Design, construction, and functional characterization of a tRNA neochromosome in yeast.

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer.

Tumor-resident intracellular microbiota is an emerging tumor component that has been documented for a variety of cancer types with unclear biological functions. Here, we explored the functional significance of these intratumor bacteria, primarily using a murine spontaneous breast-tumor model MMTV-PyMT. We found that depletion of intratumor bacteria significantly reduced lung metastasis without affecting primary tumor growth. During metastatic colonization, intratumor bacteria carried by circulating tumor cells promoted host-cell survival by enhancing resistance to fluid shear stress by reorganizing actin cytoskeleton. We further showed that intratumor administration of selected bacteria strains isolated from tumor-resident microbiota promoted metastasis in two murine tumor models with significantly different levels of metastasis potential. Our findings suggest that tumor-resident microbiota, albeit at low biomass, play an important role in promoting cancer metastasis, intervention of which might therefore be worth exploring for advancing oncology care.

When cleaning facilitates cluttering - genome editing in ciliates.

Many organisms remove DNA from their genomes during development. This has foremost been characterized as a means of defending genomes against mobile elements. However, genome editing actually hides such elements from purifying selection, with the survivors evolving approximately neutrally, 'cluttering' the germline genome, enabling it to enlarge over time.

Two paralogous PHD finger proteins participate in natural genome editing in Paramecium tetraurelia.

The unicellular eukaryote Paramecium tetraurelia contains functionally distinct nuclei: germline micronuclei (MICs) and a somatic macronucleus (MAC). During sex, the MIC genome is reorganized into a new MAC genome and the old MAC is lost. Almost 45,000 unique internal eliminated sequences (IESs) distributed throughout the genome require precise excision to guarantee a functional new MAC genome. Here, we characterize a pair of paralogous PHD finger proteins involved in DNA elimination. DevPF1, the early-expressed paralog, is present in only some of the gametic and post-zygotic nuclei during meiosis. Both DevPF1 and DevPF2 localize in the new developing MACs, where IES excision occurs. Upon DevPF2 knockdown (KD), long IESs are preferentially retained and late-expressed small RNAs decrease; no length preference for retained IESs was observed in DevPF1-KD and development-specific small RNAs were abolished. The expression of at least two genes from the new MAC with roles in genome reorganization seems to be influenced by DevPF1- and DevPF2-KD. Thus, both PHD fingers are crucial for new MAC genome development, with distinct functions, potentially via regulation of non-coding and coding transcription in the MICs and new MACs.

The midbody component Prc1-like is required for microtubule reorganization during cytokinesis and dorsal determinant segregation in the early zebrafish embryo.

We show that the zebrafish maternal-effect mutation too much information (tmi) corresponds to zebrafish prc1-like (prc1l), which encodes a member of the MAP65/Ase1/PRC1 family of microtubule-associated proteins. Embryos from tmi homozygous mutant mothers display cytokinesis defects in meiotic and mitotic divisions in the early embryo, indicating that Prc1l has a role in midbody formation during cell division at the egg-to-embryo transition. Unexpectedly, maternal Prc1l function is also essential for the reorganization of vegetal pole microtubules required for the segregation of dorsal determinants. Whereas Prc1 is widely regarded to crosslink microtubules in an antiparallel conformation, our studies provide evidence for an additional function of Prc1l in the bundling of parallel microtubules in the vegetal cortex of the early embryo during cortical rotation and prior to mitotic cycling. These findings highlight common yet distinct aspects of microtubule reorganization that occur during the egg-to-embryo transition, driven by maternal product for the midbody component Prc1l and required for embryonic cell division and pattern formation.

Visual Deprivation during Mouse Critical Period Reorganizes Network-Level Functional Connectivity.

A classic example of experience-dependent plasticity is ocular dominance (OD) shift, in which the responsiveness of neurons in the visual cortex is profoundly altered following monocular deprivation (MD). It has been postulated that OD shifts also modify global neural networks, but such effects have never been demonstrated. Here, we use wide-field fluorescence optical imaging (WFOI) to characterize calcium-based resting-state functional connectivity during acute (3 d) MD in female and male mice with genetically encoded calcium indicators (Thy1-GCaMP6f). We first establish the fundamental performance of WFOI by computing signal to noise properties throughout our data processing pipeline. Following MD, we found that Δ band (0.4-4 Hz) GCaMP6 activity in the deprived visual cortex decreased, suggesting that excitatory activity in this region was reduced by MD. In addition, interhemispheric visual homotopic functional connectivity decreased following MD, which was accompanied by a reduction in parietal and motor homotopic connectivity. Finally, we observed enhanced internetwork connectivity between the visual and parietal cortex that peaked 2 d after MD. Together, these findings support the hypothesis that early MD induces dynamic reorganization of disparate functional networks including the association cortices.

