The Phospho-Code Determining Circadian Feedback Loop Closure and Output in Neurospora.

In the negative feedback loop driving fungal and animal circadian oscillators, negative elements (FREQUENCY [FRQ], PERIODS [PERs], and CRYPTOCHROMES [CRYs]) are understood to inhibit their own expression, in part by promoting the phosphorylation of their heterodimeric transcriptional activators (e.g., White Collar-1 [WC-1]-WC-2 [White Collar complex; WCC] and BMAL1/Circadian Locomotor Output Cycles Kaput [CLOCK]). However, correlations between heterodimer activity and phosphorylation are weak, contradictions exist, and mechanistic details are almost wholly lacking. We report mapping of 80 phosphosites on WC-1 and 15 on WC-2 and elucidation of the time-of-day-specific code, requiring both a group of phosphoevents on WC-1 and two distinct clusters on WC-2, that governs circadian repression, leading to feedback loop closure. Combinatorial control via phosphorylation also governs rhythmic WCC binding to the promoters of clock-controlled genes mediating the essential first step in circadian output, a group encoding both transcription factors and signaling proteins. These data provide a basic mechanistic understanding for fundamental events underlying circadian negative feedback and output, key aspects of circadian biology.

Lipid shape and packing are key for optimal design of pH-sensitive mRNA lipid nanoparticles.

The ionizable-lipid component of RNA-containing nanoparticles controls the pH-dependent behavior necessary for an efficient delivery of the cargo-the so-called endosomal escape. However, it is still an empirical exercise to identify optimally performing lipids. Here, we study two well-known ionizable lipids, DLin-MC3-DMA and DLin-DMA using a combination of experiments, multiscale computer simulations, and electrostatic theory. All-atom molecular dynamics simulations, and experimentally measured polar headgroup pKa values, are used to develop a coarse-grained representation of the lipids, which enables the investigation of the pH-dependent behavior of lipid nanoparticles (LNPs) through Monte Carlo simulations, in the absence and presence of RNA molecules. Our results show that the charge state of the lipids is determined by the interplay between lipid shape and headgroup chemistry, providing an explanation for the similar pH-dependent ionization state observed for lipids with headgroup pKa values about one-pH-unit apart. The pH dependence of lipid ionization is significantly influenced by the presence of RNA, whereby charge neutrality is achieved by imparting a finite and constant charge per lipid at intermediate pH values. The simulation results are experimentally supported by measurements of α-carbon 13C-NMR chemical shifts for eGFP mRNA LNPs of both DLin-MC3-DMA and DLin-DMA at various pH conditions. Further, we evaluate the applicability of a mean-field Poisson-Boltzmann theory to capture these phenomena.

A randomized trial of behavioral interventions yielding sustained reductions in distracted driving.

Distracted driving is responsible for nearly 1 million crashes each year in the United States alone, and a major source of driver distraction is handheld phone use. We conducted a randomized, controlled trial to compare the effectiveness of interventions designed to create sustained reductions in handheld use while driving (NCT04587609). Participants were 1,653 consenting Progressive® Snapshot® usage-based auto insurance customers ages 18 to 77 who averaged at least 2 min/h of handheld use while driving in the month prior to study invitation. They were randomly assigned to one of five arms for a 10-wk intervention period. Arm 1 (control) got education about the risks of handheld phone use, as did the other arms. Arm 2 got a free phone mount to facilitate hands-free use. Arm 3 got the mount plus a commitment exercise and tips for hands-free use. Arm 4 got the mount, commitment, and tips plus weekly goal gamification and social competition. Arm 5 was the same as Arm 4, plus offered behaviorally designed financial incentives. Postintervention, participants were monitored until the end of their insurance rating period, 25 to 65 d more. Outcome differences were measured using fractional logistic regression. Arm 4 participants, who received gamification and competition, reduced their handheld use by 20.5% relative to control (P < 0.001); Arm 5 participants, who additionally received financial incentives, reduced their use by 27.6% (P < 0.001). Both groups sustained these reductions through the end of their insurance rating period.

Dissonant music engages early visual processing.

