bluuebunny/crossref_metadata_2025 huggingface dataset:
MRI Guided Fresh Tissue Procurement in Radical Prostatectomy Specimens: An Evolutionary Paradigm
Authors: Freidlin Raiza Z , Garmendia Marcial A , Krynitsky Jonathan , Bastian Cody , Schultz Connor , Custer Julian , Young Sarah , Mehralivand Sherif , Merino Maria J , Valera Vladimir A , Pinto Peter , Turkbey Baris , Peter Choyke L , Pohida Thomas J
Published: May 2022

Purpose

The aim of this work is to assess the feasibility of novel targeted methods for fresh tissue procurement that facilitate accurate identification of lesion location and tissue sample extraction in an excised prostate gland, while minimizing the tissue deformation or damage.

Methods and Results

Proposed fresh tissue procurement methods are based on utilizing a custom 3D printed patient-specific prostate mold. In the semi-freehand method, the access cut is guided by a set of markers on the bottom of the mold that define the boundary of a lesion in two orthogonal directions. The procurement site is identified by palpation. In the guided cut with-locator card method the procurement sites are identified by a cutout in the locator cards inserted into the access cuts. The two approaches were utilized to procure >50 samples. In the biopsy needle method guiding channels were designed into the prostate mold to procure fresh tissue. This method was used 4 times. Finally, a mixed-reality biopsy needle method was developed for phantom testing.

Conclusions

Preliminary results of fresh tissue procurement utilizing novel methods showed improve precision of obtaining cancerous fresh tissue for molecular and genomic cancer research compared to current methods which rely on surgical estimation of lesion location.
Deciphering the Impact of Genetic Variation on Human Polyadenylation
Authors: Linder Johannes , Kundaje Anshul , Seelig Georg
Published: May 2022

Genetic variants that disrupt polyadenylation can cause or contribute to genetic disorders. Yet, due to the complex cis-regulation of polyadenylation, variant interpretation remains challenging. Here, we introduce a residual neural network model,APARENT2, that can infer 3’-cleavage and polyadenylation from DNA sequence more accurately than any previous model. This model generalizes to the case of alternative polyadenylation (APA) for a variable number of polyadenylation signals. We demonstrate APARENT2’s performance on several variant datasets, including functional reporter data and human 3’ aQTLs from GTEx. We apply neural network interpretation methods to gain insights into disrupted or protective higher-order features of polyadenylation. We fine-tune APARENT2 on human tissue-resolved transcriptomic data to elucidate tissue-specific variant effects. Finally, we perform in-silico saturation mutagenesis of all human polyadenylation signals and compare the predicted effects of >44 million variants against gnomAD. While loss-of-function variants were generally selected against, we also find specific clinical conditions linked to gain-of-function mutations. For example, using APARENT2’s predictions we detect an association between gain-of-function mutations in the 3’-end and Autism Spectrum Disorder.
Decoding The Nuclear Genome of The Human Pathogen Babesia duncani Shed Light on its Virulence, Drug Susceptibility and Evolution among Apicomplexa
Authors: Lonardi Stefano , Singh Pallavi , Liang Qihua , Vydyam Pratap , Khabirova Eleonora , Fang Tiffany , Gihaz Shalev , Thekkiniath Jose , Munshi Muhammad , Abel Steven , Batugedara Gayani , Gupta Mohit , Lu Xueqing Maggie , Lenz Todd , Chakravarty Sakshar , Cornillot Emmanuel , Hu Yangyang , Ma Wenxiu , Gonzalez Luis Miguel , Sánchez Sergio , Montero Estrella , Estrada Karel , Sánchez-Flores Alejandro , Harb Omar S. , Le Roch Karine G. , Ben Mamoun Choukri
Published: May 2022

Babesia species are tick-transmitted apicomplexan pathogens and the causative agents of babesiosis, a malaria-like disease of major medical and veterinary importance. Of the different species of Babesia reported so far, Babesia duncani causes severe to lethal infection in patients. Despite the highly virulent nature of this parasite and the risk it may pose as an emerging pathogen, little is known about its biology, metabolic requirements, and pathogenesis. B. duncani is unique among apicomplexan parasites that infect red blood cells in that it can be continuously cultured in vitro in human erythrocytes but can also infect mice leading to fulminant babesiosis infection and death. Here we have taken advantage of the recent advances in the propagation of this parasite in vitro and in vivo to conduct detailed molecular, genomic and transcriptomic analyses and to gain insights into its biology. We report the assembly, 3D structure, and annotation of the nuclear genome of this parasite as well as its transcriptomic profile and an atlas of its metabolism during its intraerythrocytic life cycle. Detailed examination of the B. duncani genome and comparative genomic analyses identified new classes of candidate virulence factors, suitable antigens for diagnosis of active infection, and several attractive drug targets. Translational analyses and efficacy studies identified highly potent inhibitors of B. duncani thus enriching the pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.
Evolution of thermal performance curves: a meta-analysis of selection experiments
Authors: Malusare Sarthak P. , Zilio Giacomo , Fronhofer Emanuel A.
Published: May 2022

Temperatures are increasing due to global changes, putting biodiversity at risk. Organisms are faced with a limited set of options to cope with this situation: adapt, disperse or die. We here focus on the first possibility, more specifically, on evolutionary adaptations to temperature. Ectotherms are usually characterized by a hump-shaped relationship between fitness and temperature, a non-linear reaction norm that is referred to as thermal performance curve (TPC). To understand and predict impacts of global change, we need to know whether and how such TPCs evolve.

