bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular And Molecular Effects Of Understudied Kinase Pregnancy-Upregulated Non-Ubiquitous Calcium-Calmodulin Dependent Kinase (PNCK) In Renal Cell Carcinoma
Authors: Essegian Derek J. , Chavez Valery , Bustamante Floritza , Schürer Stephan C. , Merchan Jaime R.
Published: May 2022

Renal Cell Carcinoma (RCC) is a uniformly fatal disease when advanced. While immunotherapy and tyrosine kinase inhibitor-based combinations are associated with improved progression-free and overall survival, the majority of patients eventually develop treatment resistance and succumb to progressive, refractory disease. This underscores the urgent need to identify novel, non-canonical RCC targets for drug development. Through a comprehensive pan-cancer, pan-kinome analysis of the Cancer Genome Atlas (TCGA), the understudied kinase, pregnancy upregulated non-ubiquitous calcium-calmodulin dependent kinase (PNCK) was identified as the most differentially overexpressed kinase in RCC. PNCK mRNA was significantly overexpressed in RCC tissues compared to adjacent normal tissue, and its overexpression correlated with tumor T-stage grade and poor disease specific survival in both clear cell and papillary RCCs. PNCK overexpression in VHL mutant and VHL wild type RCC cell lines was associated with increased CREB phosphorylation, as well as increased cell proliferation and cell cycle progression. PNCK down-regulation, conversely, was associated with inhibition of CREB phosphorylation, decreased cell proliferation, cell cycle arrest and increased apoptosis, with differential effects observed between VHL mutant and VHL wild type cell lines. Pathway analyses in PNCK knockdown cells showed significant down regulation of hypoxia and angiogenesis pathways, as well as modulation of pathways promoting cell cycle arrest and apoptosis. The above results demonstrate for the first time the biological role of PNCK, an understudied kinase, in renal cell carcinoma and validate PNCK as a potential novel target for drug development in this fatal disease.
A requirement for Krüppel-Like Factor-4 in the maintenance of endothelial cell quiescence
Authors: Mastej Victoria , Axen Cassondra , Wary Anita , Minshall Richard D. , Wary Kishore K.
Published: May 2022

Rationale and Goal

Endothelial cells (ECs) are quiescent and critical for maintaining homeostatic functions of the mature vascular system, while disruption of quiescence is at the heart of endothelial to mesenchymal transition (EndMT) and tumor angiogenesis. Here, we addressed the hypothesis that KLF4 maintains the EC quiescence.

Methods and Results

In ECs, KLF4 bound to KLF2, and the KLF4-transctivation domain (TAD) interacted directly with KLF2. KLF4-depletion increased KLF2 expression, accompanied by phosphorylation of SMAD3, increased expression of alpha-smooth muscle actin (αSMA), VCAM-1, TGF-β1 and ACE2, but decreased VE-cadherin expression. In the absence of Klf4, Klf2 bound to the Klf2-promoter/enhancer region and autoregulated its own expression. Loss of EC-Klf4 in RosamT/mG::Klf4fl/fl::Cdh5CreERT2 engineered mice, increased Klf2 levels and these cells underwent EndMT.

Conclusion

In quiescent ECs, KLF2 and KLF4 partnered to regulate a combinatorial mechanism. The loss of KLF4 disrupted this combinatorial mechanism, thereby upregulating KLF2 as an adaptive response. However, increased KLF2 expression overdrives for the loss of KLF4, giving rise to an EndMT phenotype.

Key Points

Adult endothelial cells (ECs) are quiescent in that these cells are arrested at G0-phase of the cell cycle, but mechanisms of EC quiescence are not well understood.

The Krüppel-like factors (KLFs) -2 and -4 are transcriptional regulators, highly expressed in quiescent ECs, however, their roles in this process have not been addressed.

Elucidation of the mechanisms of KLF function in quiescent ECs should provide clues to the translational discoveries intended for the treatment of EC-dysfunction, such as endothelial to mesenchymal transition (EndMT) associated with several vascular diseases including tumor angiogenesis.

Graphical Abstract
Differentiation of semi-transparent tissue phantom inclusions using optical coherence tomography towards label-free neurography and lymphography
Authors: Qureshi Muhammad Mohsin , Peters Taylor , Allam Nader , Demidov Valentin , Vitkin Alex
Published: May 2022

Significance

Lymphatic and peripheral nervous system imaging is of prime importance for monitoring various important pathologic processes including cancer development, metastasis, and response to therapy.

Aim

Optical coherence tomography (OCT) is a promising approach for this imaging task but is challenged by the near-transparent nature of these structures. Our aim is to detect and differentiate semi-transparent materials using OCT texture analysis, towards label-free neurography and lymphography.

Approach

We have recently demonstrated a novel OCT texture analysis-based approach that used speckle statistics to image lymphatics and nerves in-vivo that does not rely on negative contrast. However, these two near-transparent structures could not be differentiated from each other easily in the texture analysis parameter space. Here we perform a rigorous follow-up study to improve upon this differentiation in controlled phantoms mimicking the optical properties of these tissues.

Results

The results of the three-parameter Rayleigh distribution fit to the OCT images of six types of tissue-mimicking materials varying in transparency and biophysical properties demonstrate clear differences between them, suggesting routes for improved lymphatics-nerves differentiation.