The neuroscientific examination of music processing in audio-visual contexts offers a valuable framework to assess how auditory information influences the emotional encoding of visual information. Using fMRI during naturalistic film viewing, we investigated the neural mechanisms underlying the effect of music on valence inferences during mental state attribution. Thirty-eight participants watched the same short-film accompanied by systematically controlled consonant or dissonant music. Subjects were instructed to think about the main character's intentions. The results revealed that increasing levels of dissonance led to more negatively valenced inferences, displaying the profound emotional impact of musical dissonance. Crucially, at the neuroscientific level and despite music being the sole manipulation, dissonance evoked the response of the primary visual cortex (V1). Functional/effective connectivity analysis showed a stronger coupling between the auditory ventral stream (AVS) and V1 in response to tonal dissonance and demonstrated the modulation of early visual processing via top-down feedback inputs from the AVS to V1. These V1 signal changes indicate the influence of high-level contextual representations associated with tonal dissonance on early visual cortices, serving to facilitate the emotional interpretation of visual information. Our results highlight the significance of employing systematically controlled music, which can isolate emotional valence from the arousal dimension, to elucidate the brain's sound-to-meaning interface and its distributive crossmodal effects on early visual encoding during naturalistic film viewing.

The Caenorhabditis elegans anchor cell transcriptome: ribosome biogenesis drives cell invasion through basement membrane.

Cell invasion through basement membrane (BM) barriers is important in development, immune function and cancer progression. As invasion through BM is often stochastic, capturing gene expression profiles of actively invading cells in vivo remains elusive. Using the stereotyped timing of Caenorhabditis elegans anchor cell (AC) invasion, we generated an AC transcriptome during BM breaching. Through a focused RNAi screen of transcriptionally enriched genes, we identified new invasion regulators, including translationally controlled tumor protein (TCTP). We also discovered gene enrichment of ribosomal proteins. AC-specific RNAi, endogenous ribosome labeling and ribosome biogenesis analysis revealed that a burst of ribosome production occurs shortly after AC specification, which drives the translation of proteins mediating BM removal. Ribosomes also enrich near the AC endoplasmic reticulum (ER) Sec61 translocon and the endomembrane system expands before invasion. We show that AC invasion is sensitive to ER stress, indicating a heightened requirement for translation of ER-trafficked proteins. These studies reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration.

Ring-opening metathesis polymerization of N-methylpyridinium-fused norbornenes to access antibacterial main-chain cationic polymers.

Cationic polymers have been identified as a promising type of antibacterial molecules, whose bioactivity can be tuned through structural modulation. Recent studies suggest that the placement of the cationic groups close to the core of the polymeric architecture rather than on appended side chains might improve both their bioactivity and selectivity for bacterial cells over mammalian cells. However, antibacterial main-chain cationic polymers are typically synthesized via polycondensations, which do not afford precise and uniform molecular design. Therefore, accessing main-chain cationic polymers with high degrees of molecular tunability hinges upon the development of controlled polymerizations tolerating cationic motifs (or cation progenitors) near the propagating species. Herein, we report the synthesis and ring-opening metathesis polymerization (ROMP) of N-methylpyridinium-fused norbornene monomers. The identification of reaction conditions leading to a well-controlled ROMP enabled structural diversification of the main-chain cationic polymers and a study of their bioactivity. This family of polyelectrolytes was found to be active against both Gram-negative (Escherichia coli) and Gram-positive (Methicillin-resistant Staphylococcus aureus) bacteria with minimal inhibitory concentrations as low as 25 µg/mL. Additionally, the molar mass of the polymers was found to impact their hemolytic activity with cationic polymers of smaller degrees of polymerization showing increased selectivity for bacteria over human red blood cells.

Programming hydrogel adhesion with engineered polymer network topology.

Hydrogel adhesion that can be easily modulated in magnitude, space, and time is desirable in many emerging applications ranging from tissue engineering and soft robotics to wearable devices. In synthetic materials, these complex adhesion behaviors are often achieved individually with mechanisms and apparatus that are difficult to integrate. Here, we report a universal strategy to embody multifaceted adhesion programmability in synthetic hydrogels. By designing the surface network topology of a hydrogel, supramolecular linkages that result in contrasting adhesion behaviors are formed on the hydrogel interface. The incorporation of different topological linkages leads to dynamically tunable adhesion with high-resolution spatial programmability without alteration of bulk mechanics and chemistry. Further, the association of linkages enables stable and tunable adhesion kinetics that can be tailored to suit different applications. We rationalize the physics of polymer chain slippage, rupture, and diffusion at play in the emergence of the programmable behaviors. With the understanding, we design and fabricate various soft devices such as smart wound patches, fluidic channels, drug-eluting devices, and reconfigurable soft robotics. Our study presents a simple and robust platform in which adhesion controllability in multiple aspects can be easily integrated into a single design of a hydrogel network.

