bluuebunny/crossref_metadata_2025 huggingface dataset:
CRISPR/Cas9-mediated generation of biallelic G0 anemonefish (Amphiprion ocellaris) mutants
Authors: Mitchell Laurie J. , Tettamanti Valerio , Marshall Justin N. , Cheney Karen L. , Cortesi Fabio
Published: October 2020

Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish,Amphiprion ocellaris. Microinjection of eggs at the one-cell stage was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions inA. ocellarisembryos showed that uptake was as high as 50% of injected eggs. Further analysis of the subcloned mutant gene sequences revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in G0A. ocellarisembryos. Moreover, we clearly show a loss-of-function intyrmutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful resource for future experimental studies that aim to elucidate gene function in anemonefishes and reef fishes in general.
Viral mediated tethering to SEL1L facilitates ER-associated degradation of IRE1
Authors: Hinte Florian , Müller Jendrik , Brune Wolfram
Published: October 2020

The unfolded protein response (UPR) and endoplasmic reticulum (ER)-associated degradation (ERAD) are two essential components of the quality control system for proteins in the secretory pathway. When unfolded proteins accumulate in the ER, UPR sensors such as IRE1 induce the expression of ERAD genes, thereby increasing protein export from the ER to the cytosol and subsequent degradation by the proteasome. Conversely, IRE1 itself is an ERAD substrate, indicating that the UPR and ERAD regulate each other. Viruses are intracellular parasites that exploit the host cell for their own benefit. Cytomegaloviruses selectively modulate the UPR to take advantage of beneficial and inhibit detrimental effects on viral replication. We have previously shown that murine and human cytomegaloviruses express homologous proteins (M50 and UL50, respectively) that dampen the UPR at late times post infection by inducing IRE1 degradation. However, the degradation mechanism has remained uncertain. Here we show that the cytomegalovirus M50 protein mediates IRE1 degradation by the proteasome. M50-dependent IRE1 degradation can be blocked by pharmacological inhibition of p97/VCP or by genetic ablation of SEL1L, both of which are component of the ERAD machinery. SEL1L acts as a cofactor of the E3 ubiquitin ligase HRD1, while p97/VCP is responsible for the extraction of ubiquitylated proteins from the ER to the cytosol. We further show that M50 facilitates the IRE1-SEL1L interaction by binding to both, IRE1 and SEL1L. These results indicate that the viral M50 protein dampens the UPR by tethering IRE1 to SEL1L, thereby promoting its degradation by the ERAD machinery.

Importance

Viruses infect cells of their host and force them to produce virus progeny. This can impose stress on the host cell and activate counter-regulatory mechanisms. Protein overload in the endoplasmic reticulum (ER) leads to ER stress and triggers the unfolded protein response, which in turn upregulates protein folding and increases the degradation of proteins in the ER. Previous work has shown that cytomegaloviruses interfere with the unfolded protein response by degrading the sensor molecule IRE1. Herein we demonstrate how the viral M50 protein exploits the ER-associated degradation machinery to dispose of IRE1. Degradation of IRE1 curbs the unfolded protein response and helps the virus to increase the synthesis of its own proteins.
Sequence evaluation and comparative analysis of novel assays for intact proviral HIV-1 DNA
Authors: Gaebler Christian , Falcinelli Shane D. , Stoffel Elina , Read Jenna , Murtagh Ross , Oliveira Thiago Y. , Ramos Victor , Lorenzi Julio C. C. , Kirchherr Jennifer , James Katherine S. , Allard Brigitte , Baker Caroline , Kuruc JoAnn D. , Caskey Marina , Archin Nancie M. , Siliciano Robert F. , Margolis David M. , Nussenzweig Michel C.
Published: October 2020

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon ART-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The Intact Proviral DNA Assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR), is a lower-throughput assay that uses limiting dilution long distance PCR amplification followed by qPCR and near-full length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size.