Conclusions

We demonstrate a novel OCT texture analysis based lymphatics-nerves differentiation methodology in tissue-simulating phantoms. Future work will focus on in-vivo lymphangiography and neurography studies of longitudinal treatment monitoring for therapy feedback and optimization.
A single helix repression domain is functional across eukaryotes
Authors: Leydon Alexander R. , Baez Román Ramos , Nemhauser Jennifer L.
Published: May 2022

The corepressor TOPLESS (TPL) and its paralogs coordinately regulate a large number of genes critical to plant development and immunity. As in many members of the larger pan-eukaryotic Tup1/TLE/Groucho corepressor family, TPL contains a Lis1 Homology domain (LisH), whose function is not well understood. We have previously found that the LisH in TPL—and specifically the N-terminal 18 amino acid alpha-helical region (TPL-H1) —can act as an autonomous repression domain. We hypothesized that homologous domains across diverse LisH-containing proteins could share the same function. To test that hypothesis, we built a library of H1s that broadly sampled the sequence and evolutionary space of LisH domains, and tested their activity in a synthetic transcriptional repression assay in Saccharomyces cerevisiae. Using this approach, we found that repression activity was highly conserved and likely the ancestral function of this motif. We also identified key residues that contribute to repressive function. We leveraged this new knowledge for two applications. First, we tested the role of mutations found in somatic cancers on repression function in two human LisH-containing proteins. Second, we validated function of many of our repression domains in plants, confirming that these sequences should be of use to synthetic biology applications across eukaryotes.
Orphan C/D box snoRNAs modulate fear-related memory processes in mice
Authors: Leighton Laura J. , Zhao Qiongyi , Marshall Paul R. , Madugalle Sachithrani U. , Chan Angelo H. C. , Musgrove Mason R. B. , Ren Haobin , Periyakaruppiah Ambika , Edson Janette , Wei Wei , Li Xiang , Spitale Robert C. , Bredy Timothy W.
Published: May 2022

C/D box small nucleolar RNAs (snoRNAs) comprise a class of small noncoding RNAs with important regulatory effects on cellular RNA function. Although it is well established that snoRNAs coordinate the post-transcriptional modification of pre-ribosomal and small nuclear RNAs by 2’-O-methylation, which leads to enhanced RNA stability, whether they are necessary for memory-related processes remains relatively unexplored. Using targeted sequencing, we have identified more than 150 C/D box snoRNAs in the prefrontal cortex of male C57BL/6J mice, 31 of which are differentially expressed in response to fear extinction learning. We have also discovered a subset of snoRNAs, including many orphans, that are enriched in the synaptic compartment, including the orphan snoRNA snord64. An extinction learning-induced increase in synapse-enriched snord64 led to increased 2’-O-methylation within the 3-UTR of the mRNA encoding the ubiquitin ligase RNF146. This effect was blocked by snord64 knockdown and was accompanied by attenuated forgetting of conditioned fear and the enhanced retrieval of fear extinction memory. Localized activity of orphan snoRNAs therefore represents a novel mechanism associated with fear-related learning and memory.

Significance statement

We have discovered a population of experience-dependent small nucleolar RNAs (snoRNAs), and found that several of these are recruited to the synaptic compartment in response to fear extinction learning. In particular, the orphan snoRNA, snord64, drives the methylation of the mRNA encoding the ubiquitin ligase, RNF146, with a reduction in snord64 attenuating forgetting of conditioned fear and enhancing the retrieval of fear extinction memory. This study reveals a new mechanism of gene regulation associated with fear-related memory that involves the activity of synapse-enriched snoRNAs.
Delayed antigen-specific CD4+ T-cell induction correlates with impaired immune responses to SARS-COV-2 mRNA vaccination in the elderly
Authors: Jo Norihide , Hidaka Yu , Kikuchi Osamu , Fukahori Masaru , Sawada Takeshi , Aoki Masahiko , Yamamoto Masaki , Nagao Miki , Morita Satoshi , Nakajima Takako E , Muto Manabu , Hamazaki Yoko
Published: May 2022

Despite the clinical efficacy of coronavirus disease 2019 mRNA vaccines, the elderly demonstrate lower IgG levels and neutralizing titers and a higher risk of severe diseases. CD4+ T cells play a central role in regulating antigen-specific antibody and CD8+ T-cell responses; however, because their composition and functionality change significantly with age, relationships between age-associated defects in T cells and the immunogenicity of or reactogenicity to mRNA vaccines are unclear. Using a vaccine cohort (n = 216), we found that the elderly (aged ≥65 years) showed delayed induction and early contraction of vaccine-specific CD4+ T cells, and that the compromised C–X–C motif chemokine receptor 3+ circulating T follicular helper cell response after the first dose was associated with the lower IgG levels. Additionally, the elderly experienced significantly fewer systemic adverse effects (AEs) after the second dose, with those exhibiting few AEs showing lower cytokine+ CD4+ T cells after the first dose and lower antibody levels after the second dose. Furthermore, T helper 1 cells in the elderly expressed higher levels of programmed cell death protein-1, a negative regulator of the T-cell response, which was associated with less production of vaccine-specific CD4+ T cells and impaired CD8+ T-cell expansion. Thus, efficient induction of vaccine-specific effector/memory CD4+ T cells after the first dose may trigger robust cytokine production after the second dose, leading to effective vaccine responses and higher systemic reactogenicity. These results suggested that an enhanced CD4+ T-cell response after the first dose is key to improved vaccination efficacy in the elderly.