Self-powered, light-controlled, bioresorbable platforms for programmed drug delivery.

Degradable polymer matrices and porous scaffolds provide powerful mechanisms for passive, sustained release of drugs relevant to the treatment of a broad range of diseases and conditions. Growing interest is in active control of pharmacokinetics tailored to the needs of the patient via programmable engineering platforms that include power sources, delivery mechanisms, communication hardware, and associated electronics, most typically in forms that require surgical extraction after a period of use. Here we report a light-controlled, self-powered technology that bypasses key disadvantages of these systems, in an overall design that is bioresorbable. Programmability relies on the use of an external light source to illuminate an implanted, wavelength-sensitive phototransistor to trigger a short circuit in an electrochemical cell structure that includes a metal gate valve as its anode. Consequent electrochemical corrosion eliminates the gate, thereby opening an underlying reservoir to release a dose of drugs by passive diffusion into surrounding tissue. A wavelength-division multiplexing strategy allows release to be programmed from any one or any arbitrary combination of a collection of reservoirs built into an integrated device. Studies of various bioresorbable electrode materials define the key considerations and guide optimized choices in designs. In vivo demonstrations of programmed release of lidocaine adjacent the sciatic nerves in rat models illustrate the functionality in the context of pain management, an essential aspect of patient care that could benefit from the results presented here.

Experiment aversion does not appear to generalize.

Over the past decade, governments and organizations around the world have established behavioral insights teams advocating for randomized experiments. However, recent findings by M. N. Meyer et al., Proc. Natl. Acad. Sci. U.S.A. 116, 10723-10728 (2019) and P. R. Heck, C. F. Chabris, D. J. Watts, M. N. Meyer, Proc. Natl. Acad. Sci. U.S.A. 117, 18948-18950 (2020) suggest that people often rate randomized experiments as less appropriate than the policies they contain even when approving the implementation of either policy untested and when none of the individual policies is clearly superior. The authors warn that this could cause policymakers to avoid running large-scale field experiments or being transparent about running them and might contribute to an adverse heterogeneity bias in terms of who is participating in experiments. In one direct and six conceptual preregistered replications (total N = 5,200) of the previously published larger-effect studies, using the same main dependent variable but with variations in scenario wordings, recruitment platforms, and countries, and the addition of further measures to assess people's views, we test the generalizability and robustness of these findings. Together, we find that the original results do not appear to generalize. That is, our triangulation reveals insufficient evidence to conclude that people exhibit a common pattern of behavior that would be consistent with relative experiment aversion, thereby supporting recent findings by R. Mislavsky, B. Dietvorst, U. Simonsohn, Mark. Sci. 39, 1092-1104 (2020). Thus, policymakers may not need to be concerned about employing evidence-based practices more so than about universally implementing policies.

Unconditional cash transfers reduce homelessness.

Homelessness is an economic and social crisis. In a cluster-randomized controlled trial, we address a core cause of homelessness-lack of money-by providing a one-time unconditional cash transfer of CAD$7,500 to each of 50 individuals experiencing homelessness, with another 65 as controls in Vancouver, BC. Exploratory analyses showed that over 1 y, cash recipients spent fewer days homeless, increased savings and spending with no increase in temptation goods spending, and generated societal net savings of $777 per recipient via reduced time in shelters. Additional experiments revealed public mistrust toward the ability of homeless individuals to manage money and demonstrated interventions to increase public support for a cash transfer policy using counter-stereotypical or utilitarian messaging. Together, this research offers a new approach to address homelessness and provides insights into homelessness reduction policies.

Neisseria gonorrhoeae vaccines: a contemporary overview.