Therefore, we performed a systematic literature search and a statistical meta-analysis focusing on experimental evolution and artificial selection studies. This focus allows us to directly quantify relative fitness responses to temperature selection by calculating fitness differences between TPCs from ancestral and derived populations after thermal selection.

Out of 7561 publications screened, we found 47 studies corresponding to our search criteria representing taxa across the tree of life, from bacteria, to plants and vertebrates. We show that, independently of species identity, the studies we found report a positive response to temperature selection. Considering entire TPC shapes, adaptation to higher temperatures traded off with fitness at lower temperatures, leading to niche shifts. Effects were generally stronger in unicellular organisms. By contrast, we do not find statistical support for the often discussed “Hotter is better” hypothesis.

While our meta-analysis provides evidence for adaptive potential of TPCs across organisms, it also highlights that more experimental work is needed, especially for under-represented taxa, such as plants and non-model systems.
Corynebacterium matruchotii fitness enhancement of adjacent streptococci by multiple mechanisms
Authors: Almeida Eric , Puri Surendra , Elangovan Subhashini , Kim Jiyeon , Ramsey Matthew
Published: May 2022

Polymicrobial biofilms are present in many environments particularly in the human oral cavity where they can prevent or facilitate the onset of disease. While recent advances have provided a clear picture of both the constituents and their biogeographical arrangement, it is still unclear what mechanisms of interaction occur between individual species in close proximity within these communities. In this study we investigated two mechanisms of interaction between the highly abundant supragingival plaque (SUPP) commensal Corynebacterium matruchotii and Streptococcus mitis which are directly adjacent in vivo. We discovered that C. matruchotii enhanced the fitness of streptococci dependent on its ability to detoxify streptococcal-produced hydrogen peroxide and its ability to oxidize lactate also produced by streptococci. We demonstrate that the fitness of adjacent streptococci was linked to that of C. matruchotii and that these mechanisms support the previously described “corncob” arrangement between these species but that this is favorable only in aerobic conditions. Further we utilized scanning electrochemical microscopy (SECM) to quantify lactate production and consumption between individual bacterial cells for the 1st time, revealing that lactate oxidation provides a fitness benefit to S. mitis and not pH mitigation. This study describes mechanistic interactions between two highly abundant human commensals that can explain their observed in vivo spatial arrangements and suggest a way by which they may help preserve a healthy oral bacterial community.
Opportunistic binding of EcR to open chromatin drives tissue-specific developmental responses
Authors: Uyehara Christopher M. , Leatham-Jensen Mary , McKay Daniel J.
Published: May 2022

Steroid hormones perform diverse biological functions in developing and adult animals. However, the mechanistic basis for their tissue specificity remains unclear. InDrosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its nuclear receptor, a heterodimer of the EcR and Usp proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. Here, we investigate the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates metamorphosis. We find that EcR functions bimodally, with both gene repressive and activating functions, even at the same developmental stage. EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Finally, despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, we find that EcR does not control chromatin accessibility at genomic targets. We conclude that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors.

Significance

Hormones affect an incredible array of biological processes in both normal development and in disease. In insects, the steroid hormone ecdysone controls processes ranging from neuronal diversification to morphogenesis. Despite its importance, the mechanisms through which ecdysone generates wide-ranging yet tissue-specific responses remain incompletely understood. Like many hormones, ecdysone triggers a cascade of gene expression. At the top of this hierarchy is a nuclear receptor, EcR, which functions both as a hormone receptor and as a transcription factor. However, EcR is not the only transcription factor that functions in the ecdysone cascade; multiple other transcription factors are induced by ecdysone. As a result, the extent to which EcR plays a direct role in regulating tissue-specific responses to ecdysone remains unclear.
Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation
Authors: Knox Ashley N. , Medina Eva M. , Oldenburg Darby G. , Clambey Eric T. , van Dyk Linda F.
Published: May 2022

Gammaherpesvirus (γHV) non-coding RNAs (ncRNAs) are integral modulators of viral infection. The γHVs engage RNA polymerase III (pol III)-dependent transcription of both host and viral ncRNAs, which contribute to viral establishment, gene expression, and pathogenesis. Viral ncRNAs, such as the EBV-encoded RNAs (EBERs), reportedly interact with multiple host RNA-binding proteins (RBPs) and contribute to inflammatory responses implicated in the development of malignancies. Here, we examined RBP interactions of the pol III-transcribed tRNA-miRNA encoded non-coding RNAs (TMERs) of murine γHV68, and the potential contributions of these and the related EBERs to in vivo pathogenesis. Using sequential enzymatic treatments, we found that several TMER1 forms retain a 5’-triphosphate, lending the possibility of recognition by the innate immune sensor RIG-I. We further examined the interactions of TMERs and EBERs with host RBPs, and found that multiple TMERs and EBERs interact with the La protein, though minimal interaction was detected with RIG-I during primary virus infection. Finally, we investigated the contributions of the TMERs and EBERs to disease in an immune-compromised mouse model with a series of viral recombinants. We found that expression of multiple single TMERs, or the EBERs expressed in place of the TMERs, was capable of restoring virulence to a viral recombinant lacking expression of all TMERs. Ultimately, these studies demonstrate that divergent pol III-transcribed γHV ncRNAs share interaction characteristics with two host RBPs and conserved contributions to disease, despite little to no significant sequence conservation. These findings support a model of convergent functions of the sequence-variable pol III-transcribed γHV ncRNAs.