One Sentence Summary

We compared immunogenicity and reactogenicity to COVID-19 mRNA vaccine in 107 adults (aged <65 years) and 109 elderly (aged ≥65) individuals.
Chromosome-level genome sequence of the Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus) highlights regions of introgression with O. mossambicus
Authors: Etherington GJ , Nash W , Ciezarek A , Mehta TK , Barria A , Peñaloza C , Khan MGQ , Durrant A , Forrester N , Fraser F , Irish N , Kaithakottil GG , Lipscombe J , Trong T , Watkins C , Swarbreck D , Angiolini E , Cnaani A , Gharbi K , Houston RD , Benzie JAH , Haerty W
Published: May 2022

Background

The Nile tilapia (Oreochromis niloticus) is the third most important freshwater fish for aquaculture. Its success is directly linked to continuous breeding efforts focusing on production traits such as growth rate and weight. Among those elite strains, the Genetically Improved Farmed Tilapia (GIFT) programme initiated by WorldFish is now distributed worldwide. To accelerate the development of the GIFT strain through genomic selection, a high-quality reference genome is necessary.

Findings

Using a combination of short (10X Genomics) and long read (PacBio HiFi, PacBio CLR) sequencing and a genetic map for the GIFT strain, we generated a chromosome level genome assembly for the GIFT. Using genomes of two closely related species (O. mossambicus, O. aureus), we characterised the extent of introgression between these species and O. niloticus that has occurred during the breeding process. Over 11Mb of O. mossambicus genomic material could be identified within the GIFT genome, including genes associated with immunity but also with traits of interest such as growth rate.

Conclusion

Because of the breeding history of elite strains, current reference genomes might not be the most suitable to support further studies into the GIFT strain. We generated a chromosome level assembly of the GIFT strain, characterising its mixed origins, and the potential contributions of introgressed regions to selected traits.
Transposable elements are associated with the variable response to influenza infection
Authors: Chen Xun , Pacis Alain Sarabia , Aracena Katherine A , Gona Saideep , Kwan Tony , Groza Cristian , Lin Yen Lung , Sindeaux Renata Helena Monteiro , Yotova Vania , Pramatarova Albena , Simon Marie-Michelle , Pastinen Tomi M. , Barreiro Luis , Bourque Guillaume
Published: May 2022

Influenza A virus (IAV) infections are frequent every year and result in a range of disease severity. Given that transposable elements (TEs) contribute to the activation of innate immunity, we wanted to explore their potential role in this variability. Transcriptome profiling in monocyte-derived macrophages from 39 individuals following IAV infection revealed significant inter-individual variation in viral load post-infection. Using ATAC-seq we identified a set of TE families with either enhanced or reduced accessibility upon infection. Of the enhanced families, 15 showed high variability between individuals and had distinct epigenetic profiles. Motif analysis showed an association with known immune regulators in stably enriched TE families and with other factors in variable families, including KRAB-ZNFs. We also observed a strong association between basal TE transcripts and viral load post infection. Finally, we built a predictive model suggesting that TEs, and host factors regulating TEs, contribute to the variable response to infection.
Multiple omic investigations of freeze tolerance adaptation in the aquatic ectothermic vertebrate, the Amur sleeper
Authors: Lv Wenqi , Jiang Haifeng , Qian Yuting , Meng Minghui , Wang Cheng , Sun Ning , Lu Yongrui , Bing Houhua , Fang Chengchi , Irwin David M. , He Shunping , Yang Liandong
Published: May 2022

Freeze tolerance is an amazing overwintering strategy that enables ectotherms to occupy new niches and survive in cold climates. However, the genetic basis underpinning this ecologically relevant adaptation is largely unknown. Amur sleeper is the only known freeze-tolerant fish species that can overwinter with its entire body frozen in ice. Here, we sequenced the chromosome-level genome of the Amur sleeper and performed comparative genomic, transcriptomic, and metabolomic analyses to investigate this remarkable adaptation. Phylogenetic analyses showed that the Amur sleeper diverged from its close relative with no cold hardiness about 15.07 million years ago and revealed two unusual population expansions during the glacial epochs. Integrative omics data identified a synchronous regulation of genes and metabolites involved in hypometabolism and cellular stress response, and several related genes showed strong evidence of accelerated evolution and positive selection. Potential evolutionary innovations that might aid in freezing survival were found to be associated with the dynamic rearrangement of the cytoskeleton to maintain cell viability, redistribution of water and cryoprotectants to limit cell volume reduction, and inhibition in nerve activity to facilitate dormancy, demonstrating a coordinated evolution for this complex adaptation. Overall, our work provides valuable resources and opportunities to unveil the genetic basis of freeze tolerance adaptation in ectothermic vertebrates.
Bi-Phasic Quasistatic Brain Communication for Fully Untethered Connected Brain Implants
Authors: Chatterjee Baibhab , Nath Mayukh , Gaurav Kumar K , Xiao Shulan , Jayant Krishna , Sen Shreyas
Published: May 2022