Neisseria gonorrhoeae infection is an important public health issue, with an annual global incidence of 87 million. N. gonorrhoeae infection causes significant morbidity and can have serious long-term impacts on reproductive and neonatal health and may rarely cause life-threatening disease. Global rates of N. gonorrhoeae infection have increased over the past 20 years. Importantly, rates of antimicrobial resistance to key antimicrobials also continue to increase, with the United States Centers for Disease Control and Prevention identifying drug-resistant N. gonorrhoeae as an urgent threat to public health. This review summarizes the current evidence for N. gonorrhoeae vaccines, including historical clinical trials, key N. gonorrhoeae vaccine preclinical studies, and studies of the impact of Neisseria meningitidis vaccines on N. gonorrhoeae infection. A comprehensive survey of potential vaccine antigens, including those identified through traditional vaccine immunogenicity approaches, as well as those identified using more contemporary reverse vaccinology approaches, are also described. Finally, the potential epidemiological impacts of a N. gonorrhoeae vaccine and research priorities for further vaccine development are described.

Cucumber (Cucumis sativus L.) translationally controlled tumor protein interacts with CsRab11A and promotes activation of target of rapamycin in response to Podosphaera xanthii.

The target of rapamycin (TOR) kinase serves as a central regulator that integrates nutrient and energy signals to orchestrate cellular and organismal physiology in both animals and plants. Despite significant advancements having been made in understanding the molecular and cellular functions of plant TOR kinases, the upstream regulators that modulate TOR activity are not yet fully elucidated. In animals, the translationally controlled tumor protein (TCTP) is recognized as a key player in TOR signaling. This study reveals that two TCTP isoforms from Cucumis sativus, when introduced into Arabidopsis, are instrumental in balancing growth and defense mechanisms against the fungal pathogen Golovinomyces cichoracearum. We hypothesize that plant TCTPs act as upstream regulators of TOR in response to powdery mildew caused by Podosphaera xanthii in Cucumis. Our research further uncovers a stable interaction between CsTCTP and a small GTPase, CsRab11A. Transient transformation assays indicate that CsRab11A is involved in the defense against P. xanthii and promotes the activation of TOR signaling through CsTCTP. Moreover, our findings demonstrate that the critical role of TOR in plant disease resistance is contingent upon its regulated activity; pretreatment with a TOR inhibitor (AZD-8055) enhances cucumber plant resistance to P. xanthii, while pretreatment with a TOR activator (MHY-1485) increases susceptibility. These results suggest a sophisticated adaptive response mechanism in which upstream regulators, CsTCTP and CsRab11A, coordinate to modulate TOR function in response to P. xanthii, highlighting a novel aspect of plant-pathogen interactions.

Cognitive Effects of Body Temperature During Hypothermic Circulatory Arrest Trial (GOT ICE): A Randomized Clinical Trial Comparing Outcomes After Aortic Arch Surgery.

BACKGROUND: Deep hypothermia has been the standard for hypothermic circulatory arrest (HCA) during aortic arch surgery. However, centers worldwide have shifted toward lesser hypothermia with antegrade cerebral perfusion. This has been supported by retrospective data, but there has yet to be a multicenter, prospective randomized study comparing deep versus moderate hypothermia during HCA. METHODS: This was a randomized single-blind trial (GOT ICE [Cognitive Effects of Body Temperature During Hypothermic Circulatory Arrest]) of patients undergoing arch surgery with HCA plus antegrade cerebral perfusion at 4 US referral aortic centers (August 2016-December 2021). Patients were randomized to 1 of 3 hypothermia groups: DP, deep (≤20.0 °C); LM, low-moderate (20.1-24.0 °C); and HM, high-moderate (24.1-28.0 °C). The primary outcome was composite global cognitive change score between baseline and 4 weeks postoperatively. Analysis followed the intention-to-treat principle to evaluate if: (1) LM noninferior to DP on global cognitive change score; (2) DP superior to HM. The secondary outcomes were domain-specific cognitive change scores, neuroimaging findings, quality of life, and adverse events. RESULTS: A total of 308 patients consented; 282 met inclusion and were randomized. A total of 273 completed surgery, and 251 completed the 4-week follow-up (DP, 85 [34%]; LM, 80 [34%]; HM, 86 [34%]). Mean global cognitive change score from baseline to 4 weeks in the LM group was noninferior to the DP group; likewise, no significant difference was observed between DP and HM. Noninferiority of LM versus DP, and lack of difference between DP and HM, remained for domain-specific cognitive change scores, except structured verbal memory, with noninferiority of LM versus DP not established and structured verbal memory better preserved in DP versus HM (P = 0.036). There were no significant differences in structural or functional magnetic resonance imaging brain imaging between groups postoperatively. Regardless of temperature, patients who underwent HCA demonstrated significant reductions in cerebral gray matter volume, cortical thickness, and regional brain functional connectivity. Thirty-day in-hospital mortality, major morbidity, and quality of life were not different between groups. CONCLUSIONS: This randomized multicenter study evaluating arch surgery HCA temperature strategies found low-moderate hypothermia noninferior to traditional deep hypothermia on global cognitive change 4 weeks after surgery, although in secondary analysis, structured verbal memory was better preserved in the deep group. The verbal memory differences in the low- and high-moderate groups and structural and functional connectivity reductions from baseline merit further investigation and suggest opportunities to further optimize brain perfusion during HCA. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02834065.

