Search

Drosophila border cell clusters model collective cell migration. Airyscan super-resolution microscopy enables fine-scale description of cluster shape and texture. Here we describe how to convert Airyscan images of border cell clusters into 3D models of the surface and detect regions of convex and concave curvature. We use spectral decomposition analysis to compare surface textures across genotypes to determine how genes of interest impact cluster surface geometry. This protocol applies to border cells and could generalize to additional cell types. For complete details on the use and execution of this protocol, please refer to Gabbert et al.1.

Electroporation is a technique to introduce DNA constructs into cells using electric current. Here, we present a protocol to electroporate DNA plasmids into Ciona robusta embryos at the 1-cell stage. We describe steps for setting up and conducting electroporation. We then detail procedures for collecting, fixing, and mounting embryos and counting expression. This protocol can be used to study the expression of enhancers via reporter assays, manipulating cells using genes or modified genes such as dominant negatives, and genome editing. For complete details on the use and execution of this protocol, please refer to Song, et al.1.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

Capture of a photon by an opsin visual pigment isomerizes its 11-cis-retinaldehyde (11cRAL) chromophore to all-trans-retinaldehyde (atRAL), which subsequently dissociates. To restore light sensitivity, the unliganded apo-opsin combines with another 11cRAL to make a new visual pigment. Two enzyme pathways supply chromophore to photoreceptors. The canonical visual cycle in retinal pigment epithelial cells supplies 11cRAL at low rates. The photic visual cycle in Müller cells supplies cones with 11-cis-retinol (11cROL) chromophore precursor at high rates. Although rods can only use 11cRAL to regenerate rhodopsin, cones can use 11cRAL or 11cROL to regenerate cone visual pigments. We performed a screen in zebrafish retinas and identified ZCRDH as a candidate for the enzyme that converts 11cROL to 11cRAL in cone inner segments. Retinoid analysis of eyes from Zcrdh-mutant zebrafish showed reduced 11cRAL and increased 11cROL levels, suggesting impaired conversion of 11cROL to 11cRAL. By microspectrophotometry, isolated Zcrdh-mutant cones lost the capacity to regenerate visual pigments from 11cROL. ZCRDH therefore possesses all predicted properties of the cone 11cROL dehydrogenase. The human protein most similar to ZCRDH is RDH12. By immunocytochemistry, ZCRDH was abundantly present in cone inner segments, similar to the reported distribution of RDH12. Finally, RDH12 was the only mammalian candidate protein to exhibit 11cROL-oxidase catalytic activity. These observations suggest that RDH12 in mammals is the functional ortholog of ZCRDH, which allows cones, but not rods, to regenerate visual pigments from 11cROL provided by Müller cells. This capacity permits cones to escape competition from rods for visual chromophore in daylight-exposed retinas.

The contrast between the disruption of genome topology after cohesin loss and the lack of downstream gene expression changes instigates intense debates regarding the structure-function relationship between genome and gene regulation. Here, by analyzing transcriptome and chromatin accessibility at the single-cell level, we discover that, instead of dictating population-wide gene expression levels, cohesin supplies a general function to neutralize stochastic coexpression tendencies of cis-linked genes in single cells. Notably, cohesin loss induces widespread gene coactivation and chromatin co-opening tens of million bases apart in cis. Spatial genome and protein imaging reveals that cohesin prevents gene co-bursting along the chromosome and blocks spatial mixing of transcriptional hubs. Single-molecule imaging shows that cohesin confines the exploration of diverse enhancer and core promoter binding transcriptional regulators. Together, these results support that cohesin arranges nuclear topology to control gene coexpression in single cells.

DDX3X regulates the translation of a subset of human transcripts containing complex 5' untranslated regions (5' UTRs). In this study, we developed the helicase activity reporter for translation (HART), which uses DDX3X-sensitive 5' UTRs to measure DDX3X-mediated translational activity in cells. To directly measure RNA structure in DDX3X-dependent mRNAs, we used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then used HART to investigate how sequence alterations influence DDX3X sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3X's interaction with the translational machinery. HART revealed that both DDX3X's association with the translational machinery and its helicase activity are required for its function in promoting the translation of DDX3X-sensitive 5' UTRs. These findings suggest DDX3X plays a crucial role in regulating translation through its interaction with the translational machinery during ribosome scanning and establish the HART reporter as a robust, lentivirally encoded, colorimetric measurement of DDX3X-dependent translation in cells.

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.