IMPORTANCE

Viruses manipulate the infected cell and host inflammatory responses through expression of coding and non-coding RNAs. The gammaherpesviruses are a subfamily of herpesviruses associated with chronic inflammatory diseases and malignancies, especially in immune-compromised individuals. Among these, the human Epstein-Barr virus and murine gammaherpesvirus 68 (γHV68) express highly abundant, RNA polymerase III-dependent, short non-coding RNAs. Whether these sequence-divergent ncRNAs have conserved functional properties is unknown. By using viral recombinants to allow direct comparison of these ncRNAs during primary infection, we find that the sequence-divergent ncRNAs of γHV68 and EBV share a conserved property to bind to the host RNA binding protein, La, and function interchangeably to facilitate in vivo pathogenesis. These studies demonstrate that abundant, RNA polymerase III-dependent viral ncRNAs can potently function to alter the host cell landscape and promote disease in a sequence-independent manner.
Piezo1 activates non-canonical EGFR endocytosis and signaling
Authors: Pardo-Pastor Carlos , Rosenblatt Jody
Published: May 2022

EGFR-ERK signaling controls cell cycle progression during development, homeostasis, and disease. Both the soluble extracellular ligand, EGF, and mechanical inputs like matrix stiffness, cell adhesion, or stretch activate EGFR-ERK signaling. However, the molecules transducing mechanical forces into EGFR signaling remain unidentified. We previously found that stretch promotes mitosis by mechanically activating the ion channel Piezo1 to trigger ERK signaling. Here, we show that Piezo1 provides the missing link to mechanical EGFR-ERK activation. Both EGF ligand and mechanical or agonist activation of Piezo1 trigger clathrin-mediated endocytosis of EGFR and ERK activation, established markers of EGFR signaling. However, while EGF stimulation requires canonical EGFR tyrosine autophosphorylation, EGFR activation by Piezo1 instead requires Src-p38 kinase-dependent serine EGFR phosphorylation. Additionally, in contrast to the homeostatic signaling downstream EGF activation, direct agonist stimulation of Piezo1 promoted cell cycle re-entry and proliferation in mouse airway epitheliaex vivovia long-term nuclear accumulation of ERK, AP-1 (FOS and JUN), and YAP, typical of regenerative and malignant signaling. Our results suggest two modes of EGFR signaling: basal EGF-dependent signaling via tyrosine autophosphorylation and mechanically activated Piezo1-dependent signaling via serine phosphorylation, resulting in sustained proliferation, critical to repair, regeneration, and cancer growth. Given the limited success of tyrosine kinase inhibitors in cancer, this new axis may provide a more relevant target for cancer treatment.
Experience alters hippocampal and cortical network communication via a KIBRA-dependent mechanism
Authors: Quigley Lilyana D. , Pendry Robert , Mendoza Matthew L. , Pfeiffer Brad. E. , Volk Lenora J.
Published: May 2022

Synaptic plasticity is hypothesized to underlie “replay” of salient experience during hippocampal sharp-wave/ripple (SWR)-based ensemble activity and to facilitate systems-level memory consolidation coordinated by SWRs and cortical sleep spindles. However, it remains unclear how molecular changes at synapses contribute to experience-induced modification of network function. The synaptic protein KIBRA regulates plasticity and memory, although its impact on circuit dynamics remains unknown. Here, we recorded in vivo neural activity from WT mice and littermates lacking KIBRA to examine circuit function before, during, and after novel experience. In WT mice, experience altered network dynamics in a manner consistent with incorporation of new information content in replay and enhanced hippocampal-cortical communication. However, while baseline SWR features were normal in KIBRA cKO mice, experience-dependent alterations in SWRs were absent. Furthermore, intra-hippocampal and hippocampal-cortical communication during SWRs was disrupted following KIBRA deletion. These results reveal molecular mechanisms that underlie network-level memory formation and consolidation.
Non-Invasive monitoring of normal tissue radiation damage using quantitative ultrasound spectroscopy
Authors: Rafat Marjan , Kaffas Ahmed El , Swarnakar Ankush , Shostak Anastasia , Graves Edward E.
Published: May 2022

Background

While radiation therapy (RT) is an critical component of breast cancer therapy and is known to decrease overall local recurrence rates, recent studies have shown that normal tissue radiation damage may increase recurrence risk. Fibrosis is a well-known consequence of RT, but the specific sequence of molecular and mechanical changes induced by RT remains poorly understood.

Purpose

There is currently a need to understand the contribution of the irradiated tissue microenvironment to recurrence to improve cancer therapy outcomes. This study seeks to evaluate the use of quantitative ultrasound spectroscopy (QUS) for real time determination of the normal tissue characteristic radiation response and to correlate these results to molecular features in irradiated tissues.