Quality of Life in Patients With Chronic Limb-Threatening Ischemia Treated With Revascularization.

BACKGROUND: In the BEST-CLI trial (Best Endovascular Versus Best Surgical Therapy for Patients With Chronic Limb-Threatening Ischemia), a prespecified secondary objective was to assess the effects of revascularization strategy on health-related quality of life (HRQoL). METHODS: Patients with chronic limb-threatening ischemia were randomized to surgical bypass (Bypass) or endovascular intervention (Endo) in 2 parallel trials. Cohort 1 included patients with single-segment great saphenous vein; cohort 2 included those lacking suitable single-segment great saphenous vein. HRQoL was assessed over the trial duration using Vascular Quality-of-Life (VascuQoL), European Quality-of-Life-5D (EQ-5D), the Short Form-12 (SF-12) Physical Component Summary (SF-12 PCS), SF-12 Mental Component Summary (SF-12 MCS), Utility Index Score (SF-6D R2), and numeric rating scales of pain. HRQoL was summarized by cohort and compared within and between groups using mixed-model linear regression. RESULTS: A total of 1193 and 335 patients in cohorts 1 and 2 with a mean follow-up of 2.9 and 2.0 years, respectively, were analyzed. In cohort 1, HRQoL significantly improved from baseline to follow-up for both groups across all measures. For example, mean (SD) VascuQoL scores were 3.0 (1.3) and 3.0 (1.2) for Bypass and Endo at baseline and 4.7 (1.4) and 4.8 (1.5) over follow-up. There were significant group differences favoring Endo when assessed with VascuQoL (difference, -0.14 [95% CI, -0.25 to -0.02]; P=0.02), SF-12 MCS (difference, -1.03 [95% CI, -1.89 to -0.18]; P=0.02), SF-6D R2 (difference, -0.01 [95% CI, -0.02 to -0.001]; P=0.03), numeric rating scale pain at present (difference, 0.26 [95% CI, 0.03 to 0.49]; P=0.03), usual level during previous week (difference, 0.26 [95% CI, 0.04 to 0.48]; P=0.02), and worst level during previous week (difference, 0.29 [95% CI, 0.02 to 0.56]; P=0.04). There was no difference between treatment arms on the basis of EQ-5D (difference, -0.01 [95% CI, -0.03 to 0.004]; P=0.12) or SF-12 PCS (difference, -0.41 [95% CI, -1.2 to 0.37]; P=0.31). In cohort 2, HRQoL also significantly improved from baseline to the end of follow-up for both groups based on all measures, but there were no differences between Bypass and Endo on any measure. CONCLUSIONS: Among patients with chronic limb-threatening ischemia deemed eligible for either Bypass or Endo, revascularization resulted in significant and clinically meaningful improvements in HRQoL. In patients with an available single-segment great saphenous vein for bypass, but not among those without one, Endo was statistically superior on some HRQoL measures; however, these differences were below the threshold of clinically meaningful difference.

Degradation strategies for structural characterization of insoluble synthetic polymers by mass spectrometry.

With the advent of soft ionization techniques such as electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) to produce intact gas-phase ions from nonvolatile macromolecules, mass spectrometry has become an essential technique in the field of polymeric materials. However, (co)polymers of very high molecular weight or with reticulated architectures still escape ESI or MALDI, mainly due to solubility issues. Strategies developed to tackle such an analytical challenge all rely on sample degradation to produce low-mass species amenable to existing ionization methods. Yet, chain degradation needs to be partial and controlled to generate sufficiently large species that still contain topological or architectural information. The present article reviews the different analytical degradation strategies implemented to perform mass spectrometry of these challenging synthetic polymers, covering thermal degradation approaches in sources developed in the 2000s, off-line sample pre-treatments for controlled chemical degradation of polymeric substrates, and most recent achievements employing reactive ionization modes to perform chemolysis on-line with MS.