Adenosine Deaminases Acting on RNA (ADARs) are enzymes that catalyze the conversion of adenosine to inosine in RNA duplexes. These enzymes can be harnessed to correct disease-causing G-to-A mutations in the transcriptome because inosine is translated as guanosine. Guide RNAs (gRNAs) can be used to direct the ADAR reaction to specific sites. Chemical modification of ADAR guide strands is required to facilitate delivery, increase metabolic stability, and increase the efficiency and selectivity of the editing reaction. Here, we show the ADAR reaction is highly sensitive to ribose modifications (e.g. 4'-C-methylation and Locked Nucleic Acid (LNA) substitution) at specific positions within the guide strand. Our studies were enabled by the synthesis of RNA containing a new, ribose-modified nucleoside analog (4'-C-methyladenosine). Importantly, the ADAR reaction is potently inhibited by LNA or 4'-C-methylation at different positions in the ADAR guide. While LNA at guide strand positions -1 and -2 block the ADAR reaction, 4'-C-methylation only inhibits at the -2 position. These effects are rationalized using high-resolution structures of ADAR-RNA complexes. This work sheds additional light on the mechanism of ADAR deamination and aids in the design of highly selective ADAR guide strands for therapeutic editing using chemically modified RNA.

Aposematic organisms rely on their conspicuous appearance to signal that they are defended and unpalatable. Such phenotypes are strongly tied to survival and reproduction. Aposematic colors and patterns are highly variable; however, the genetic, biochemical, and physiological mechanisms producing this conspicuous coloration remain largely unidentified. Here, we identify genes potentially affecting color variation in two color morphs of Ranitomeya imitator: the orange-banded Sauce and the redheaded Varadero morphs. We examine gene expression in black and orange skin patches from the Sauce morph and black and red skin patches from the Varadero morph. We identified genes differentially expressed between skin patches, including those that are involved in melanin synthesis (e.g. mlana, pmel, tyrp1), iridophore development (e.g. paics, ppat, ak1), pteridine synthesis (e.g. gch1, pax3-a, xdh), and carotenoid metabolism (e.g. dgat2, rbp1, scarb2). In addition, using weighted correlation network analysis, we identified the top 50 genes with high connectivity from the most significant network associated with gene expression differences between color morphs. Of these 50 genes, 13 were known to be related to color production (gch1, gmps, gpr143, impdh1, mc1r, pax3-a, pax7, ppat, rab27a, rlbp1, tfec, trpm1, xdh).

A key feature distinguishing high-grade glioma (HG) from low-grade glioma (LG) is the extensive neovascularization and endothelial hyperproliferation. Prior work has shown that tumor-associated vasculature from HG is molecularly and functionally distinct from normal brain vasculature and expresses higher levels of protumorigenic factors that promote glioma growth and progression. However, it remains unclear whether vessels from LG also express protumorigenic factors, and to what extent they functionally contribute to glioma growth. Here, we profile the transcriptomes of glioma-associated vascular cells (GVC) from IDH-mutant (mIDH) LG and IDH-wild-type (wIDH) HG and show that they exhibit significant molecular and functional differences. LG-GVC show enrichment of extracellular matrix-related gene sets and sensitivity to antiangiogenic drugs, whereas HG-GVC display an increase in immune response-related gene sets and antiangiogenic resistance. Strikingly, conditioned media from LG-GVC inhibits the growth of wIDH glioblastoma cells, whereas HG-GVC promotes growth. In vivo cotransplantation of LG-GVC with tumor cells reduces growth, whereas HG-GVC enhances tumor growth in orthotopic xenografts. We identify ASPORIN (ASPN), a small leucine-rich repeat proteoglycan, highly enriched in LG-GVC as a growth suppressor of wIDH glioblastoma cells in vitro and in vivo. Together, these findings indicate that GVC from LG and HG are molecularly and functionally distinct and differentially regulate tumor growth. Implications: This study demonstrated that vascular cells from IDH-mutant LG and IDH-wild-type HG exhibit distinct molecular signatures and have differential effects on tumor growth via regulation of ASPN-TGFβ1-GPM6A signaling.

Tissue regeneration after damage is generally thought to involve the mobilization of adult stem cells that divide and differentiate into progressively specialized progeny. However, recent studies indicate that tissue regeneration can be accompanied by reversion to a fetal-like state. During this process, cells at the injury site reactivate programs that operate during fetal development but are typically absent in adult homeostasis. Here, we summarize our current understanding of the molecular signals and epigenetic mediators that orchestrate "fetal-like reversion" during intestinal regeneration. We also explore evidence for this phenomenon in other organs and species and highlight open questions that merit future examination.