Importance

The Intact Proviral DNA Assay (IPDA) and Quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 ART-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.
LED light gradient as a screening tool for light quality responses in model plant species
Authors: Lejeune P. , Fratamico A. , Bouché F. , Fernández S. Huerga , Tocquin P. , Périlleux C.
Published: October 2020

Current developments in light-emitting diodes (LEDs) technologies have opened new perspectives for sustainable and highly efficient indoor cultivation. The introduction of LEDs not only allows a reduction in the production costs on a quantitative level, it also offers opportunities to manipulate and optimise qualitative traits. Indeed, while plants respond strongest to red and blue lights for photosynthesis, the whole light spectrum has an effect on plant shape, development, and chemical composition. In order to evaluate LEDs as an alternative to traditional lighting sources, the species-specific plant responses to distinct wavelengths need to be evaluated under controlled conditions. Here, we tested the possibility to use light composition gradients in combination with semi-automated phenotyping to rapidly explore the phenotypic responses of different species to variations in the light spectrum provided by LED sources. Plants of seven different species (Arabidopsis thaliana, Ocimum basilicum, Solanum lycopersicum, Brachypodium distachyon, Oryza sativa, Euphorbia peplus, Setaria viridis) were grown under standard white fluorescent light for 30 days, then transferred to a Red:Blue gradient for another 30 days and finally returned to white light. In all species, differences in terms of dimension, shape, and color were rapidly observed across the gradient and the overall response was widely species-dependent. The experiment yielded large amounts of imaging-based phenotypic data and we suggest simple data analysis methods to aggregate the results and facilitate comparisons between species. Similar experimental setups will help achieve rapid environmental optimization, screen new crop species and genotypes, or develop new gene discovery strategies.
Genomic heritability of song and condition in wild singing mice
Authors: Burkhard Tracy T. , Matz Mikhail , Phelps Steven M.
Published: October 2020

Courtship displays are dramatic examples of complex behaviors that vary within and among species. Evolutionary explanations for this diversity rely upon genetic variation, yet the heritability of complex phenotypes is seldom investigated in the field. Here, we estimate genomic heritability of advertisement song and body condition in a wild population of singing mice. The heritability of song exhibits a systematic pattern, with high heritability for spectral characteristics linked to vocal morphology, intermediate heritability for rhythmic patterns, and lower but significant heritability for measures of motivation, like song length and rate. Physiological measures of condition, like hormonal markers of adiposity, exhibited intermediate heritability. Among singing mice, song rate and body condition have a strong phenotypic correlation; our estimate suggests a comparable genetic correlation that merits further study. Our results illustrate how advances in genomics and quantitative genetics can be integrated in free-living species to address longstanding challenges in behavior and evolution.
Inhibition of polo-like kinase 1 (PLK1) facilitates reactivation of gamma-herpesviruses in B-cell lymphomas and their elimination
Authors: Biswas Ayan , Fiches Guillaume N. , Zhou Dawei , Wu Zhenyu , Liu Xuefeng , Ma Qin , Zhao Weiqiang , Zhu Jian , Santoso Netty G.
Published: October 2020

Both Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and contribute to certain types of tumors, including B cell lymphomas, especially in immuno-compromised individuals, such as people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes, through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHVde novoinfection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-reactivated lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Lastly, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. Furthermore, we also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV reactivation and cell death of EBV-reactivated lymphoma cells. Additionally, we noticed the PLK1 in B cells is elevated in the context of HIV infection and by HIV Nef protein, which would favor KSHV/EBV latency. Above all, our findings illustrated that PLK1 is a novel host target that can be inhibited to benefit the viral oncolysis to eliminate KSHV/EBV-infected lymphoma cells, particularly for PLWH.
Microglia Stimulate Zebrafish Brain Repair Via a Tumor Necrosis Factor-α-Initiated Inflammatory Cascade
Authors: Kanagaraj Palsamy , Chen Jessica Y. , Skaggs Kaia , Qadeer Yusuf , Connors Meghan , Cutler Noah , Richmond Joshua , Kommidi Vineeth , Poles Allison , Affrunti Danielle , Powell Curtis , Goldman Daniel , Parent Jack M.
Published: October 2020