Cortical Reorganization after Limb Loss: Bridging the Gap between Basic Science and Clinical Recovery.

Despite the increasing incidence and prevalence of amputation across the globe, individuals with acquired limb loss continue to struggle with functional recovery and chronic pain. A more complete understanding of the motor and sensory remodeling of the peripheral and central nervous system that occurs postamputation may help advance clinical interventions to improve the quality of life for individuals with acquired limb loss. The purpose of this article is to first provide background clinical context on individuals with acquired limb loss and then to provide a comprehensive review of the known motor and sensory neural adaptations from both animal models and human clinical trials. Finally, the article bridges the gap between basic science researchers and clinicians that treat individuals with limb loss by explaining how current clinical treatments may restore function and modulate phantom limb pain using the underlying neural adaptations described above. This review should encourage the further development of novel treatments with known neurological targets to improve the recovery of individuals postamputation.Significance Statement In the United States, 1.6 million people live with limb loss; this number is expected to more than double by 2050. Improved surgical procedures enhance recovery, and new prosthetics and neural interfaces can replace missing limbs with those that communicate bidirectionally with the brain. These advances have been fairly successful, but still most patients experience persistent problems like phantom limb pain, and others discontinue prostheses instead of learning to use them daily. These problematic patient outcomes may be due in part to the lack of consensus among basic and clinical researchers regarding the plasticity mechanisms that occur in the brain after amputation injuries. Here we review results from clinical and animal model studies to bridge this clinical-basic science gap.

Quantitation of F-actin in cytoskeletal reorganization: Context, methodology and implications.

The actin cytoskeleton is involved in a large number of cellular signaling events in addition to providing structural integrity to the cell. Actin polymerization is a key event during cellular signaling. Although the role of actin cytoskeleton in cellular processes such as trafficking and motility has been extensively studied, the reorganization of the actin cytoskeleton upon signaling has been rarely explored due to lack of suitable assays. Keeping in mind this lacuna, we developed a confocal microscopy based approach that relies on high magnification imaging of cellular F-actin, followed by image reconstruction using commercially available software. In this review, we discuss the context and relevance of actin quantitation, followed by a detailed hands-on approach of the methodology involved with specific points on troubleshooting and useful precautions. In the latter part of the review, we elucidate the method by discussing applications of actin quantitation from our work in several important problems in contemporary membrane biology ranging from pathogen entry into host cells, to GPCR signaling and membrane-cytoskeleton interaction. We envision that future discovery of cell-permeable novel fluorescent probes, in combination with genetically encoded actin-binding reporters, would allow real-time visualization of actin cytoskeleton dynamics to gain deeper insights into active cellular processes in health and disease.

The evolutionary novelty of insect defensins: from bacterial killing to toxin neutralization.

Insect host defense comprises two complementary dimensions, microbial killing-mediated resistance and microbial toxin neutralization-mediated resilience, both jointly providing protection against pathogen infections. Insect defensins are a class of effectors of innate immunity primarily responsible for resistance to Gram-positive bacteria. Here, we report a newly originated gene from an ancestral defensin via genetic deletion following gene duplication in Drosophila virilis, which confers an enhanced resilience to Gram-positive bacterial infection. This gene encodes an 18-mer arginine-rich peptide (termed DvirARP) with differences from its parent gene in its pattern of expression, structure and function. DvirARP specifically expresses in D. virilis female adults with a constitutive manner. It adopts a novel fold with a 310 helix and a two CXC motif-containing loop stabilized by two disulfide bridges. DvirARP exhibits no activity on the majority of microorganisms tested and only a weak activity against two Gram-positive bacteria. DvirARP knockout flies are viable and have no obvious defect in reproductivity but they are more susceptible to the DvirARP-resistant Staphylococcus aureus infection than the wild type files, which can be attributable to its ability in neutralization of the S. aureus secreted toxins. Phylogenetic distribution analysis reveals that DvirARP is restrictedly present in the Drosophila subgenus, but independent deletion variations also occur in defensins from the Sophophora subgenus, in support of the evolvability of this class of immune effectors. Our work illustrates for the first time how a duplicate resistance-mediated gene evolves an ability to increase the resilience of a subset of Drosophila species against bacterial infection.