Sensory adaptation is the process whereby brain circuits adjust neuronal activity in response to redundant sensory stimuli. Although sensory adaptation has been extensively studied for individual neurons on timescales of tens of milliseconds to a few seconds, little is known about it over longer timescales or at the population level. We investigated population-level adaptation in the barrel field of the mouse somatosensory cortex (S1BF) using in vivo two-photon calcium imaging and Neuropixels recordings in awake mice. Among stimulus-responsive neurons, we found both adapting and facilitating neurons, which decreased or increased their firing, respectively, with repetitive whisker stimulation. The former outnumbered the latter by 2:1 in layers 2/3 and 4; hence, the overall population response of mouse S1BF was slightly adapting. We also discovered that population adaptation to one stimulus frequency (5 Hz) does not necessarily generalize to a different frequency (12.5 Hz). Moreover, responses of individual neurons to repeated rounds of stimulation over tens of minutes were strikingly heterogeneous and stochastic, such that their adapting or facilitating response profiles were not stable across time. Such representational drift was particularly striking when recording longitudinally across 8-9 days, as adaptation profiles of most whisker-responsive neurons changed drastically from one day to the next. Remarkably, repeated exposure to a familiar stimulus paradoxically shifted the population away from strong adaptation and toward facilitation. Thus, the adapting vs. facilitating response profile of S1BF neurons is not a fixed property of neurons but rather a highly dynamic feature that is shaped by sensory experience across days.

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single-cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing 8 new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nanoscale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal noncanonical amino acid tagging (BONCAT), we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle.

The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.

Precision pharmacology aims to manipulate specific cellular interactions within complex tissues. In this pursuit, we introduce DART.2 (drug acutely restricted by tethering), a second-generation cell-specific pharmacology technology. The core advance is optimized cellular specificity-up to 3,000-fold in 15 min-enabling the targeted delivery of even epileptogenic drugs without off-target effects. Additionally, we introduce brain-wide dosing methods as an alternative to local cannulation and tracer reagents for brain-wide dose quantification. We describe four pharmaceuticals-two that antagonize excitatory and inhibitory postsynaptic receptors, and two that allosterically potentiate these receptors. Their versatility is showcased across multiple mouse-brain regions, including cerebellum, striatum, visual cortex and retina. Finally, in the ventral tegmental area, we find that blocking inhibitory inputs to dopamine neurons accelerates locomotion, contrasting with previous optogenetic and pharmacological findings. Beyond enabling the bidirectional perturbation of chemical synapses, these reagents offer intersectional precision-between genetically defined postsynaptic cells and neurotransmitter-defined presynaptic partners.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Systemic low-grade inflammation is a feature of chronic disease. C-reactive protein (CRP) is a common biomarker of inflammation and used as an indicator of disease risk; however, the role of inflammation in disease is not completely understood. Methylation is an epigenetic modification in the DNA which plays a pivotal role in gene expression. In this study we evaluated differential DNA methylation patterns associated with blood CRP level to elucidate biological pathways and genetic regulatory mechanisms to improve the understanding of chronic inflammation. The racially and ethnically diverse participants in this study were included as 50% White, 41% Black or African American, 7% Hispanic or Latino/a, and 2% Native Hawaiian, Asian American, American Indian, or Alaska Native (total n = 13,433) individuals. We replicated 113 CpG sites from 87 unique loci, of which five were novel (CADM3, NALCN, NLRC5, ZNF792, and cg03282312), across a discovery set of 1,150 CpG sites associated with CRP level (p

Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the '5mCAdpBS-Seq' workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.

Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis.

With aging skeletal muscle fibers undergo repeating cycles of denervation and reinnervation. In approximately the 8th decade of life reinnervation no longer keeps pace, resulting in the accumulation of persistently denervated muscle fibers that in turn cause an acceleration of muscle dysfunction. The significance of denervation in important clinical outcomes with aging is poorly studied. The Study of Muscle, Mobility, and Aging (SOMMA) is a large cohort study with the primary objective to assess how aging muscle biology impacts clinically important traits. Using transcriptomics data from vastus lateralis muscle biopsies in 575 participants we have selected 49 denervation-responsive genes to provide insights to the burden of denervation in SOMMA, to test the hypothesis that greater expression of denervation-responsive genes negatively associates with SOMMA participant traits that included time to walk 400 meters, fitness (VO2peak), maximal mitochondrial respiration, muscle mass and volume, and leg muscle strength and power. Consistent with our hypothesis, increased transcript levels of: a calciumdependent intercellular adhesion glycoprotein (CDH15), acetylcholine receptor subunits (CHRNA1, CHRND, CHRNE), a glycoprotein promoting reinnervation (NCAM1), a transcription factor regulating aspects of muscle organization (RUNX1), and a sodium channel (SCN5A) were each negatively associated with at least 3 of these traits. VO2peak and maximal respiration had the strongest negative associations with 15 and 19 denervation-responsive genes, respectively. In conclusion, the abundance of denervationresponsive gene transcripts is a significant determinant of muscle and mobility outcomes in aging humans, supporting the imperative to identify new treatment strategies to restore innervation in advanced age.