Wireless communication using electro-magnetic (EM) fields acts as the backbone for information exchange among wearable devices around the human body. However, for Implanted devices, EM fields incur high amount of absorption in the tissue, while alternative modes of transmission including ultrasound, optical and magnetoelectric methods result in large amount of transduction losses due to conversion of one form of energy to another, thereby increasing the overall end-to-end energy loss. To solve the challenge of wirelesspowering and communication in a brain implant with low end-end channel loss, we present Bi-Phasic Quasistatic Brain Communication (BP-QBC), achieving < 60dB worst-case end-to-end channel loss at a channel length of ~55mm, by using Electro-quasistatic (EQS) Signaling thatavoids transduction losses due to no field-modality conversion. BP-QBC utilizes dipole coupling based signal transmission within the brain tissue using differential excitation in the transmitter (TX) and differential signal pick-up at the receiver (RX), while offering ~41X lower power w.r.t. traditional Galvanic Human Body Communication (G-HBC) at a carrier frequency of 1MHz, by blocking any DC current paths through the brain tissue. Since the electrical signal transfer through the human tissue is electro-quasistatic up to several 10’s of MHz range, BP-QBC allows a scalable (bps-10Mbps) duty-cycled uplink (UL) from the implant to an external wearable. The power consumption in the BP-QBC TX is only 0.52 μW at 1Mbps (with 1% duty cycling), which is within the range of harvested power in the downlink (DL) from a wearable hub to an implant through the EQS brain channel, with externally applied electric currents < 1/5th of ICNIRP safety limits. Furthermore, BP-QBCeliminates the need for sub-cranial interrogators/repeaters, as it offers better signal strength due to no field transduction. Such low end-to-end channel loss with high data rates enabled by a completely new modality of brain communication and powering has deep societal and scientific impact in the fields of neurobiological research, brain-machine interfaces, electroceuticals and connected healthcare.
Transformation of coral communities subjected to an unprecedented heatwave is modulated by local disturbance
Authors: Baum Julia K. , Claar Danielle C. , Tietjen Kristina L. , Magel Jennifer M.T. , Maucieri Dominique G. , Cobb Kim M. , McDevitt-Irwin Jamie M.
Published: May 2022

Corals are imminently threatened by climate change-amplified marine heatwaves. Yet how to conserve reef ecosystems faced with this threat remains unclear, since protected reefs often seem equally or more susceptible to thermal stress as unprotected ones. Here, we disentangle this apparent paradox, revealing that the relationship between reef disturbance and heatwave impacts depends upon the focal scale of biological organization. We document a heatwave of unprecedented duration that culminated in an 89% loss of coral cover. At the community level, losses hinged on pre-heatwave community structure, with sites dominated by competitive corals—which were predominantly protected from local disturbance—undergoing the greatest losses. In contrast, at the species level, survivorship of individual coral colonies typically decreased as local disturbance intensified, illustrating that underlying chronic disturbances can impair resilience to thermal stress at this scale. Our study advances understanding of the relationship between climate change and local disturbance, knowledge of which is crucial for coral conservation this century.
A detailed behavioral, videographic, and neural dataset on object recognition in mice
Authors: Rodgers Chris C.
Published: May 2022

Mice adeptly use their whiskers to touch, recognize, and learn about objects in their environment. This ability is enabled by computations performed by populations of neurons in the somatosensory cortex. To understand these computations, we trained mice to use their whiskers to recognize different shapes while we recorded activity in the barrel cortex, which processes whisker input. Here, we present a large dataset of high-speed video of the whiskers, along with rigorous tracking of the entire extent of multiple whiskers and every contact they made on the shape. We used spike sorting to identify individual neurons, which responded with precise timing to whisker contacts and motion. These data will be useful for understanding the behavioral strategies mice use to identify objects by touch, as well as the neuronal responses that mediate those strategies. More generally, our carefully curated labeled data could be used to develop new computer vision algorithms for tracking body posture, or for extracting responses of individual neurons from large-scale neural recordings.
Molecular dynamics simulations reveal membrane lipid interactions of the full-length lymphocyte specific kinase Lck
Authors: Prakaash Dheeraj , Cook Graham P. , Acuto Oreste , Kalli Antreas C.
Published: May 2022

The membrane-bound lymphocyte-specific protein-tyrosine kinase (Lck) triggers T cell antigen receptor signalling to initiate adaptive immune responses. Despite many structure-function studies, the mode of action of Lck and the potential role of plasma membrane lipids in regulating Lck’s activity remains elusive. Advances in molecular dynamics simulations of membrane proteins in complex lipid bilayers have opened a new perspective in gathering such information. Here, we have modelled the full-length Lck open and closed conformations available from crystallographic studies and simulated its interaction with the inner leaflet of the T cell plasma membrane. In both conformations, we found that the unstructured unique domain and the structured domains including the kinase interacted with the membrane with a preference for PIP lipids. Interestingly, our simulations suggest that the Lck-SH2 domain interacts with lipids differently in the open and closed Lck conformations, demonstrating that lipid interaction can potentially regulate Lck’s conformation and in turn modulate T cell signalling. Additionally, the Lck-SH2 and kinase domain residues that significantly contacted PIP lipids are found to be conserved among the Src family of kinases, thereby potentially representing similar PIP interactions within the family.
KaicoTracker: a robust and automated locomotory analysis for baculovirus-infected silkworm larvae
Authors: Hikida Hiroyuki , Katsuma Susumu
Published: May 2022

