide (LPS) and live Escherichia coli (E. coli) challenges and the fatigue response to an E. coli infection. In contrast, the inflammatory gene expression in response to LPS or a toll-like receptor 2 agonist of Percoll-enriched primary microglia cultures was comparable between tumor-bearing and -free mice, as were the neuroinflammatory and sickness behavioral responses to an intracerebroventricular interleukin (IL)-1ß injection. These data led to the hypothesis that Balb/c mammary tumors blunt the neuroinflammatory responses to an immune challenge via a mechanism involving tumor suppression of the peripheral humoral response. Balb/c mammary tumors modestly attenuated select circulating cytokine responses to LPS and E. coli challenges. Further, a second mammary tumor/mouse strain model (E0771 tumors in C57Bl/6 mice) displayed mildly elevated inflammatory responses to an immune challenge. Taken together, these data indicate that tumor-induced suppression of neuroinflammation and sickness behaviors may be driven by a blunted microglial phenotype, partly because of an attenuated peripheral signal to the brain, which may contribute to infection responses and behavioral side effects reported in cancer patients. Finally, these neuroimmune effects likely vary based on tumor type and/or host immune phenotype.