The adult zebrafish brain, unlike mammals, has a remarkable regenerative capacity. Although inflammation inhibits regeneration in mammals, it is necessary for zebrafish brain repair. Microglia are resident brain immune cells that regulate the inflammatory response. To explore the microglial role in repair, we used liposomal clodronate, colony stimulating factor-1 receptor (csf1r) inhibition to ablate microglia and two genetic mutants that lacks microglia during brain injury. We found that microglial ablation inhibited injury-induced neurogenesis and regeneration. Microglial suppression specifically attenuated cell proliferation at the progenitor cell amplification stage of neurogenesis. Notably, the loss of microglia impaired phospho-stat3 (signal transducer and activator of transcription 3) and ß-catenin signaling by dynamic regulation of tumor necrosis factor-a after injury, and the ectopic activation of stat3 and ß-catenin rescued neurogenesis defects caused by microglial loss. Microglial absence leads to neutrophil accumulation, hindering the resolution of inflammation and macrophages are not sufficient for regeneration. These findings reveal specific roles of microglia and inflammatory signaling during zebrafish telencephalic regeneration that should provide strategies to improve mammalian brain repair.
Efflux mediated chlorpyrifos tolerance inEscherichia coliBL21(DE3)
Authors: Aswathi Aswathi , Pandey Ashok , Sukumaran Rajeev K
Published: October 2020

Bacteria are continually challenged with variety of synthetic chemicals/xenobiotics in their immediate surroundings, including pesticides. Chlorpyrifos is one of the most commonly used organophosphate pesticides in the world. The non-environmental strain ofEscherichia coli,BL21 (DE3) displayed high tolerance to chlorpyrifos but with no/negligible degradation. The intrinsic resistance mechanisms that aid the organism in its high tolerance are probed. Efflux pumps being ubiquitous in nature and capable of conferring resistance against wide variety of xenobiotics were found to be over-expressed in the presence of CP. Also, an efflux pump inhibitor PAβN increased the susceptibility ofE. colito chlorpyrifos due to the intracellular accumulation of CP. The tripartite efflux pump EmrAB-TolC with increased expression in both transcript and protein on CP exposure, might play a major role in CP tolerance. The transcriptional regulators involved in multidrug resistance along with transporters belonging to all the major families conferring antimicrobial resistance were up-regulated. Also up-regulated were the genes involved in phopshonate metabolism and all the genes in the copper or silver export system. The common resistance mechanisms i.e, activation of efflux pumps between CP, antibacterial metals and antibiotics resistance might result in cross-resistance, ultimately increasing the prevalence of multidrug resistant strains, making infections hard to treat.
ATP biphasically modulates LLPS of TDP-43 PLD by specifically binding arginine residues
Authors: Mei Dang , Lim Liangzhong , Kang Jian , Song Jianxing
Published: October 2020

Mysteriously neurons maintain ATP concentrations of ∼3 mM but whether ATP modulates TDP-43 LLPS remains completely unexplored. Here we characterized the effect of ATP on LLPS of TDP-43 PLD and seven mutants by DIC and NMR. The results revealed: 1) ATP induces and subsequently dissolves LLPS of TDP-43 PLD by specifically binding Arg saturated at 1:100. 2) ATP modifies the conformation-specific electrostatic property beyond just imposing screening effect. 3) Reversibility of LLPS of TDP-43 PLD and further exaggeration into aggregation appear to be controlled by a delicate network composed of both attractive and inhibitory interactions. Results together establish that ATP might be a universal but specific regulator for most, if not all, R-containing intrinsically-disordered regions by altering physicochemical properties, conformations, dynamics, LLPS and aggregation. Under physiological conditions, TDP-43 is highly bound with ATP and thus inhibited for LLPS, highlighting a central role of ATP in cell physiology, pathology and aging.
Distinct human gut microbial taxonomic signatures uncovered with different sample processing and microbial cell disruption methods for metaproteomic analysis
Authors: García-Durán Carmen , Martínez López Raquel , Zapico Inés , Pérez Enrique , Romeu Eduardo , Arroyo Javier , Hernáez María Luisa , Pitarch Aida , Monteoliva Lucía , Gil Concha
Published: October 2020