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen of the domestic silkworm, Bombyx mori, whose infection drastically alters larval locomotory patterns and activity. As B. mori is the only insect that has been completely domesticated, its larvae are typically inactive. In contrast, BmNPV-infected larvae exhibit vigorous movement. Recently, we developed a method for locomotory analysis of BmNPV-infected B. mori larvae, which yielded high-resolution data on BmNPV-induced host locomotory alteration. However, this method had a disadvantage when analyzing the behavior of uninfected larvae, as they were typically inactive and difficult to distinguish from the background. In addition, the analysis processes involve many manual steps, which reduces analysis throughput. We have developed KaicoTracker, a new tool for automated locomotory analysis of BmNPV-infected B. mori larvae, using Python and OpenCV. This tool is an all-inclusive package that tracks and quantifies larval locomotion and verifies the accuracy of the tracking. Larval locomotion is tracked by motion tracking function based on k-Nearest Neighbor implemented in OpenCV. Based on the tracking results, the positions of the larvae are determined, and locomotory distance, speed, duration, and frequency are calculated. The larval positions are marked on the original video, allowing tracking precision to be examined. By analyzing previously obtained data, we confirmed that this tool provides a robust analysis of inactive larvae with fewer manual steps.
The neighboring genes AvrLm10A and AvrLm10B are part of a large multigene family of cooperating effector genes conserved in Dothideomycetes and Sordariomycetes
Authors: Talbi Nacera , Fokkens Like , Audran Corinne , Petit-Houdenot Yohann , Pouzet Cécile , Blaise Françoise , Gay Elise , Rouxel Thierry , Balesdent Marie-Hélène , Rep Martijn , Fudal Isabelle
Published: May 2022

With only a few exceptions, fungal effectors (small secreted proteins) have long been considered as species- or even isolate-specific. With the increasing availability of high-quality fungal genomes and annotations, trans-species or trans-genera families of effectors are being uncovered. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus responsible for stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighboring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome show that AvrLm10A/AvrLm10B belong to a multigene family comprising five pairs of genes with a similar tail-to-tail organization. The two genes in a pair always had the same expression pattern and two expression profiles were distinguished, associated with the biotrophic colonization of cotyledons and / or petioles and stems. Of the two protein pairs further investigated Lmb_jn3_08094/Lmb_jn3_08095 and Lmb_jn3_09745 / Lmb_jn3_09746, one (Lmb_jn3_09745 / Lmb_jn3_09746) had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair. AvrLm10A homologues are present in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi whereas fewer AvrLm10B homologues were identified. One of the AvrLm10A homologues, SIX5, is an effector from Fusarium oxysporum f.sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A homologues were associated with at least eight distinct putative effector families, suggesting an ability of AvrLm10A/SIX5 to cooperate with diverse effectors. These results point to a general role of the AvrLm10A/SIX5 protein as a ‘cooperator protein’, able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighboring AvrLm10A homologue.
Non-cell-autonomous HSC70.1 chaperone displays homeostatic feed-back regulation by binding its own mRNA
Authors: Yang Lei , Zhou Yuan , Wang Shuangfeng , Xu Ying , Ostendorp Steffen , Tomkins Melissa , Kehr Julia , Morris Richard J. , Kragler Friedrich
Published: May 2022

Heat shock proteins of the HSC70/HSP70 family are evolutionarily conserved chaperones that are involved in protein folding, protein transport and RNA binding. Arabidopsis HSC70 chaperones are thought to act as housekeeping chaperones and as such are involved in many growth-related pathways. Whether Arabidopsis HSC70 binds RNA and its function has remained an open question. Here, we show that the HSC70.1 chaperone binds its own mRNA via its C-terminal Short Variable Region (SVR) and inhibits its own translation. We propose that this negative protein-transcript feedback loop may establish an on-demand chaperone pool that allows for a rapid response to stress. Furthermore, we show that the SVR encoding RNA region is necessary for HSC70.1 transcript mobility to distant tissues and that HSC70.1 transcript and not protein mobility is required to rescue root growth and flowering time of hsc70 mutants. In summary, it seems that the Arabidopsis HSC70.1 chaperone can form a complex with its own transcript to regulate its translation and that both protein and transcript can act in a non-cell-autonomous manner maintaining chaperone homestasis between tissues.
E-SNPs&GO: Embedding of protein sequence and function improves the annotation of human pathogenic variants
Authors: Manfredi Matteo , Savojardo Castrense , Martelli Pier Luigi , Casadio Rita
Published: May 2022

Motivation

The advent of massive DNA sequencing technologies is producing a huge number of human single-nucleotide polymorphisms occurring in protein-coding regions and possibly changing protein sequences. Discriminating harmful protein variations from neutral ones is one of the crucial challenges in precision medicine. Computational tools based on artificial intelligence provide models for protein sequence encoding, bypassing database searches for evolutionary information. We leverage the new encoding schemes for an efficient annotation of protein variants.