Language and developmental plasticity after perinatal stroke.

The mature human brain is lateralized for language, with the left hemisphere (LH) primarily responsible for sentence processing and the right hemisphere (RH) primarily responsible for processing suprasegmental aspects of language such as vocal emotion. However, it has long been hypothesized that in early life there is plasticity for language, allowing young children to acquire language in other cortical regions when LH areas are damaged. If true, what are the constraints on functional reorganization? Which areas of the brain can acquire language, and what happens to the functions these regions ordinarily perform? We address these questions by examining long-term outcomes in adolescents and young adults who, as infants, had a perinatal arterial ischemic stroke to the LH areas ordinarily subserving sentence processing. We compared them with their healthy age-matched siblings. All participants were tested on a battery of behavioral and functional imaging tasks. While stroke participants were impaired in some nonlinguistic cognitive abilities, their processing of sentences and of vocal emotion was normal and equal to that of their healthy siblings. In almost all, these abilities have both developed in the healthy RH. Our results provide insights into the remarkable ability of the young brain to reorganize language. Reorganization is highly constrained, with sentence processing almost always in the RH frontotemporal regions homotopic to their location in the healthy brain. This activation is somewhat segregated from RH emotion processing, suggesting that the two functions perform best when each has its own neural territory.

Atomic Reconstruction of Au Thin Films through Interfacial Strains.

Interfaces play a critical thermodynamic role in the existence of multilayer systems. Due to their utility in bridging energetic and compositional differences between distinct species, the formation of interfaces inherently creates internal strain in the bulk due to the reorganization needed to accommodate such a change. We report the effect of scaling interfacial stress by deposition of different adlayers on a host thin metal film. Intrinsic property differences between host and deposited metal atoms result in varying degree of composition and energy gradient within the interface. Interfacial stress can increase defects in the host leading to (i) energy dissipation and reorganization to minimize surface energy, and (ii) increased material strength. We infer that dissipation of interfacial stress induces defect migration, hence bulk and surface atomic reconstruction as captured by the surface roughness and grain size reduction coupled with a concomitant increase in material strength.

Motor mapping of the hand muscles using peripheral innervation-based navigated transcranial magnetic stimulation to identify functional reorganization of primary motor regions in malignant tumors.

Tumor-related motor reorganization remains unclear. Navigated transcranial magnetic stimulation (nTMS) can investigate plasticity non-invasively. nTMS-induced motor-evoked potentials (MEPs) of different muscles are commonly used to measure the center of gravity (CoG), the location with the highest density of corticospinal neurons in the precentral gyrus. We hypothesized that a peripheral innervation-based MEP analysis could outline the tumor-induced motor reorganization with a higher clinical and oncological relevance. Then, 21 patients harboring tumors inside the left corticospinal tract (CST) or precentral gyrus were enrolled in group one (G1), and 24 patients with tumors outside the left CST or precentral gyrus were enrolled in Group 2 (G2). Median- and ulnar-nerve-based MEP analysis combined with diffusion tensor imaging fiber tracking was used to explore motor function distribution. There was no significant difference in CoGs or size of motor regions and underlying tracts between G1 and G2. However, G1 involved a sparser distribution of motor regions and more motor-positive sites in the supramarginal gyrus-tumors inside motor areas induced motor reorganization. We propose an "anchor-and-ship theory" hypothesis for this process of motor reorganization: motor CoGs are stably located in the cortical projection area of the CST, like a seated anchor, as the core area for motor output. Primary motor regions can relocate to nearby gyri via synaptic plasticity and association fibers, like a ship moving around its anchor. This principle can anticipate functional reorganization and be used as a neuro-oncological tool for local therapy, such as radiotherapy or surgery.

Modeling the neurocognitive dynamics of language across the lifespan.