Metaproteomics is as a promising technique for studying the human gut microbiota, because it can reveal the taxonomic profile and also shed light on the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing and microbial cell disruption methods for metaproteomic analyses of human gut microbiota. An unsupervised principal component analysis showed that different methods produced similar human gut microbial taxonomic profiles. An unsupervised two-way hierarchical clustering analysis identified the microbial taxonomic signatures associated with each method. Proteobacteria and Bacteroidetes identification was favored by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads. Ascomycota identification was enhanced by using large-sized glass beads during sample processing for stool dispersion. Euryarchaeota identification was improved with a combination of small and medium-sized glass beads for cell disruption. Assessments of the relative abundance of Firmicutes, Actinobacteria and Spirochaetes improved when ultrasonication was performed before cell disruption with glass beads. The latter method also increased the overall number of identified proteins. Taxonomic and protein functional analyses of metaproteomic data derived from stool samples from six healthy individuals showed common taxonomic profiles. We also detected certain proteins involved in microbial functions relevant to the host and related mostly to particular taxa, such as B12 biosynthesis and short chain fatty acid production carried out mainly by members in thePrevotellagenus and the Firmicutes phylum, respectively. Finally, in this metaproteomic study we identified several human proteins, mostly related to the anti-microbial response, which could contribute to determining the beneficial and detrimental relationships between gut microbiota and human cells in particular human diseases.
Effects of High Magnetic Fields on Diffusion of Biologically Active Molecules
Authors: Zablotskii Vitalii , Polyakova Tatyana , Dejneka Alexandr
Published: October 2020

The diffusion of biologically active molecules is a ubiquitous process, controlling many mechanisms and the characteristic time scales for pivotal processes in living cells. Here, we show how a high static magnetic field (MF) affects the diffusion of paramagnetic and diamagnetic species, including oxygen, hemoglobin, ROS and drugs. We derive and solve the equation describing diffusion of such biologically active molecules in the presence of a MF as well as reveal the underlying mechanism of the MF effect on diffusion. We find that a high MF accelerates diffusion of diamagnetic species while slowing the diffusion of paramagnetic molecules in cell cytoplasm. When applied to oxygen and hemoglobin diffusion in red blood cells, our results suggest that a MF may significantly alter the gas exchange in an erythrocyte and cause swelling. Our prediction that the diffusion rate and characteristic time can be controlled by a MF opens new avenues for experimental studies foreseeing numerous biomedical applications.
Psychiatric Genomics Research During the COVID-19 Pandemic: A Survey of Psychiatric Genomics Consortium Researchers
Authors: Guintivano Jerry , Dick Danielle , Bulik Cynthia M
Published: October 2020

Between April 20, 2020 and June 19, 2020 we conducted a survey of the membership of the Psychiatric Genomics Consortium (PGC) to explore the impact of COVID-19 on their research and academic careers. A total of 123 individuals responded representing academic ranks from trainee to full professor, tenured and fixed-term appointments, and all genders. The survey included both quantitative and free text responses. Results revealed considerable concern about the impact of COVID-19 on research with the greatest concern reported by individuals in non-permanent positions and female researchers. Concerns about the availability of funding and the impact of the pandemic on career progression were commonly reported by early career researchers. We provide recommendations for institutions, organizations such as the PGC, as well as individual senior investigators to ensure that the futures of early career investigators, especially those underrepresented in academic medicine such as women and underrepresented minorities, are not disproportionately disadvantaged by the COVID-19 pandemic.
Bi-directional electron transfer between H2and NADPH mitigates the response to light fluctuations in green algae
Authors: Milrad Yuval , Schweitzer Shira , Feldman Yael , Yacoby Iftach
Published: October 2020