Methods

Murine mammary fat pads (MFPs) were irradiated to 20 Gy, and QUS was used to analyze tissue physical properties pre-irradiation as well as at 1, 5, and 10 days post-irradiation. Tissues were processed for scanning electron microscopy imaging as well as histological and immunohistochemical staining to evaluate morphology and structure.

Results

Tissue morphological and structural changes were observed non-invasively following radiation using mid-band fit (MBF), spectral slope (SS), and spectral intercept (SI) measurements obtained from QUS. Statistically significant shifts in MBF and SI indicate structural tissue changes in real time, which matched histological observations. Radiation damage was indicated by increased adipose tissue density and extracellular matrix (ECM) deposition.

Conclusions

Our findings demonstrate the potential of using QUS to non-invasively evaluate normal tissue changes resulting from radiation damage. This supports further pre-clinical studies to determine how the tissue microenvironment and physical properties change in response to therapy, which may be important for improving treatment strategies.
Functional brain-wide network mapping during acute stress exposure in rats: Interaction between the lateral habenula and cortical, amygdalar, hypothalamic and monoaminergic regions
Authors: Durieux Laura , Herbeaux Karine , Borcuk Christopher , Hildenbrand Cécile , Andry Virginie , Goumon Yannick , Barbelivien Alexandra , Mathis Chantal , Bataglia Demian , Majchrzak Monique , Lecourtier Lucas
Published: May 2022

Upon stress exposure a broad network of structures comes into play in order to provide adequate responses and restore homeostasis. It has been known for decades that the main structures engaged during the stress response are the medial prefrontal cortex, the amygdala, the hippocampus, the hypothalamus, the monoaminergic systems (noradrenaline, dopamine, serotonin), and the periaqueductal gray. The lateral habenula (LHb) is an epithalamic structure directly connected to prefrontal cortical areas and to the amygdala, whereas it functionally interacts with the hippocampus. Also, it is a main modulator of monoaminergic systems. The LHb is activated upon exposure to basically all types of stressors, suggesting it is also involved in the stress response. However, it remains unknown if and how the LHb functionally interacts with the broad stress response network. In the current study we performed in rats a restraint stress procedure followed by immunohistochemical staining of the c-Fos protein throughout the brain. Using Graph Theory-based functional connectivity analyses, we confirm the principal hubs of the stress network (e.g. prefrontal cortex, amygdala, periventricular hypothalamus), and show that the LHb is engaged during stress exposure in close interaction with the medial prefrontal cortex, the lateral septum, and the medial habenula. In addition, we performed DREADD-induced LHb inactivation during the same restraint paradigm in order to explore its consequences on the stress response network. This last experiment gave contrasting results as the DREADD ligand alone, clozapine-N-oxide, was able to modify the network.

GRAPHICAL ABSTRACT

GRAPHICAL ABSTRACT TEXT

In this study, using immunohistochemical staining of the immediate early gene c-fos and graph theory-based functional correlational analyses, we aimed at unravelling the possible engagement of the lateral habenula (LHb) within the stress response network during acute stress exposure (10-min restraint) in rats. We found that the medial part of the LHb (LHbM) was preferentially engaged, and that this engagement was concomitant to this of structures such as the medial prefrontal cortex (mPFC), the insular cortex (Ins), hypothalamic (PVH) and thalamic (PVT) paraventricular nuclei, the extended amygdala, comprising the Bed nucleus of the stria terminalis (BNST) and the entire amygdala (AMG), as well as the dopaminergic ventral tegmental area (VTA) and the serotonergic dorsal raphe nucleus (RD). This suggests upon stressful situations the LHbM serves as a relay of cortical, thalamic, hypothalamic and temporal information, further transmitted to midbrain monoaminergic systems to probably initiate coping strategies.
Topology of gene regulatory compartments relative to the nuclear matrix
Authors: Park Gunhee , Ryu Kwangmin , Lee Junho , Kim Geunhee , Park Tae Lim , Shim Hwanyong , Lee YigJi , Kim Changyeop , Cho Won-Ki
Published: May 2022

Summary Paragraph

Inside the nucleus, there are special compartments with characterized functions, some of which are involved in gene expression1. These compartments include transcriptional condensates and nuclear speckles, which contain factors required for transcription and splicing, respectively. While the characteristics of these intranuclear compartments were extensively investigated, spatial relationship between them is yet unclear. Meanwhile, RNA-protein structural network named the nuclear matrix has been observed inside the nucleus and suggested as a framework for the spatial segregation of the intranuclear space2–6. However, concept of the nuclear matrix was not widely accepted due to its lack of in vivo evidence7. Here, we report visualization of the nuclear matrix and intranuclear compartments using super-resolution fluorescence microscopy. We found the nuclear matrix is dynamic in live cells and easily disrupted upon transcription inhibition. Remarkably, we observed an orderly layered distribution of transcriptional condensates and nuclear speckles relative to the nuclear matrix. We also observed a separation of chromosome territories from transcriptional condensates and nuclear speckles by the nuclear matrix. Based on our findings, we propose a topological model for the regulation of transcription across the nuclear matrix.
Neural signals encoded by specific frequencies of closed-loop electrical stimulation structurally and functionally reconstruct spinal sensorimotor circuits after spinal cord injury
Authors: Zhou Kai , Yang Dan , Wei Wei , Zhang Hui , Yang Wei , Zhang Yunpeng , Nie Yingnan , Hao Mingming , Zhang Ting , Wang Shouyan , Liu Yaobo
Published: May 2022