Results

E-SNPs&GO is a novel method that, given an input protein sequence and a single residue variation, can predict whether the variation is related to diseases or not. The proposed method, for the first time, adopts an input encoding completely based on protein language models and embedding techniques, specifically devised to encode protein sequences and GO functional annotations. We trained our model on a newly generated dataset of 65,888 human protein single residue variants derived from public resources. When tested on a blind set comprising 6,541 variants, our method outperforms recent approaches released in literature for the same task, reaching a MCC score of 0.71. We propose E-SNPs&GO as a suitable, efficient and accurate large-scale annotator of protein variant datasets.

Contact

pierluigi.martelli@unibo.it
The gastric mucosa of Atlantic salmon (Salmo salar) is abundant in highly active chitinases
Authors: Holen Matilde Mengkrog , Tuveng Tina Rise , Kent Matthew Peter , Vaaje-Kolstad Gustav
Published: May 2022

The Atlantic salmon (Salmo salar) genome contains 10 chitinase encoding genes, but little is known about the function of these chitinases. We show that the protein products of three genes, the family 18 glycoside hydrolase (GH18) chitinases Chia.3, Chia.4, and Chia.7 are secreted in the stomach mucosa and are amongst the most abundant proteins in this matrix. Chia.3 and Chia.4, sharing 95% sequence identity, were not possible to separate by standard chromatographic methods and were thus purified as a chitinase pair. Biochemical analysis revealed chitinolytic activity towards β-chitin for up to 24 hours at pH 2-6. Furtherin vitroanalysis showed that this chitinase pair efficiently degraded various chitin-containing substrates to chitobiose (GlcNAc2) suggesting that Atlantic salmon has the potential to utilize novel chitin-containing feed sources.
Roles and interactions of the specialized initiation factors EIF4E2, EIF4E5 and EIF4E6 inTrypanosoma brucei: EIF4E2 maintains the abundances of S-phase mRNAs
Authors: Falk Franziska , Melo Palhares Rafael , Waithaka Albina , Clayton Christine
Published: May 2022

Trypanosoma bruceihas six versions of the cap-binding translation initiation factor EIF4E. We investigated the functions of EIF4E2, EIF4E3, EIF4E5 and EIF4E6 in bloodstream forms. We confirmed the protein associations previously found in procyclic forms, and detected specific co-purification of some RNA-binding proteins. Bloodstream forms lacking EIF4E5 grew normally and differentiated to replication-incompetent procyclic forms. Depletion of EIF4E6 inhibited bloodstream-form trypanosome growth and translation. EIF4E2 co-purified only the putative RNA binding protein SLBP2. Bloodstream forms lacking EIF4E2 multiplied slowly, had a low maximal cell density, and expressed the stumpy-form marker PAD1, but showed no evidence for enhanced stumpy-form signalling. EIF4E2 knock-out cells differentiated readily to replication-competent procyclic forms. EIF4E2 was strongly associated with mRNAs that are maximally abundant in S-phase, three of which are bound and stabilized by the Pumilio domain protein PUF9. The same mRNAs had decreased abundances in EIF4E2 knock-out cells. Yeast 2-hybrid results suggested that PUF9 interacts directly with SLBP2, but PUF9 was not detected in EIF4E2 pull-downs. We suggest that the EIF4E2-SLBP2 complex might interact with PUF9, and its bound RNAs, only early during G1/S, stabilizing the mRNAs in preparation for translation later in S-phase or in early G2.
CAMML with the Integration of Marker Proteins (ChIMP)
Authors: Schiebout Courtney , Frost H. Robert
Published: May 2022

Motivation

Cell typing is a critical task in the analysis of single cell data, particularly when studying diseased tissues that contain a complex mixture of normal tissue and infiltrating immune cells. Unfortunately, the sparsity and noise of single cell data make accurate cell typing at the level of individual cells extremely difficult. To address these challenges, we previously developed the CAMML method for multi-label cell typing of single cell RNA-sequencing (scRNA-seq) data. CAMML uses weighted gene sets to score each profiled cell for multiple potential cell types. While CAMML outperforms other scRNA-seq cell typing techniques, it only leverages transcriptomic data so cannot take advantage of newer multi-omic single cell assays that jointly profile gene expression and protein abundance (e.g., joint scRNA-seq/CITE-seq).

Result

We developed the ChIMP (CAMML with the Integration of Marker Proteins) method to support multi-label cell typing of individual cells jointly profiled via scRNA-seq and CITE-seq. ChIMP combines cell type scores computed on scRNA-seq data via the CAMML approach with discretized CITE-seq measurements for cell type marker proteins. The multi-omic cell type scores generated by ChIMP allow researchers to more precisely and conservatively cell type joint scRNA-seq/CITE-seq data.
On tumoural growth and treatment under cellular dedifferentiation
Authors: Fischer Matthias M. , Blüthgen Nils
Published: May 2022