Pain, detected by nociceptors, is an integral part of injury, yet whether and how it can impact tissue physiology and recovery remain understudied. Here, we applied chemogenetics in mice to locally activate dermal TRPV1 innervations in naive skin and found that it triggered new regenerative cycling by dormant hair follicles (HFs). This was preceded by rapid apoptosis of dermal macrophages, mediated by the neuropeptide calcitonin gene-related peptide (CGRP). TRPV1 activation also triggered a macrophage-dependent induction of osteopontin (Spp1)-expressing dermal fibroblasts. The neuropeptide CGRP and the extracellular matrix protein Spp1 were required for the nociceptor-triggered hair growth. Finally, we showed that epidermal abrasion injury induced Spp1-expressing dermal fibroblasts and hair growth via a TRPV1 neuron and CGRP-dependent mechanism. Collectively, these data demonstrated a role for TRPV1 nociceptors in orchestrating a macrophage and fibroblast-supported mechanism to promote hair growth and enabling the efficient restoration of this mechano- and thermo-protective barrier after wounding.

Somatic and sympathetic tones fluctuate together seamlessly across daily behaviors. In this issue of Cell, Zhang et al. describe populations of spinal projecting neurons in the rostral ventromedial medulla (rVMM) that harmonize somatic motor function and sympathetic activation. The coordinated regulation plays a vital role in supporting behaviors associated with various arousal states.

Intratumor heterogeneity underlies cancer evolution and treatment resistance, but targetable mechanisms driving intratumor heterogeneity are poorly understood. Meningiomas are the most common primary intracranial tumors and are resistant to all medical therapies, and high-grade meningiomas have significant intratumor heterogeneity. Here we use spatial approaches to identify genomic, biochemical and cellular mechanisms linking intratumor heterogeneity to the molecular, temporal and spatial evolution of high-grade meningiomas. We show that divergent intratumor gene and protein expression programs distinguish high-grade meningiomas that are otherwise grouped together by current classification systems. Analyses of matched pairs of primary and recurrent meningiomas reveal spatial expansion of subclonal copy number variants associated with treatment resistance. Multiplexed sequential immunofluorescence and deconvolution of meningioma spatial transcriptomes using cell types from single-cell RNA sequencing show decreased immune infiltration, decreased MAPK signaling, increased PI3K-AKT signaling and increased cell proliferation, which are associated with meningioma recurrence. To translate these findings to preclinical models, we use CRISPR interference and lineage tracing approaches to identify combination therapies that target intratumor heterogeneity in meningioma cell co-cultures.

In plant vegetative tissues, cell division employs a mitotic microtubule array called the preprophase band (PPB) that marks the cortical division site. This transient cytoskeletal array imprints the spatial information to be read by the cytokinetic phragmoplast at later stages of mitotic cell division. In Arabidopsis thaliana, we discovered that the PPB recruited the Myosin XI motor MYA1/Myo11F to the cortical division site, where it joined microtubule-associated proteins and motors to form a ring of prominent cytoskeletal assemblies that received the expanding phragmoplast. Such a myosin localization pattern at the cortical division site was dependent on the POK1/2 Kinesin-12 motors. This regulatory function of MYA1/Myo11F in phragmoplast guidance was dependent on intact actin filaments. The discovery of these cytoskeletal motor assemblies pinpoints a mechanism underlying how two dynamic cytoskeletal networks work in concert to govern PPB-dependent division plane orientation in flowering plants.

Playing two-dimensional video games has been shown to result in improvements in a range of visual and cognitive tasks, and these improvements appear to generalize widely1,2,3,4,5,6. Here we report that young adults with healthy vision, surprisingly, showed a dramatic improvement in stereo vision after playing three-dimensional, but not two-dimensional, video games for a relatively short period of time. Intriguingly, neither group showed any significant improvement in binocular contrast sensitivity. This dissociation suggests that the visual enhancement was specific to genuine stereoscopic processing, not indirectly resulting from enhanced contrast processing, and required engaging in a disparity cue-rich three-dimensional environment.

Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28, CTLA4 and ICOS, encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells, both conventional and regulatory subsets, uncovered gene-, cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNAGly and BioRNALeu, respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

Chronic conditions associated with aging have proven difficult to prevent or treat. Senescence is a cell fate defined by loss of proliferative capacity and the development of a pro-inflammatory senescence-associated secretory phenotype comprised of cytokines/chemokines, proteases, and other factors that promotes age-related diseases. Specifically, an increase in senescent peripheral blood mononuclear cells (PBMCs), including T cells, is associated with conditions like frailty, rheumatoid arthritis, and bone loss. However, it is unknown if the percentage of senescent PBMCs associated with age-associated orthopedic decline could be used for potential diagnostic or prognostic use in orthopedics. Here, we report senescent cell detection using the fluorescent compound C12FDG to quantify PBMCs senescence across a large cohort of healthy and osteoarthritic patients. There is an increase in the percent of circulating C12FDG+ PBMCs that is commensurate with increases in age and senescence-related serum biomarkers. Interestingly, C12FDG+ PBMCs and T cells also were found to be elevated in patients with mild to moderate osteoarthritis, a progressive joint disease that is strongly associated with inflammation. The percent of C12FDG+ PBMCs and age-related serum biomarkers were decreased in a small subgroup of study participants taking the senolytic drug fisetin. These results demonstrate quantifiable measurements in a large group of participants that could create a composite score of healthy aging sensitive enough to detect changes following senolytic therapy and may predict age-related orthopedic decline. Detection of peripheral senescence in PBMCs and subsets using C12FDG may be clinically useful for quantifying cellular senescence and determining how and if it plays a pathological role in osteoarthritic progression.

The coppery titi monkey (Plecturocebus cupreus) is an emerging nonhuman primate model system for behavioral and neurobiological research. At the same time, the almost entire absence of genomic resources for the species has hampered insights into the genetic underpinnings of the phenotypic traits of interest. To facilitate future genotype-to-phenotype studies, we here present a high-quality, fully annotated de novo genome assembly for the species with chromosome-length scaffolds spanning the autosomes and chromosome X (scaffold N50 = 130.8 Mb), constructed using data obtained from several orthologous short- and long-read sequencing and scaffolding techniques. With a base-level accuracy of ∼99.99% in chromosome-length scaffolds as well as benchmarking universal single-copy ortholog and k-mer completeness scores of >99.0% and 95.1% at the genome level, this assembly represents one of the most complete Pitheciidae genomes to date, making it an invaluable resource for comparative evolutionary genomics research to improve our understanding of lineage-specific changes underlying adaptive traits as well as deleterious mutations associated with disease.

The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

Zygnematophyceae are the algal sisters of land plants. Here we sequenced four genomes of filamentous Zygnematophyceae, including chromosome-scale assemblies for three strains of Zygnema circumcarinatum. We inferred traits in the ancestor of Zygnematophyceae and land plants that might have ushered in the conquest of land by plants: expanded genes for signaling cascades, environmental response, and multicellular growth. Zygnematophyceae and land plants share all the major enzymes for cell wall synthesis and remodifications, and gene gains shaped this toolkit. Co-expression network analyses uncover gene cohorts that unite environmental signaling with multicellular developmental programs. Our data shed light on a molecular chassis that balances environmental response and growth modulation across more than 600 million years of streptophyte evolution.

Cyanobacteria uniquely contain a primitive water-soluble carotenoprotein, the orange carotenoid protein (OCP). Nearly all extant cyanobacterial genomes contain genes for the OCP or its homologs, implying an evolutionary constraint for cyanobacteria to conserve its function. Genes encoding the OCP and its two constituent structural domains, the N-terminal domain, helical carotenoid proteins (HCPs), and its C-terminal domain, are found in the most basal lineages of extant cyanobacteria. These three carotenoproteins exemplify the importance of the protein for carotenoid properties, including protein dynamics, in response to environmental changes in facilitating a photoresponse and energy quenching. Here, we review new structural insights for these carotenoproteins and situate the role of the protein in what is currently understood about their functions.

Inosine (I), resulting from the deamination of adenosine (A), is a prominent modification in the human transcriptome. The enzymes responsible for the conversion of adenosine to inosine in human mRNAs are the ADARs (adenosine deaminases acting on RNA). Inosine modification introduces a layer of complexity to mRNA processing and function, as it can impact various aspects of RNA biology, including mRNA stability, splicing, translation, and protein binding. The relevance of this process is emphasized in the growing number of human disorders associated with dysregulated A-to-I editing pathways. Here, we describe the impact of the A-to-I conversion on the structure and stability of duplex RNA and on the consequences of this modification at different locations in mRNAs. Furthermore, we highlight specific open questions regarding the interplay between inosine formation in duplex RNA and the innate immune response.