Epidural electrical stimulation restores locomotion in animals and humans with spinal cord injury (SCI). However, the coding rules underlying electrical stimulation remain poorly understood, which has greatly limited the application of such electrical neuromodulation techniques in SCI clinical treatment. To elucidate the coding rules of electrical stimulation on spinal sensorimotor circuit reconstruction after complete SCI, we initially developed a spinal−muscle closed-loop stimulation protocol to mimic feedforward and feedback electrical signals in spinal sensorimotor circuits. Afterwards, using methods of sensorimotor function evaluation, neural circuit tracing and neural signal recording, we discovered a unique stimulus frequency of 10−20 Hz under closed-loop conditions was required for structural and functional reconstruction of spinal sensorimotor circuits. The single-cell transcriptome analysis of activated motoneurons characterized molecular networks involved in spinal sensorimotor circuit reconstruction. This study provides insights into neural signal decoding during spinal sensorimotor circuit reconstruction, and indicates a technological approach for the clinical treatment of SCI.
Signaling protein abundance modulates the strength of the Spindle Assembly Checkpoint
Authors: Chen Chu , Humphrey Lauren , Jema Soubhagyalaxmi , Karmarkar Shriya , Ferrari Frank , Joglekar Ajit P.
Published: May 2022

During mitosis, unattached kinetochores in a dividing cell signal to the Spindle Assembly Checkpoint to delay anaphase onset and prevent chromosome missegregation1–4. The signaling activity of these kinetochores and the likelihood of chromosome missegregation both depend on the amount of SAC signaling proteins that each kinetochore recruits5–8. Therefore, factors that control SAC protein recruitment to signaling kinetochores must be thoroughly understood. Phosphoregulation of kinetochore and SAC signaling proteins emerging from the concerted action of many kinases and phosphatases is a major determinant of SAC protein recruitment to signaling kinetochores9. Whether the abundance of SAC proteins also influences their recruitment and signaling activity at human kinetochores has not been studied8, 10. Here, we reveal that the low cellular abundance of the SAC signaling protein Bub1 limits kinetochore recruitment of Bub1 and BubR1 and reduces the SAC signaling activity of the kinetochore. Conversely, Bub1 overexpression results in higher protein recruitment and SAC activity producing longer delays in anaphase onset. We also find that the number of SAC proteins recruited by a signaling kinetochore is inversely correlated with the total number of signaling kinetochores in the cell. This correlation likely arises from the competition among the signaling kinetochores to recruit from a limited pool of signaling proteins. The inverse correlation between the number of signaling kinetochores in the cell and the signaling activity of individual kinetochores may allow the dividing cell to prevent the large number of signaling kinetochores in prophase from generating an unnecessarily large signal, while enabling the last unaligned kinetochore to signal at the maximum possible strength.
A Cdh3-Lam332 signaling axis in a leader cell subpopulation controls protrusion dynamics and tumor organoid collective migration
Authors: Hwang Priscilla Y , Mathur Jairaj , Cao Yanyang , Almeida Jose , Cornish Daphne , Clarke Maria , Pathak Amit , Longmore Gregory D
Published: May 2022

Carcinoma dissemination can occur when heterogeneous tumor and tumor stromal cells clusters migrate together via collective migration. Cells at the front lead and direct collective migration, yet how these leader cells form and interact with the microenvironment to direct migration are not fully appreciated. From live videos of primary mouse and human breast tumor organoids in a 3D microfluidic system that mimics the native breast tumor microenvironment, we developed 3D computational models which hypothesize that leader cells generate high protrusive forces and overcome extracellular matrix (ECM) resistance. Using single cell sequencing, we reveal leader cells are heterogeneous, and identify and isolate a unique Cadherin-3 (Cdh3) positive leader cell subpopulation that is necessary and sufficient to lead migration. Cdh3 controls leader cell protrusion dynamics through the local production of Laminin-332 which is required for integrin/focal adhesion function. Our findings highlight how a subset of leader cells interact with the microenvironment to direct collective migration.

Teaser

Higher protrusions of Cdh3+ leader cells polarize tumor organoids that then invade collagen via Lam332 adhesion feedback.
Calcium-activated big conductance (BK) potassium channels traffic through nuclear envelopes into kinocilia in skate electrosensory cells
Authors: Chen Abby L. , Wu Ting-Hsuan , Shi Lingfang , Clusin William T. , Kao Peter N.
Published: May 2022