Healthy aging is associated with a heterogeneous decline across cognitive functions, typically observed between language comprehension and language production (LP). Examining resting-state fMRI and neuropsychological data from 628 healthy adults (age 18-88) from the CamCAN cohort, we performed state-of-the-art graph theoretical analysis to uncover the neural mechanisms underlying this variability. At the cognitive level, our findings suggest that LP is not an isolated function but is modulated throughout the lifespan by the extent of inter-cognitive synergy between semantic and domain-general processes. At the cerebral level, we show that default mode network (DMN) suppression coupled with fronto-parietal network (FPN) integration is the way for the brain to compensate for the effects of dedifferentiation at a minimal cost, efficiently mitigating the age-related decline in LP. Relatedly, reduced DMN suppression in midlife could compromise the ability to manage the cost of FPN integration. This may prompt older adults to adopt a more cost-efficient compensatory strategy that maintains global homeostasis at the expense of LP performances. Taken together, we propose that midlife represents a critical neurocognitive juncture that signifies the onset of LP decline, as older adults gradually lose control over semantic representations. We summarize our findings in a novel synergistic, economical, nonlinear, emergent, cognitive aging model, integrating connectomic and cognitive dimensions within a complex system perspective.

Amygdala structural and functional reorganization as an indicator of affective dysfunction in patients with tinnitus.

The aim of this study was to systematically investigate structural and functional alterations in amygdala subregions using multimodal magnetic resonance imaging (MRI) in patients with tinnitus with or without affective dysfunction. Sixty patients with persistent tinnitus and 40 healthy controls (HCs) were recruited. Based on a questionnaire assessment, 26 and 34 patients were categorized into the tinnitus patients with affective dysfunction (TPAD) and tinnitus patients without affective dysfunction (TPWAD) groups, respectively. MRI-based measurements of gray matter volume, fractional anisotropy (FA), fractional amplitude of low-frequency fluctuations (fALFF), regional homogeneity (ReHo), degree centrality (DC), and functional connectivity (FC) were conducted within 14 amygdala subregions for intergroup comparisons. Associations between the MRI properties and clinical characteristics were estimated via partial correlation analyses. Compared with that of the HCs, the TPAD and TPWAD groups exhibited significant structural and functional changes, including white matter integrity (WMI), fALFF, ReHo, DC, and FC alterations, with more pronounced WMI changes in the TPAD group, predominantly within the left auxiliary basal or basomedial nucleus (AB/BM), right central nucleus, right lateral nuclei (dorsal portion), and left lateral nuclei (ventral portion containing basolateral portions). Moreover, the TPAD group exhibited decreased FC between the left AB/BM and left middle occipital gyrus and right superior frontal gyrus (SFG), left basal nucleus and right SFG, and right lateral nuclei (intermediate portion) and right SFG. In combination, these amygdalar alterations exhibited a sensitivity of 65.4% and specificity of 96.9% in predicting affective dysfunction in patients with tinnitus. Although similar structural and functional amygdala remodeling were observed in the TPAD and TPWAD groups, the changes were more pronounced in the TPAD group. These changes mainly involved alterations in functionality and white matter microstructure in various amygdala subregions; in combination, these changes could serve as an imaging-based predictor of emotional disorders in patients with tinnitus.

More widespread functionality of posterior language area in patients with brain tumors.

Damage to the posterior language area (PLA), or Wernicke's area causes cortical reorganization in the corresponding regions of the contralateral hemisphere. However, the details of reorganization within the ipsilateral hemisphere are not fully understood. In this context, direct electrical stimulation during awake surgery can provide valuable opportunities to investigate neuromodulation of the human brain in vivo, which is difficult through the non-invasive approaches. Thus, in this study, we aimed to investigate the characteristics of the cortical reorganization of the PLA within the ipsilateral hemisphere. Sixty-two patients with left hemispheric gliomas were divided into groups depending on whether the lesion extended to the PLA. All patients underwent direct cortical stimulation with a picture-naming task. We further performed functional connectivity analyses using resting-state functional magnetic resonance imaging (MRI) in a subset of patients and calculated betweenness centrality, an index of the network importance of brain areas. During direct cortical stimulation, the regions showing positive (impaired) responses in the non-PLA group were localized mainly in the posterior superior temporal gyrus (pSTG), whereas those in the PLA group were widely distributed from the pSTG to the posterior supramarginal gyrus (pSMG). Notably, the percentage of positive responses in the pSMG was significantly higher in the PLA group (47%) than in the non-PLA group (8%). In network analyses of functional connectivity, the pSMG was identified as a hub region with high betweenness centrality in both the groups. These findings suggest that the language area can spread beyond the PLA to the pSMG, a hub region, in patients with lesion progression to the pSTG. The change in the pattern of the language area may be a compensatory mechanism to maintain efficient brain networks.

When colors mislead: Genomics and bioacoustics prompt re-classification of Asian flycatcher radiation (Aves: Niltavinae).