Because derivation of retinal organoids (ROs) and transplantation are frequently split between geographically distant locations, we developed a special shipping device and protocol capable of the organoids' delivery to any location. Human embryonic stem cell (hESC)-derived ROs were differentiated from the hESC line H1 (WA01), shipped overnight to another location, and then transplanted into the subretinal space of blind immunodeficient retinal degeneration (RD) rats. Development of transplants was monitored by spectral-domain optical coherence tomography. Visual function was accessed by optokinetic tests and superior colliculus (SC) electrophysiology. Cryostat sections through transplants were stained with hematoxylin and eosin; or processed for immunohistochemistry to label human donor cells, retinal cell types, and synaptic markers. After transplantation, ROs integrated into the host RD retina, formed functional photoreceptors, and improved vision in rats with advanced RD. The survival and vision improvement are comparable with our previous results of hESC-ROs without a long-distance delivery. Furthermore, for the first time in the stem cell transplantation field, we demonstrated that the response heatmap on the SC showed a similar shape to the location of the transplant in the host retina, which suggested the point-to-point projection of the transplant from the retina to SC. In conclusion, our results showed that using our special device and protocol, the hESC-derived ROs can be shipped over long distance and are capable of survival and visual improvement after transplantation into the RD rats. Our data provide a proof-of-concept for stem cell replacement as a therapy for RD patients.

Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli 'helper' strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 μg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability, scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA.

Studies suggest that ketogenic diets (KD) may improve memory in mouse models of aging and Alzheimer's disease (AD). This study determined whether a continuous or intermittent KD (IKD) enhanced cognitive behavior in the TgF344-AD rat model of AD. At 6 months-old, TgF344-AD and wild-type (WT) littermates were placed on a control (CD), KD, or IKD (morning CD and afternoon KD) provided as two meals per day for 2 or 6 months. Cognitive and motor behavior and circulating β-hydroxybutyrate (BHB), AD biomarkers and blood lipids were assessed. Animals on a KD diet had elevated circulating BHB, with IKD levels intermediate to CD and KD. TgF344-AD rats displayed impaired spatial learning memory in the Barnes maze at 8 and 12 months of age and impaired motor coordination at 12 months of age. Neither KD nor IKD improved performance compared to CD. At 12 months of age, TgF344-AD animals had elevated blood lipids. IKD reduced lipids to WT levels with KD further reducing cholesterol below WT levels. This study shows that at 8 or 12 months of age, KD or IKD intervention did not improve measures of cognitive or motor behavior in TgF344-AD rats; however, both IKD and KD positively impacted circulating lipids.

Meiosis is a key step during germ cell differentiation, accompanied by the activation of thousands of genes through germline-specific chromatin reorganization. The chromatin remodeling mechanisms underpinning early meiotic stages remain poorly understood. Here we focus on the function of one of the major autism genes, CHD8, in spermatogenesis, based on the epidemiological association between autism and low fertility rates. Specific ablation of Chd8 in germ cells results in gradual depletion of undifferentiated spermatogonia and the failure of meiotic double-strand break (DSB) formation, leading to meiotic prophase I arrest and cell death. Transcriptional analyses demonstrate that CHD8 is required for extensive activation of spermatogenic genes in spermatogonia, necessary for spermatogonial proliferation and meiosis. CHD8 directly binds and regulates genes crucial for meiosis, including H3K4me3 histone methyltransferase genes, meiotic cohesin genes, HORMA domain-containing genes, synaptonemal complex genes, and DNA damage response genes. We infer that CHD8 contributes to meiotic DSB formation and subsequent meiotic progression through combined regulation of these meiosis-related genes. Our study uncovers an essential role of CHD8 in the proliferation of undifferentiated spermatogonia and the successful progression of meiotic prophase I.

The genus Xanthomonas has been primarily studied for pathogenic interactions with plants. However, besides host and tissue-specific pathogenic strains, this genus also comprises nonpathogenic strains isolated from a broad range of hosts, sometimes in association with pathogenic strains, and other environments, including rainwater. Based on their incapacity or limited capacity to cause symptoms on the host of isolation, nonpathogenic xanthomonads can be further characterized as commensal and weakly pathogenic. This study aimed to understand the diversity and evolution of nonpathogenic xanthomonads compared to their pathogenic counterparts based on their cooccurrence and phylogenetic relationship and to identify genomic traits that form the basis of a life history framework that groups xanthomonads by ecological strategies. We sequenced genomes of 83 strains spanning the genus phylogeny and identified eight novel species, indicating unexplored diversity. While some nonpathogenic species have experienced a recent loss of a type III secretion system, specifically the hrp2 cluster, we observed an apparent lack of association of the hrp2 cluster with lifestyles of diverse species. We performed association analysis on a large data set of 337 Xanthomonas strains to explain how xanthomonads may have established association with the plants across the continuum of lifestyles from commensals to weak pathogens to pathogens. Presence of distinct transcriptional regulators, distinct nutrient utilization and assimilation genes, transcriptional regulators, and chemotaxis genes may explain lifestyle-specific adaptations of xanthomonads.