Electroreception through ampullae of Lorenzini in the little skate, Leucoraja erinacea, involves functional coupling between voltage-activated calcium channels (CaV1.3, cacna1d) and calcium-activated big conductance potassium (BK) channels (BK, kcnma1). Whole mount confocal microscopy was used to characterize expression of BK and CaV1.3 in intact ampullae. BK and CaV1.3 are present in electrosensory cell plasma membranes and colocalize with lamin B1 in nuclear envelopes and kinocilia. Predicted nuclear localization signals (NLS) were identified in BK and CaV1.3 by bioinformatic sequence analyses. The BK NLS is bipartite, occurs at an alternative splice site for the mammalian STREX exon and is modified by post-translational phosphorylation. Nuclear localization of skate BK channels was characterized in heterologously-transfected HEK293 cells. Double point mutations in the bipartite NLS (KR to AA, or SVLS to AVLA) independently attenuated BK channel nuclear localization, illustrating that BK trafficking into nuclei and electrosensory cell kinocilia is regulated through a newly identified bipartite NLS.
Expanding luciferase reporter systems for cell-free protein expression
Authors: Sato Wakana , Rasmussen Melanie , Deich Christopher , Engelhart Aaron E. , Adamala Katarzyna P.
Published: May 2022

Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.
Three ALS genes regulate expression of the MHC class II antigen presentation pathway
Authors: Chi Binkai , Öztürk Muhammet M. , Paraggio Christina L. , Leonard Claudia E. , Sanita Maria E. , Dastpak Mahtab , O’Connell Jeremy D. , Coady Jordan A. , Zhang Jiuchun , Gygi Steven P. , Lopez-Gonzalez Rodrigo , Reed Robin
Published: May 2022

Here we report that the major histocompatibility complex II (MHC II) antigen presentation pathway is regulated by the ALS-causative genes, FUS, TAF15, or MATR3. Of >6000 proteins detected by quantitative mass spectrometry, the subunits of the MHC II heterodimer, HLA-DR, were the top 2 downregulated proteins in HeLa knock outs (KO) of these ALS genes, but not the related gene, EWSR1. Moreover, CD74, which is the 3rd essential component of HLA-DR, was downregulated in the 3 KOs. We show that the downregulations are due to loss of CIITA, a transcription factor dedicated to expression of MHC II genes. Thus, our results reveal the 1st shared cellular pathway regulated by multiple ALS genes, and this pathway is ALS genes -> CIITA -> MHC II genes. We obtained the same results in HMC3 cells, a microglia cell line, showing that loss of the MHC II pathway extends to an ALS-relevant cell type in the central nervous system (CNS). Furthermore, the MHC II pathway is downregulated in hematopoietic progenitor cells (HPCs) bearing the ALS FUSR495X mutation. This observation may be highly significant to ALS pathogenesis as HPCs give rise to a multitude of CNS-specific and systemic immune cells, both of which have known or suspected roles in ALS. Together, our data raise the possibility that loss of the MHC II pathway in a large range of immune cells results in global failure of the immune system to protect motor neurons from damage that leads to the disease. Consequently, CIITA and the other genes in the MHC II pathway may be important new therapeutic targets for the disease.
Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform
Authors: Gaillochet Christophe , Pena Fernandez Alexandra , Goossens Vera , D’Halluin Katelijn , Drozdzecki Andrzej , Shafie Myriam , Van Duyse Julie , Van Isterdael Gert , Gonzalez Camila , Vermeersch Mattias , De Saeger Jonas , Develtere Ward , Audenaert Dominique , De Vleesschauwer David , Meulewaeter Frank , Jacobs Thomas B.
Published: May 2022

The ever-increasing number of CRISPR components creates a significant burden when developing new genome engineering tools. Plant biotechnology in particular has few high-throughput options to perform iterative design-build-test-learn cycles when creating new gene-editing reagents. We have established ITER (Iterative Testing of Editing Reagents) based on arrayed protoplast transfections and high-content imaging, allowing one optimization cycle – from design to results– within three weeks. We validated ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors. Given that previous LbCas12a-ABEs have low or no activity in plants, we used ITER to develop an optimized LbCas12a-ABE. We show that the sequential improvement of five components –NLS, crRNA, LbCas12a, adenine deaminase and linker– led to a remarkable increase in ABE activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirmed the activity of LbCas12a-ABE at endogenous targets and in stable wheat transformants and leveraged these improvements to develop a highly mutagenic LbCas12a nuclease and LbCas12a-CBE. Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants.
Extending of imaging volume in soft x-ray tomography
Authors: Ekman Axel , Chen Jian-Hua , Vanslembrouck Bieke , Larabell Carolyn A , Le Gros Mark A , Weinhardt Venera
Published: May 2022

Soft x-ray tomography offers rapid whole single cell imaging with a few tens of nanometers spatial resolution without fixation or labelling. At the moment, this technique is limited to 10 µm thick specimens, such that applications of soft x-ray tomography to large human cells or multicellular specimens are not possible. We have developed a theoretical and experimental framework for soft x-ray tomography to enable extension of imaging volume to 18 µm thick specimens. This approach, based on long depth of field and half-acquisition tomography, is easily applicable to existing full-rotation based microscopes. This opens applications for imaging of large human cells, which are often observed in cancer research and cell to cell interactions.
Mutual repression between JNK/AP-1 and JAK/STAT stratifies cell behaviors during tissue regeneration
Authors: Jaiswal Janhvi , Engesser Raphael , Peyroton Andrea Armengol , Weichselberger Vanessa , Crucianelli Carlo , Grass Isabelle , Timmer Jens , Classen Anne-Kathrin
Published: May 2022