Traditional classification of many animals, including birds, has been highly dependent on external morphological characters like plumage coloration. However, both bioacoustics and genetic or genomic data have revolutionized our understanding of the relationships of certain lineages and led to sweeping taxonomic re-organizations. In this study, we present a case of erroneous delimitation of genus boundaries in the species-rich flycatcher subfamily Niltavinae. Genera within this subfamily have historically been delineated based on blue versus brown male body plumage until recent studies based on a few mitochondrial and nuclear loci unearthed several cases of generic misclassification. Here we use extensive bioacoustic data from 43 species and genomic data from 28 species for a fundamental reclassification of species in the Niltavinae. Our study reveals that song is an important trait to classify these birds even at the genus level, whereas plumage traits exhibit ample convergence and have led to numerous historic misattributions. Our taxonomic re-organization leads to new biogeographic limits of major genera, such that the genus Cyornis now only extends as far east as the islands of Sulawesi, Sula, and Banggai, whereas Eumyias is redefined to extend far beyond Wallace's Line to the islands of Seram and Timor. Our conclusions advise against an over-reliance on morphological traits and underscore the importance of integrative datasets.

Beyond resilience: Responses to changing climate and disturbance regimes in temperate forest landscapes across the Northern Hemisphere.

Climate change has profound impacts on forest ecosystem dynamics and could lead to the emergence of novel ecosystems via changes in species composition, forest structure, and potentially a complete loss of tree cover. Disturbances fundamentally shape those dynamics: the prevailing disturbance regime of a region determines the inherent variability of a system, and its climate-mediated change could accelerate forest transformation. We used the individual-based forest landscape and disturbance model iLand to investigate the resilience of three protected temperate forest landscapes on three continents-selected to represent a gradient from low to high disturbance activity-to changing climate and disturbance regimes. In scenarios of sustained strong global warming, natural disturbances increased across all landscapes regardless of projected changes in precipitation (up to a sevenfold increase in disturbance rate over the 180-year simulation period). Forests in landscapes with historically high disturbance activity had a higher chance of remaining resilient in the future, retaining their structure and composition within the range of variability inherent to the system. However, the risk of regime shift and forest loss was also highest in these systems, suggesting forests may be vulnerable to abrupt change beyond a threshold of increasing disturbance activity. Resilience generally decreased with increasing severity of climate change. Novelty in tree species composition was more common than novelty in forest structure, especially under dry climate scenarios. Forests close to the upper tree line experienced high novelty in structure across all three study systems. Our results highlight common patterns and processes of forest change, while also underlining the diverse and context-specific responses of temperate forest landscapes to climate change. Understanding past and future disturbance regimes can anticipate the magnitude and direction of forest change. Yet, even across a broad gradient of disturbance activity, we conclude that climate change mitigation is the most effective means of maintaining forest resilience.

Direct Comparative Studies Revealing the Contribution of TADF Activity of Organic Emitters Towards Efficient Electrochemiluminescence.

Electrochemiluminescence (ECL) featuring thermally activated delayed fluorescence (TADF) properties has attracted considerable interest, showcasing their potential for 100 % exciton harvesting, which marks a significant advancement in the realm of organic ECL. However, the challenge of elucidating the precise contribution of TADF to the enhanced ECL efficiency arises due to the lack of comparative studies of organic compounds with or without efficient TADF properties. In this study, we present four carbazole-benzonitrile molecules possessing similar chemical structures and comparable exchange energy (ΔEST). Despite their comparable properties, these compounds exhibited varying TADF efficiencies, warranting a closer examination of their underlying structural and electronic characteristics governing the optical properties. Consequently, intense ECL emission was only observed from 4CzBN with a remarkable TADF efficiency, underscoring the substantial difference in the ECL signal among molecules with comparable ΔEST and similar spectral properties but varying TADF activity.

Facilitating Electron Transfer by Resizing Cyclocarbon Acceptor from C18 to C16.

Recent advances in synthetic methods, combined with tip-induced on-surface chemistry, have enabled the formation of numerous cyclocarbon molecules. Here, we investigate computationally the experimentally studied C16 and C18 molecules as well as their van der Waals (vdW) complexes with several typical donor and acceptor molecules. Our results demonstrate a remarkable electron-withdrawing ability of cyclocarbon molecules. The vdW complexes of C16 and C18 exhibit a thermodynamically favorable photoinduced electron transfer (ET) from the donor partner to the cyclocarbons that occurs on a picosecond time scale. The lower reorganization energy of C16 compared to C18 leads to a significant acceleration of the ET reactions.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.