Abstract: The glymphatic system functions in the removal of potentially harmful metabolites and proteins from the brain. Dynamic, contrast-enhanced MRI was used in fully awake rats to follow the redistribution of intraventricular contrast agent entrained to the light–dark cycle and its hypothetical relationship to the sleep–waking cycle, blood flow, and brain temperature in specific brain areas. Brain areas involved in circadian timing and sleep–wake rhythms showed the lowest redistribution of contrast agent during the light phase or time of inactivity and sleep in rats. Global brain redistribution of contrast agent was heterogeneous. The redistribution was highest along the dorsal cerebrum and lowest in the midbrain/pons and along the ventral surface of the brain. This heterogeneous redistribution of contrast agent paralleled the gradients and regional variations in brain temperatures reported in the literature for awake animals. Three-dimensional quantitative ultrashort time-to-echo contrast-enhanced imaging was used to reconstruct small, medium, and large arteries and veins in the rat brain and revealed areas of lowest redistribution overlapped with this macrovasculature. This study raises new questions and theoretical considerations of the impact of the light–dark cycle, brain temperature, and blood flow on the function of the glymphatic system.

Abstract: Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century. Expanse of SARS-CoV-2 sequencing capacity in Africa. ( A ) African countries (shaded in gray) and institutions (red circles) with on-site sequencing facilities that are capable of producing SARS-CoV-2 whole genomes locally. ( B ) The number of SARS-CoV-2 genomes produced per country and the proportion of those genomes that were produced locally, regionally within Africa, or abroad. ( C ) Decreased turnaround time of sequencing output in Africa to an almost real-time release of genomic data.

Abstract: Adolescence is a time of profound changes in the physical wiring and function of the brain. Here, we analyzed structural and functional brain network development in an accelerated longitudinal cohort spanning 14 to 25 y ( n = 199). Core to our work was an advanced in vivo model of cortical wiring incorporating MRI features of corticocortical proximity, microstructural similarity, and white matter tractography. Longitudinal analyses assessing age-related changes in cortical wiring identified a continued differentiation of multiple corticocortical structural networks in youth. We then assessed structure–function coupling using resting-state functional MRI measures in the same participants both via cross-sectional analysis at baseline and by studying longitudinal change between baseline and follow-up scans. At baseline, regions with more similar structural wiring were more likely to be functionally coupled. Moreover, correlating longitudinal structural wiring changes with longitudinal functional connectivity reconfigurations, we found that increased structural differentiation, particularly between sensory/unimodal and default mode networks, was reflected by reduced functional interactions. These findings provide insights into adolescent development of human brain structure and function, illustrating how structural wiring interacts with the maturation of macroscale functional hierarchies.