Transposable elements (TE) play critical roles in shaping genome evolution. Highly repetitive TE sequences are also a major source of assembly gaps making it difficult to fully understand the impact of these elements on host genomes. The increased capacity of long-read sequencing technologies to span highly repetitive regions promises to provide new insights into patterns of TE activity across diverse taxa. Here we report the generation of highly contiguous reference genomes using PacBio long-read and Omni-C technologies for three species of Passerellidae sparrow. We compared these assemblies to three chromosome-level sparrow assemblies and nine other sparrow assemblies generated using a variety of short- and long-read technologies. All long-read based assemblies were longer (range: 1.12 to 1.41 Gb) than short-read assemblies (0.91 to 1.08 Gb) and assembly length was strongly correlated with the amount of repeat content. Repeat content for Bell's sparrow (31.2% of genome) was the highest level ever reported within the order Passeriformes, which comprises over half of avian diversity. The highest levels of repeat content (79.2% to 93.7%) were found on the W chromosome relative to other regions of the genome. Finally, we show that proliferation of different TE classes varied even among species with similar levels of repeat content. These patterns support a dynamic model of TE expansion and contraction even in a clade where TEs were once thought to be fairly depauperate and static. Our work highlights how the resolution of difficult-to-assemble regions of the genome with new sequencing technologies promises to transform our understanding of avian genome evolution.

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here we investigate the three-dimensional (3D) conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. Sixty-one percent of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general importance of enhancer-promoter physical proximity for developmental gene activation in mammals.

Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules derived from unsaturated fatty acids. To better understand the basis of the substrate selectivity of MRP4, we used cryogenic-electron microscopy to determine six structures of nanodisc-reconstituted MRP4 at various stages throughout its transport cycle. Substrate-bound structures of MRP4 in complex with PGE1, PGE2 and the sulfonated-sterol DHEA-S reveal a common binding site that accommodates a diverse set of organic anions and suggest an allosteric mechanism for substrate-induced enhancement of MRP4 ATPase activity. Our structure of a catalytically compromised MRP4 mutant bound to ATP-Mg2+ is outward-occluded, a conformation previously unobserved in the MRP subfamily and consistent with an alternating-access transport mechanism. Our study provides insights into the endogenous function of this versatile efflux transporter and establishes a basis for MRP4-targeted drug design.

The global panzootic lineage (GPL) of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly available Bd genome sequences, we identified multiple non-GPL Bd isolates that contain a circular Rep-encoding single-stranded (CRESS)-like DNA virus, which we named Bd DNA virus 1 (BdDV-1). We further sequenced and constructed genome assemblies with long read sequences to find that the virus is integrated into the nuclear genome in some strains. Attempts to cure virus-positive isolates were unsuccessful; however, phenotypic differences between naturally virus-positive and virus-negative Bd isolates suggested that BdDV-1 decreases the growth of its host in vitro but increases the virulence of its host in vivo. BdDV-1 is the first-described CRESS DNA mycovirus of zoosporic true fungi, with a distribution inversely associated with the emergence of the panzootic lineage.

The Genome Aggregation Database (gnomAD), widely recognized as the gold-standard reference map of human genetic variation, has largely overlooked tandem repeat (TR) expansions, despite the fact that TRs constitute ∼6% of our genome and are linked to over 50 human diseases. Here, we introduce the TR-gnomAD (https://wlcb.oit.uci.edu/TRgnomAD), a biobank-scale reference of 0.86 million TRs derived from 338,963 whole-genome sequencing (WGS) samples of diverse ancestries (39.5% non-European samples). TR-gnomAD offers critical insights into ancestry-specific disease prevalence using disparities in TR unit number frequencies among ancestries. Moreover, TR-gnomAD is able to differentiate between common, presumably benign TR expansions, which are prevalent in TR-gnomAD, from those potentially pathogenic TR expansions, which are found more frequently in disease groups than within TR-gnomAD. Together, TR-gnomAD is an invaluable resource for researchers and physicians to interpret TR expansions in individuals with genetic diseases.

The relationship between the early-age activity of Mediterranean fruit flies (medflies) or other fruit flies and their lifespan has not been much studied, in contrast to the connections between lifespan and diet, sexual signaling, and reproduction. The objective of this study is to assess intra-day and day-to-day activity profiles of female Mediterranean fruit flies and their role as biomarker of longevity as well as to explore the relationships between these activity profiles, diet, and age-at-death throughout the lifespan. We use advanced statistical methods from functional data analysis (FDA). Three distinct patterns of activity variations in early-age activity profiles can be distinguished. A low-caloric diet is associated with a delayed activity peak, while a high-caloric diet is linked with an earlier activity peak. We find that age-at-death of individual medflies is connected to their activity profiles in early life. An increased risk of mortality is associated with increased activity in early age, as well as with a higher contrast between daytime and nighttime activity. Conversely, medflies are more likely to have a longer lifespan when they are fed a medium-caloric diet and when their daily activity is more evenly distributed across the early-age span and between daytime and nighttime. The before-death activity profile of medflies displays two characteristic before-death patterns, where one pattern is characterized by slowly declining daily activity and the other by a sudden decline in activity that is followed by death.