Epithelial repair relies on the activation of stress signaling pathways to coordinate cellular repair behaviors. Their deregulation is implicated in chronic wound and cancer pathologies. Despite such translational importance, an understanding of how spatial patterns of signaling pathways and repair behaviors arise in damaged tissues remains elusive. Using TNF-α/Eiger-mediated inflammatory damage to Drosophila imaginal discs, we uncover that JNK/AP-1 signaling cells act as paracrine organizers and initiate a mutual repression network that spatially segregates JNK/AP-1 and JAK/STAT signaling cells into distinct populations. While JNK/AP-1 signaling cells produce JAK/STAT-activating Upd ligands, these signal-sending cells suppress activation of JAK/STAT via Ptp61F. Conversely, responding cells with activated JAK/STAT suppress JNK activation via Zfh2. The resulting bistable segregation of signaling domains is associated with distinct cellular tasks and regenerative potential. While JNK/AP-1 signaling cells at the wound center act as paracrine organizers, their cell cycle is senescently arrested. Thus, compensatory proliferation occurs exclusively in JAK/STAT signaling cells at the wound periphery. This spatial stratification is essential for proper tissue repair, as co-activation of JNK/AP-1 and JAK/STAT in the same cells creates conflicting inputs on cell cycle progression, leading to excess apoptosis of senescently arrested organizer cells. Finally, we demonstrate that bistable spatial segregation of JNK/AP-1 and JAK/STAT drives senescent and proliferative behaviors in transient as well as chronic tissue damage models, and importantly, in RasV12, scrib tumors under the influence of JNK/AP-1 activity. Revealing this previously uncharacterized regulatory network between JNK/AP-1, JAK/STAT and associated cell behaviors have important implications for our conceptual understanding of tissue repair, chronic wound pathologies and tumor microenvironments, where both pathways are strongly implicated.
The association of MEG3 lncRNA with nuclear speckles in living cells
Authors: Hasenson Sarah E. , Alkalay Ella , Atrash Mohammad K. , Boocholez Alon , Gershbaum Julianna , Hochberg-Laufer Hodaya , Shav-Tal Yaron
Published: May 2022

Nuclear speckles are nuclear bodies containing RNA-binding proteins as well as RNAs including long non-coding RNAs (lncRNAs). MEG3 is a nuclear retained lncRNA that was identified to be associated with nuclear speck-les. To understand the association dynamics of MEG3 lncRNA with nuclear speckles in living cells we generated a fluorescently-tagged MEG3 transcript that could be detected in real-time. Under regular conditions, transient association of MEG3 with nuclear speckles was observed, including a nucleoplasmic fraction. Conditions under which transcription or splicing were inactive, which are known to affect nuclear speckle structure, showed prominent and increased association of MEG3 lncRNA with the nuclear speckles, specifically forming a ring-like structure around the nuclear speckles. This contrasted with MALAT1 lncRNA that is normally highly associated with nuclear speckles, which was released and dispersed in the nucleoplasm. Under normal conditions MEG3 dynamically associated with the periphery of the nuclear speckles, but under transcription or splicing inhibition, MEG3 could also enter the center of the nuclear speckle. Altogether, using live-cell imaging approaches we find that MEG3 lncRNA is a transient resident of nuclear speckles and that its association with this nuclear body is modulated by the levels of transcription and splicing activities in the cell.
MAP1LC3C regulates lysosomal exocytosis and induces zinc reprogramming in renal cancer cells
Authors: Verma Rita , Aggarwal Parul , Reigle James , Secic Dina , Wetzel Collin , Bischoff Megan E. , VandenHeuvel Katherine , Biesiada Jacek , Ehmer Birgit , Landero Figueroa Julio A. , Plas David R. , Medvedovic Mario , Meller Jarek , Czyzyk-Krzeska Maria F.
Published: May 2022

MAP1LC3C (LC3C) is a member of the microtubule associated family of proteins that are essential in the formation of autophagosomes and lysosomal degradation of cargo. LC3C has tumor suppressing activity and its expression is dependent on kidney cancer tumor suppressors, such as VHL and FLCN. Recently we demonstrated that LC3C autophagy is regulated by noncannonical upstream regulatory complexes and targets for degradation postdivision midbody rings associated with cancer cells stemness. Here we show that loss of LC3C leads to peripheral positioning of the lysosomes and lysosomal exocytosis (LE) in a subset of cells. This process is independent of the autophagic activity of LC3C. Analysis of isogenic cells with low and high LE shows substantial transcriptomic reprogramming with altered expression of Zn-related genes and activity of Polycomb Repressor Complex 2 (PRC2), accompanied by a robust decrease in intracellular Zn. Metabolomic analysis revealed alterations in amino acid steady-state levels. Cells with augmented LE show tumor initiation properties and form aggressive tumors in xenograft models. Immunocytochemistry identified high levels of LAMP1 on the plasma membrane of cancer cells in human ccRCC and reduced levels of Zn, an indication that LE is a frequent event in ccRCC, potentially contributing to the loss of Zn. Overall, these data indicate that an important tumor suppressing activity of LC3C is contributing to the reprogramming of lysosomal activity and Zn metabolism with implication for epigenetic remodeling in a subpopulation of tumor propagating properties of cancer cells.
Impaired dendritic spike generation in the Fragile X prefrontal cortex is due to loss of dendritic sodium channels
Authors: Brandalise Federico , Kalmbach Brian E. , Cook Erik P. , Brager Darrin H.
Published: May 2022