Differentiated cancer cells may regain stem cell characteristics; however, the effects of such a cellular dedifferentiation on tumoural growth and treatment are currently understudied. Thus, we here extend a mathematical model of cancer stem cell (CSC) driven tumour growth to also include dedifferentiation. We show that dedifferentiation increases the likelihood of tumorigenenis and the speed of tumoural growth, both modulated by the proliferative potential of the non-stem cancer cells (NSCCs). We demonstrate that dedifferentiation also may lead to treatment evasion, especially when a treatment solely targets CSCs. Conversely, targeting both CSCs and NSCCs in parallel is shown to be more robust to dedifferentiation. Despite dedifferentiation, perturbing CSC-related parameters continues to exert the largest relative effect on tumoural growth; however, we show the existence of synergies between specific CSC- and NSCC-directed treatments which cause superadditive reductions of tumoural growth. Overall, our study demonstrates various effects of dedifferentiation on growth and treatment of tumoural lesions, and we anticipate our results to be helpful in guiding future molecular and clinical research on limiting tumoural growth in vivo.
Lifelong regeneration of cerebellar Purkinje neurons after induced cell ablation in zebrafish
Authors: Pose-Méndez Sol , Schramm Paul , Winter Barbara , Meier Jochen C. , Ampatzis Konstantinos , Köster Reinhard W.
Published: May 2022

Zebrafish have an impressive capacity to regenerate neurons in the central nervous system. However, regeneration of the principal neuron of the evolutionary conserved cerebellum, the Purkinje cell (PC), is believed to be limited to developmental stages based on invasive lesions. In contrast, non-invasive cell type specific ablation by induced apoptosis closer represents processes of neurodegeneration. We demonstrate that the ablated larval PC population entirely recovers in number, quickly reestablishes electrophysiological properties and properly integrates into circuits to regulate cerebellum-controlled behavior. PC progenitors are present in larvae and adults, and PC ablation in adult cerebelli results in an impressive PC regeneration of different PC subtypes able to restore behavioral impairments. Interestingly, caudal PCs are more resistant to ablation and regenerate more efficiently, suggesting a rostro-caudal pattern of de- and regeneration properties. These findings demonstrate that the zebrafish cerebellum is able to regenerate functional PCs during all stages of the animal’s life.
Phase Separation and Correlated Motions in Motorized Genome
Authors: Jiang Zhongling , Qi Yifeng , Kamat Kartik , Zhang Bin
Published: May 2022

The human genome is arranged in the cell nucleus non-randomly, and phase separation has been proposed as an important driving force for genome organization. However, the cell nucleus is an active system, and the contribution of non-equilibrium activities to phase separation and genome structure and dynamics remains to be explored. We simulated the genome using an energy function parameterized with chromosome conformation capture (Hi-C) data with the presence of active, nondirectional forces that break the detailed balance. We found that active forces that may arise from transcription and chromatin remodeling can dramatically impact the spatial localization of heterochromatin. When applied to euchromatin, active forces can drive heterochromatin to the nuclear envelope and compete with passive interactions among heterochromatin that tend to pull them in opposite directions. Furthermore, active forces induce long-range spatial correlations among genomic loci beyond single chromosome territories. We further showed that the impact of active forces could be understood from the effective temperature defined as the fluctuation-dissipation ratio. Our study suggests that non-equilibrium activities can significantly impact genome structure and dynamics, producing unexpected collective phenomena.
Machine learning directed organoid morphogenesis uncovers an excitable system driving human axial elongation
Authors: Anand Giridhar M. , Megale Heitor C. , Murphy Sean H. , Weis Theresa , Lin Zuwan , He Yichun , Wang Xiao , Liu Jia , Ramanathan Sharad
Published: May 2022

The human embryo breaks symmetry to form the anterior-posterior axis of the body. As the embryo elongates along this axis, progenitors in the tailbud give rise to axial tissues that generate the spinal cord, skeleton, and musculature. The mechanisms underlying human axial elongation are unknown. While ethics necessitate in vitro studies, the variability of human organoid systems has hindered mechanistic insights. Here we developed a bioengineering and machine learning framework that optimizes symmetry breaking by tuning the spatial coupling between human pluripotent stem cell-derived organoids. This framework enabled the reproducible generation of hundreds of axially elongating organoids, each possessing a tailbud and an epithelial neural tube with a single lumen. We discovered that an excitable system composed of WNT and FGF signaling drives axial elongation through the induction of a signaling center in the form of neuromesodermal progenitor (NMP)-like cells. The ability of NMP-like cells to function as a signaling center and drive elongation is independent of their potency to generate mesodermal cell types. We further discovered that the instability of the underlying excitable system is suppressed by secreted WNT inhibitors of the secreted frizzled-related protein (SFRP) family. Absence of these inhibitors led to the formation of ectopic tailbuds and branches. Our results identify mechanisms governing stable human axial elongation to achieve robust morphogenesis.
Bcl-xL is a key mediator of apoptosis following KRASG12C inhibition in KRASG12C mutant colorectal cancer
Authors: Khawaja Hajrah , Briggs Rebecca , Latimer Cheryl , Rassel Md A.M.B. , Griffin Daryl , Hanson Lyndsey , Bardelli Alberto , Di Nicolantonio Frederica , McDade Simon , Scott Christopher J. , Lambe Shauna , Maurya Manisha , Lindner Andreas , Prehn Jochen H.M. , Sousa Jose , Winnington Chris , LaBonte Melissa J. , Ross Sarah , Van Schaeybroeck Sandra
Published: May 2022

Purpose

Novel covalent inhibitors of KRASG12C have shown limited response rates in KRASG12C mutant (MT) colorectal cancer (CRC) patients. Thus, novel KRASG12C inhibitor combination strategies that can achieve deep and durable responses are needed.