Disruption of central and peripheral circadian clocks and circadian controlled estrogen receptor rhythms in night shift nurses in working environments.

Chronic disruption of circadian rhythms by night shift work is associated with an increased breast cancer risk. However, little is known about the impact of night shift on peripheral circadian genes (CGs) and circadian-controlled genes (CCGs) associated with breast cancer. Hence, we assessed central clock markers (melatonin and cortisol) in plasma, and peripheral CGs (PER1, PER2, PER3, and BMAL1) and CCGs (ESR1 and ESR2) in peripheral blood mononuclear cells (PBMCs). In day shift nurses (n = 12), 24-h rhythms of cortisol and melatonin were aligned with day shift-oriented light/dark schedules. The mRNA expression of PER2, PER3, BMAL1, and ESR2 showed 24-h rhythms with peak values in the morning. In contrast, night shift nurses (n = 10) lost 24-h rhythmicity of cortisol with a suppressed morning surge but retained normal rhythmic patterns of melatonin, leading to misalignment between cortisol and melatonin. Moreover, night shift nurses showed disruption of rhythmic expressions of PER2, PER3, BMAL1, and ESR2 genes, resulting in an impaired inverse correlation between PER2 and BMAL1 compared to day shift nurses. The observed trends of disrupted circadian markers were recapitulated in additional day (n = 20) and night (n = 19) shift nurses by measurement at early night and midnight time points. Taken together, this study demonstrated the misalignment of cortisol and melatonin, associated disruption of PER2 and ESR2 circadian expressions, and internal misalignment in peripheral circadian network in night shift nurses. Morning plasma cortisol and PER2, BMAL1, and ESR2 expressions in PBMCs may therefore be useful biomarkers of circadian disruption in shift workers.

Transient brain structure changes after high phenylalanine exposure in adults with phenylketonuria.

Phenylketonuria is a rare metabolic disease resulting from a deficiency of the enzyme phenylalanine hydroxylase. Recent cross-sectional evidence suggests that early-treated adults with phenylketonuria exhibit alterations in cortical grey matter compared to healthy peers. However, the effects of high phenylalanine exposure on brain structure in adulthood need to be further elucidated. In this double-blind, randomised, placebo-controlled crossover trial, we investigated the impact of a four-week high phenylalanine exposure on the brain structure and its relationship to cognitive performance and metabolic parameters in early-treated adults with phenylketonuria. Twenty-eight adult patients with early-treated classical phenylketonuria (19-48 years) underwent magnetic resonance imaging before and after the four-week phenylalanine and placebo interventions (four timepoints). Structural T1-weighted images were preprocessed and evaluated using DL+DiReCT, a deep-learning-based tool for brain morphometric analysis. Cortical thickness, white matter volume, and ventricular volume were compared between the phenylalanine and placebo periods. Brain phenylalanine levels were measured using 1H spectroscopy. Blood levels of phenylalanine, tyrosine, and tryptophan were assessed at each of the four timepoints, along with performance in executive functions and attention. Blood phenylalanine levels were significantly higher after the phenylalanine period (1441µmol/L) than after the placebo period (873µmol/L, P<0.001). Morphometric analyses revealed a statistically significant decrease in cortical thickness in 17 out of 60 brain regions after the phenylalanine period compared to placebo. The largest decreases were observed in the right pars orbitalis (point estimate=-0.095mm, P<0.001) and the left lingual gyrus (point estimate=-0.070mm, P<0.001). Bilateral white matter and ventricular volumes were significantly increased after the phenylalanine period. However, the structural alterations in the Phe-placebo group returned to baseline measures following the washout and placebo period. Additionally, elevated blood and brain phenylalanine levels were related to increased bilateral white matter volume (rs=0.43 to 0.51, P≤0.036) and decreased cortical thickness (rs=-0.62 to -0.39, not surviving FDR correction) after the phenylalanine and placebo periods. Moreover, decreased cortical thickness was correlated with worse cognitive performance after both periods (rs=-0.54 to -0.40, not surviving FDR correction). These findings provide evidence that a four-week high phenylalanine exposure in adults with phenylketonuria results in transient reductions of the cortical grey matter and increases in white matter volume. Further research is needed to determine the potential long-term impact of high phenylalanine levels on brain structure and function in adults with phenylketonuria.