Abstract: This study introduces a tool for studying the unfolded protein response and suggests that IRE1 inhibitors may find their greatest clinical utility in circumstances of on-going differentiation in pathogenic secretory cells, exemplified by malignant plasma cells in multiple myeloma or mucous-producing cells in chronic obstructive pulmonary disease. We used the inhibitor to unequivocally link the IRE1 RNase to the phenomenon of ER stress-induced mRNA decay, because 4μ8C rapidly blocks this process both in vitro and in cell culture. Moreover, our studies revealed that in mammalian cells, unlike in yeast and worms, IRE1 has no measureable role in cell survival during acute ER stress. Rather, loss of IRE1 function following 4μ8C treatment caused a pronounced block in ER expansion and secretory output and attenuated the growth of multiple myeloma cells. These observations suggest that in animals the IRE1 branch of the unfolded protein response has specialized to adapt cells to a heavy secretory load and is no longer important in day-to-day ER protein folding homeostasis. We demonstrate that Lysine 907 is the target of all extant IRE inhibitors and explored this unifying mechanism for IRE1 inhibition with docking and molecular dynamics simulations. In contrast to other lysine residues on the molecule, Lysine 907 is buried in the enzyme active site. The inhibitor is held in place by hydrophobic and stacking interactions with protein side chains in the active site to specify an unusually stable Schiff base at Lysine 907 and accounting for the surprising selectivity of the drug for IRE1. In this study, we used a fluorescence-based high-throughput screening program adapted from our studies of the yeast enzyme ( 5 ) to search for small molecule inhibitors of the mammalian IRE1. We found and characterized 8-formyl-7-hydroxy-4-methylcoumarin (abbreviated herein as 4μ8C) as a potent and selective inhibitor of IRE1. Using the unique spectroscopic properties of 4μ8C to trace its interaction with IRE1 in vitro, we discovered that the compound reacts covalently with the enzyme to form a stable Schiff base at two critical lysines in both the kinase and RNase pockets of the molecule. In cells this modification is limited to the RNase site at Lysine 907, dissecting the catalytic moieties of the IRE RNase and inactivating the enzyme (see Fig. P1 ). Fig. P1. Targeted inhibition of IRE1. 8-formyl-7-hydroxy-4-methylcoumarin (abbreviated 4μ8C, see inset to graph), identified by high-throughput screening, was found to inhibit the endonuclease activity of mammalian IRE1 with high selectivity in both an in vitro FRET-derepression assay (see graph) and cultured cells. The compound binds to a critical lysine in the endonuclease active site of IRE1 by formation of an unusually stable Schiff base. IRE1 K 907 -4μ8C modification constrains the flexibility of the endonuclease site by formation of stacking interactions with F 889 and interjects between essential catalytic residues, inactivating the enzyme. Structure shows human IRE1 protomer (Protein Data Bank ID code 3P23, Left ) and the detail ( Lower Right , residues 870 to 939) shows computationally docked 4μ8C (green) at K 907 , with residues F 889 , Y 892 , N 906 , K 907 , and H 910 highlighted. Although the unfolded protein response in simple eukaryotes comprises only IRE1, in animals two other regulatory pathways arise from the ER when proteins fail to fold in this compartment. The translation initiation factor 2α (eIF2α) kinase PERK constitutes a second branch and acts to attenuate ER load by inhibiting protein synthesis, and a third branch is mediated through transcriptional regulation by ATF6. For many years, redundancy between the long-term effects mediated by the three arms of the unfolded protein response has obscured the interpretation of genetic experiments to ascertain the unique role of each of these three components, IRE1, PERK, and ATF6 ( 1 ). These considerations, among others, have generated an interest in selective inhibitors of unfolded protein response components, both as tools for fundamental research and as potential anticancer, antiinflammatory, and antiviral therapeutic agents. In animals, IRE1 activity splices the latent messenger RNA that encodes XBP1. This triggers a cascade of events that activate XBP1, a potent transcription factor that upregulates genes that will enhance the ability of the ER to cope with unfolded proteins and upregulate secretory capacity ( 2 , 3 ). Thus, IRE1 activity results in rectifying gene expression changes that include the enhanced expression of specialist protein folding enzymes and degradation components to clear misfolded proteins, expanding the ER apparatus to cope with the protein folding load. In animal cells, IRE1 has also been linked to the promiscuous degradation of ER-localized mRNAs in a process known as regulated IRE1-dependent degradation (or RIDD), but the mechanistic basis and functional consequences of this process are presently incompletely understood ( 4 ). It is estimated that up to one-third of all proteins synthesized by eukaryotic cells are initially trafficked through the specialized environment of the endoplasmic reticulum (ER). In this subcellular compartment, conditions are optimized for protein folding and entry to the ER represents the first commitment step toward the secretory pathway. Diseases as diverse as cancer, neurodegeneration, and metabolic disorders such as insulin resistance and type II diabetes mellitus are associated and often caused or exacerbated by the failure of proteins to fold correctly in the ER. The evolutionary response to this potential toxicity is a tight regulation of components of this cellular compartment. Perturbances in the protein-folding environment of the ER are detected and corrected by a cellular stress response known as the unfolded protein response, the most ancient component of which is initiated by an ER-localized transmembrane protein called IRE1 ( 1 ). IRE1 has an ER lumenal domain that senses unfolded proteins and transmits a signal across the ER membrane to the effector domain of the protein in the cytosol. This effector domain is endowed with two enzymatic activities: a protein kinase and an RNase, both of which are activated in response to the accumulation of unfolded proteins in the ER (so-called ER stress).