Patients with Fragile X syndrome, the leading monogenetic cause of autism, suffer from impairments related to the prefrontal cortex including working memory and attention. Synaptic inputs to the distal dendrites of layer 5 pyramidal neurons in the prefrontal cortex have a weak influence on the somatic membrane potential. To overcome this filtering, distal inputs are transformed into local dendritic Na+ spikes, which propagate to the soma and trigger action potential output. Layer 5 extratelencephalic (ET) PFC neurons project to the brainstem and various thalamic nuclei and are therefore well positioned to integrate task-relevant sensory signals and guide motor actions. We used current clamp and outside-out patch clamp recording to investigate dendritic spike generation in ET neurons from male wild type and Fmr1 knockout (FX) mice. The threshold for dendritic spikes was more depolarized in FX neurons compared to wild type. Analysis of voltage responses to simulated in vivo “noisy” current injections showed that a larger dendritic input stimulus was required to elicit dendritic spikes in FX ET dendrites compared wild type. Patch clamp recordings revealed that the dendritic Na+ conductance was significantly smaller in FX ET dendrites. Taken together, our results suggest that input-output transformation is impaired in ET neurons of the PFC in FX mice. Considering our prior findings that somatic D-type K+ and dendritic HCN-channel function is reduced in ET neurons, we suggest that the integration of information by PFC circuits is fundamentally altered in Fragile X syndrome.

KEY POINTS

Dendritic spike threshold is depolarized in Layer 5 PFC neurons in FX mice

Simultaneous somatic and dendritic recording with white noise current injections revealed that larger dendritic stimuli were required to elicit dendritic spikes in FX ET neurons

Outside-out patch clamp recording revealed that dendritic sodium conductance density was lower in FX ET neurons
Multimodal disease transmission as a limiting factor for the spatial extent of a host plant
Authors: Uricchio Lawrence H. , Bruns Emily L. , Hood Michael , Boots Mike , Antonovics Janis
Published: May 2022

Theoretical models suggest that infectious diseases could play a substantial role in determining species’ ranges, but few studies have collected the empirical data required to test this hypothesis. Pathogens that sterilize their hosts or spread through frequency-dependent transmission could have especially strong effects on the limits of species’ distributions because sterilized hosts can serve as long-lived disease reservoirs and frequency-dependent transmission mechanisms are effective even at very low population densities. We collected spatial disease prevalence data and population abundance data for alpine carnations infected by the sterilizing pathogen M. violaceum, a disease that is spread through both frequency-dependent (vector-borne) and density-dependent (aerial spore transmission) mechanisms. Our 13-year study reveals rapid declines in population abundance without a compensatory decrease in disease prevalence. We apply a stochastic, spatial model of disease spread that accommodates spatial habitat heterogeneity to investigate how the population dynamics depend on multimodal (frequency-dependent and density-dependent) transmission. We found that the observed rate of population decline can be readily explained by multimodal transmission, but is unlikely to be explained by either frequency-dependent or density-dependent mechanisms alone. Multimodal disease transmission rates high enough to explain the observed decline predicted that eventual local extinction of the host species is highly likely. Our results add to a growing body of literature showing how multimodal transmission can constrain species distributions in nature.

Open Research

All scripts associated with the analyses in this manuscript, as well as the data we collected, are available at https://github.com/uricchio/antherSmutDis.
A dynamicin vitromodel of Down Syndrome neurogenesis with Trisomy 21 gene dosage correction
Authors: Bansal Prakhar , Banda Erin. C , Glatt-Deeley Heather R. , Stoddard Christopher E. , Linsley Jeremy W. , Arora Neha , Ahern Darcy T. , Kondaveeti Yuvabharath , Nicouleau Michael , Sabariego-Navarro Miguel , Dierssen Mara , Finkbeiner Steven , Pinter Stefan F.
Published: May 2022

ABSTRACT/SUMMARY

Excess gene dosage from human chromosome 21 (chr21) causes Down syndrome (DS), spanning developmental as well as acute phenotypes in terminal cell types. Which phenotypes remain amenable to intervention after development is unknown. To address this question in a model of DS neurogenesis, we generated trisomy 21 (T21) human induced pluripotent stem cells (hiPSCs) alongside otherwise isogenic euploid controls from mosaic DS fibroblasts, and integrated an inducibleXISTtransgene on one chr21 copy. Monoallelic chr21 silencing byXISTwas near-complete and irreversible in hiPSCs. Differential expression reveals T21 neural lineages and T21 hiPSCs suppress similar translation and mitochondrial pathways, and activate cellular stress responses. WhenXISTis induced before the neural progenitor stage, T21 dosage correction mitigates a pronounced skew towards astrogenesis in differentiation. Because our transgene remained inducible in post-mitotic T21 neurons and astrocytes, we demonstrateXISTefficiently represses genes even after terminal differentiation, which will empower exploration of cell type-specific T21 phenotypes that remain responsive to chr21 dosage.