Traumatic brain injury disrupts state-dependent functional cortical connectivity in a mouse model.

Traumatic brain injury (TBI) is the leading cause of death in young people and can cause cognitive and motor dysfunction and disruptions in functional connectivity between brain regions. In human TBI patients and rodent models of TBI, functional connectivity is decreased after injury. Recovery of connectivity after TBI is associated with improved cognition and memory, suggesting an important link between connectivity and functional outcome. We examined widespread alterations in functional connectivity following TBI using simultaneous widefield mesoscale GCaMP7c calcium imaging and electrocorticography (ECoG) in mice injured using the controlled cortical impact (CCI) model of TBI. Combining CCI with widefield cortical imaging provides us with unprecedented access to characterize network connectivity changes throughout the entire injured cortex over time. Our data demonstrate that CCI profoundly disrupts functional connectivity immediately after injury, followed by partial recovery over 3 weeks. Examining discrete periods of locomotion and stillness reveals that CCI alters functional connectivity and reduces theta power only during periods of behavioral stillness. Together, these findings demonstrate that TBI causes dynamic, behavioral state-dependent changes in functional connectivity and ECoG activity across the cortex.

Hitchhiking of Cas9 with nucleus-localized proteins impairs its controllability and leads to efficient genome editing of NLS-free Cas9.

CRISPR-Cas9 is the most commonly used genome-editing tool in eukaryotic cells. To modulate Cas9 entry into the nucleus to enable control of genome editing, we constructed a light-controlled CRISPR-Cas9 system to control exposure of the Cas9 protein nuclear localization signal (NLS). Although blue-light irradiation was found to effectively control the entry of Cas9 protein into the nucleus with confocal microscopy observation, effective gene editing occurred in controls with next-generation sequencing analysis. To further clarify this phenomenon, a CRISPR-Cas9 editing system without the NLS and a CRISPR-Cas9 editing system containing a nuclear export signal were also constructed. Interestingly, both Cas9 proteins could achieve effective editing of target sites with significantly reduced off-target effects. Thus, we speculated that other factors might mediate Cas9 entry into the nucleus. However, NLS-free Cas9 was found to produce effective target gene editing even following inhibition of cell mitosis to prevent nuclear import caused by nuclear membrane disassembly. Furthermore, multiple nucleus-localized proteins were found to interact with Cas9, which could mediate the "hitchhiking" of NLS-free Cas9 into the nucleus. These findings will inform future attempts to construct controllable gene-editing systems and provide new insights into the evolution of the nucleus and compatible protein functions.

Household Air Pollution Interventions to Improve Health in Low- and Middle-Income Countries: An Official American Thoracic Society Research Statement.

Background: An estimated 3 billion people, largely in low- and middle-income countries, rely on unclean fuels for cooking, heating, and lighting to meet household energy needs. The resulting exposure to household air pollution (HAP) is a leading cause of pneumonia, chronic lung disease, and other adverse health effects. In the last decade, randomized controlled trials of clean cooking interventions to reduce HAP have been conducted. We aim to provide guidance on how to interpret the findings of these trials and how they should inform policy makers and practitioners.Methods: We assembled a multidisciplinary working group of international researchers, public health practitioners, and policymakers with expertise in household air pollution from within academia, the American Thoracic Society, funders, nongovernmental organizations, and global organizations, including the World Bank and the World Health Organization. We performed a literature search, convened four sessions via web conference, and developed consensus conclusions and recommendations via the Delphi method.Results: The committee reached consensus on 14 conclusions and recommendations. Although some trials using cleaner-burning biomass stoves or cleaner-cooking fuels have reduced HAP exposure, the committee was divided (with 55% saying no and 45% saying yes) on whether the studied interventions improved measured health outcomes.Conclusions: HAP is associated with adverse health effects in observational studies. However, it remains unclear which household energy interventions reduce exposure, improve health, can be scaled, and are sustainable. Researchers should engage with policy makers and practitioners working to scale cleaner energy solutions to understand and address their information needs.