Experimental design

Small molecule KRASG12C inhibitors AZ’1569 and AZ’8037 were employed. To identify novel candidate combination strategies for AZ’1569, we performed RNA sequencing, siRNA and high-throughput drug screening. Top hits were validated in a panel of KRASG12CMT CRC cells and in vivo. AZ’1569-resistant CRC cells were generated and characterised.

Results

Response to AZ’1569 was heterogeneous across the KRASG12CMT models. AZ’1569 was ineffective at inducing apoptosis when used as single-agent or combined with chemotherapy or agents targeting the EGFR/KRAS/AKT axis. Using a systems biology approach, we identified the anti-apoptotic BH3-family member BCL2L1/Bcl-xL as a top hit mediating resistance to AZ’1569. Further analyses identified acute increases in the pro-apoptotic protein BIM following AZ’1569 treatment. ABT-263 (Navitoclax), a pharmacological Bcl-2 family-inhibitor that blocks the ability of Bcl-xL to bind and inhibit BIM, led to dramatic and universal apoptosis when combined with AZ’1569. Furthermore, this combination also resulted in dramatically attenuated tumour growth in KRASG12CMT xenografts. Finally, AZ’1569-resistant cells showed amplification of KRASG12C, EphA2/c-MET activation, increased pro-inflammatory chemokine profile and cross-resistance to several targeted agents. Importantly, KRAS amplification and AZ’1569-resistance were reversible upon drug withdrawal, arguing strongly for the use of drug holidays in the case of KRAS amplification.

Conclusions

Combinatorial targeting of Bcl-xL and KRASG12C is highly effective, suggesting a novel therapeutic strategy for KRASG12CMT CRC patients.
The statistical power of three monkeys
Authors: Laurens Jean
Published: May 2022

Neuroscience studies in non-human primates (NHP) often follow the rule of thumb that results observed in one animal must be replicated in at least one other. However, we lack a statistical justification for this rule of thumb, or an analysis of whether including three or more animals is better than including two. Yet, a formal statistical framework for experiments with few subjects would be crucial for experimental design, ethical justification, and data analysis. Also, including three or four animals in a study creates the possibility that the results observed in one animal will differ from those observed in the others: we need a statistically justified rule to resolve such situations. Here, I present a statistical framework to address these issues. This framework assumes that conducting an experiment will produce a similar result for a large proportion of the population (termed ‘representative’), but will produce spurious results for a substantial proportion of animals (termed ‘outliers’); the fractions of ‘representative’ and ‘outliers’ animals being defined by a prior distribution. I propose a procedure in which experimenters collect results from M animals and accept results that are observed in at least N of them (‘N-out-of-M’ procedure). I show how to compute the risks α (of reaching an incorrect conclusion) and β (of failing to reach a conclusion) for any prior distribution, and as a function of N and M. Strikingly, I find that the N-out-of-M model leads to a similar conclusion across a wide range of prior distributions: recordings from two animals lowers the risk α and therefore ensures reliable result, but leaves a large risk β; and recordings from three animals and accepting results observed in two of them strikes an efficient balance between acceptable risks α and β. This framework gives a formal justification for the rule of thumb of using at least two animals in NHP studies, suggests that recording from three animals when possible markedly improves statistical power, provides a statistical solution for situations where results are not consistent between all animals, and may apply to other types of studies involving few animals.
Inferring Density-Dependent Population Dynamics Mechanisms through Rate Disambiguation for Logistic Birth-Death Processes
Authors: Huynh Linh , Scott Jacob G. , Thomas Peter J.
Published: May 2022

Density dependence is important in the ecology and evolution of microbial and cancer cells. Typically, we can only measure net growth rates, but the underlying density-dependent mechanisms that give rise to the observed dynamics can manifest in birth processes, death processes, or both. Therefore, we utilize the mean and variance of cell number fluctuations to separately identify birth and death rates from time series that follow stochastic birth-death processes with logistic growth. Our method provides a novel perspective on stochastic parameter identifiability, which we validate by analyzing the accuracy in terms of the discretization bin size. We apply our method to the scenario where a homogeneous cell population goes through three stages: (1) grows naturally to its carrying capacity, (2) is treated with a drug that reduces its carrying capacity, and (3) overcomes the drug effect to restore its original carrying capacity. In each stage, we disambiguate whether it happens through the birth process, death process, or some combination of the two, which contributes to understanding drug resistance mechanisms. In the case of limited data sets, we provide an alternative method based on maximum likelihood and solve a constrained nonlinear optimization problem to identify the most likely density dependence parameter for a given cell number time series. Our methods can be applied to other biological systems at different scales to disambiguate density-dependent mechanisms underlying the same net growth rate.

Mathematics Subject Classifications

60J27 · 92D25 · 62M10 · 60J25
Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system
Authors: Fung Herman K.H. , Grimes Shelley , Huet Alexis , Duda Robert L. , Chechik Maria , Gault Joseph , Robinson Carol V. , Hendrix Roger W. , Jardine Paul J. , Conway James F. , Baumann Christoph G. , Antson Alfred A.
Published: May 2022

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for both this phage and a cos system, reveal unexpected mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.