Spermatogonial stem cells functionality reside in the slow-cycling and heterogeneous undifferentiated spermatogonia cell population. This pool of cells supports lifelong fertility in adult males by balancing self-renewal and differentiation to produce haploid gametes. However, the molecular mechanisms underpinning long-term stemness of undifferentiated spermatogonia during adulthood remain unclear. Here, we discover that an epigenetic regulator, Polycomb repressive complex 1 (PRC1), shields adult undifferentiated spermatogonia from differentiation, maintains slow cycling, and directs commitment to differentiation during steady-state spermatogenesis in adults. We show that PRC2-mediated H3K27me3 is an epigenetic hallmark of adult undifferentiated spermatogonia. Indeed, spermatogonial differentiation is accompanied by a global loss of H3K27me3. Disruption of PRC1 impairs global H3K27me3 deposition, leading to precocious spermatogonial differentiation. Therefore, PRC1 directs PRC2-H3K27me3 deposition to maintain the self-renewing state of undifferentiated spermatogonia. Importantly, in contrast to its role in other tissue stem cells, PRC1 negatively regulates the cell cycle to maintain slow cycling of undifferentiated spermatogonia. Our findings have implications for how epigenetic regulators can be tuned to regulate the stem cell potential, cell cycle and differentiation to ensure lifelong fertility in adult males.

The loading of RecA onto ssDNA by RecBCD is an essential step of RecBCD-mediated homologous recombination. RecBCD facilitates RecA-loading onto ssDNA in a χ-dependent manner via its RecB nuclease domain (RecBn). Before recognition of χ, RecBn is sequestered through interactions with RecBCD. It was proposed that upon χ-recognition, RecBn undocks, allowing RecBn to swing out via a contiguous 70 amino acid linker to reveal the RecA-loading surface, and then recruit and load RecA onto ssDNA. We tested this hypothesis by examining the interactions between RecBn (RecB928-1180) and truncated RecBCD (RecB1-927CD) lacking the nuclease domain. The reconstituted complex of RecB1-927CD and RecBn is functional in vitro and in vivo. Our results indicate that despite being covalently severed from RecB1-927CD, RecBn can still load RecA onto ssDNA, establishing that RecBn does not function while only remaining tethered to the RecBCD complex via the linker. Instead, RecBCD undergoes a χ-induced intramolecular rearrangement to reveal the RecA-loading surface.

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP's enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

Transcriptomic contributions to the anatomical, functional, and network layout of the human cerebral cortex (HCC) have become a major interest in cognitive and systems neuroscience. Here, we tested if transcriptomic differences support a modern, algorithmic cytoarchitectonic parcellation of HCC. Using a data-driven approach, we identified a sparse subset of genes that differentially contributed to the cytoarchitectonic parcellation of HCC. A combined metric of cortical thickness and myelination (CT/M ratio), as well as cell density, correlated with gene expression. Enrichment analyses showed that genes specific to the cytoarchitectonic parcellation of the HCC were related to molecular functions such as transmembrane transport and ion channel activity. Together, the relationship between transcriptomics and cytoarchitecture bridges the gap among (i) gradients at the macro-scale (including thickness and myelination), (ii) areas at the meso-scale, and (iii) cell density at the microscale, as well as supports the recently proposed cortical spectrum theory and structural model.

Thrombospondin-4 (TSP-4) belongs to the extracellular matrix glycoprotein family of thrombospondins (TSPs). The multidomain, pentameric structure of TSP-4 allows its interactions with numerous extracellular matrix components, proteins and signaling molecules that enable its modulation to various physiological and pathological processes. Characterization of TSP-4 expression under development and pathogenesis of disorders has yielded important insights into mechanisms underlying the unique role of TSP-4 in mediating various processes including cell-cell, cell-extracellular matrix interactions, cell migration, proliferation, tissue remodeling, angiogenesis, and synaptogenesis. Maladaptation of these processes in response to pathological insults and stress can accelerate the development of disorders including skeletal dysplasia, osteoporosis, degenerative joint disease, cardiovascular diseases, tumor progression/metastasis and neurological disorders. Overall, the diverse functions of TSP-4 suggest that it may be a potential marker or therapeutic target for prognosis, diagnosis, and treatment of various pathological conditions upon further investigations. This review article highlights recent findings on the role of TSP-4 in both physiological and pathological conditions with a focus on what sets it apart from other TSPs.