The metabolism of green algae has been the focus of much research over the last century. These photosynthetic organisms can thrive under various conditions and adapt quickly to changing environments by concomitant usage of several metabolic apparatuses. The main electron coordinator in their chloroplasts, nicotinamide adenine dinucleotide phosphate (NADPH), participates in many enzymatic activities and is also responsible for interorganelle communication. Under anaerobic conditions, green algae also accumulate molecular hydrogen (H2), a promising alternative for fossil fuels. However, in order to scale-up its accumulation, a firm understanding of its integration in the photosynthetic apparatus is still lacking. While it is generally accepted that NADPH metabolism correlates to H2accumulation, the mechanism of this collaboration is still vague and rely on indirect measurements. Here, we investigated this connection using simultaneous measurements of both dissolved gases concentration, NADPH fluorescence and electrochromic shifts at 520-546 nm. Our results indicate that energy transfer between H2and NADPH is bi-directional and crucial for the maintenance of redox balance under light fluctuations. At light onset, NADPH consumption is initially eventuated in H2evolution, which initiate the photosynthetic electron flow. Later on, as illumination continues the majority of NADPH is recycled by Nda2 rather than consumed by terminal sinks such as CBB cycle and H2production. Dark onset triggers re-assimilation of H2, which produces NADPH and so, enables initiation of dark fermentative metabolism.

One sentence summary

Energy transfer between H2and NADPH is bi-directional and crucial for the maintenance of redox balance under light fluctuations.
Multi-Atlas Image Soft Segmentation via Computation of the Expected Label Value
Authors: Aganj Iman , Fischl Bruce
Published: October 2020

The use of multiple atlases is common in medical image segmentation. This typically requires deformable registration of the atlases (or the average atlas) to the new image, which is computationally expensive and susceptible to entrapment in local optima. We propose to instead consider the probability of all possible atlas-to-image transformations and compute theexpected label value (ELV), thereby not relying merely on the transformation deemed “optimal” by the registration method. Moreover, we do so without actually performing deformable registration, thus avoiding the associated computational costs. We evaluate our ELV computation approach by applying it to brain, liver, and pancreas segmentation on datasets of magnetic resonance and computed tomography images.
Utilizing Q-Learning to Generate 3D Vascular Networks for Bioprinting Bone
Authors: Sedigh Ashkan , Tulipan Jacob E. , Rivlin Michael R. , Tomlinson Ryan E.
Published: October 2020

Bioprinting is an emerging tissue engineering method used to generate cell-laden scaffolds with high spatial resolution. Bioprinted vascularized bone grafts are a potential application of this technology that would meet a critical clinical need, since current approaches to volumetric bone repair have significant limitations. However, generation of vascular networks suitable for bioprinting is challenging. Here, we propose a novel Q-learning approach to quickly generate 3D vascular networks within patient-specific bone geometry that are optimized for bioprinting. First, the inlet and outlet locations are specified and the scenario is modeled using a grid world for initial agent training. Next, the path planned in the grid world environment is converted to a Bezier curve, which is then used to generate the final 3D vascularized bone model. The vessels generated using this procedure have minimal tortuosity, which increases the likelihood of successful bioprinting. Furthermore, the ability to specify inlet and outlet position is necessary for both surgical feasibility as well as generation of more complex vascular networks. In total, this study demonstrates the reliability of our reinforcement learning method for automated generation of 3D vascular networks within patient-specific geometry that can be used for bioprinting vascularized bone grafts.
Protein diffusion on membrane domes, tubes and pearling structures
Authors: Molina R. Rojas , Liese S. , Carlson A.
Published: October 2020

Diffusion is a fundamental mechanism for protein distribution in cell membranes. These membranes often exhibit complex shapes, which range from shallow domes to elongated tubular or pearl-like structures. Shape complexity of the membrane influences the diffusive spreading of proteins and molecules. Despite the importance membrane geometry plays in these diffusive processes, it is challenging to establish the dependence between diffusion and membrane morphology. We solve the diffusion equation numerically on various curved shapes representative for experimentally observed membrane shapes. Our results show that membrane necks become diffusion barriers. We determine the diffusive half time,i.e., the time that is required to reduce the amount of proteins in the budded region by one half and find a quadratic relation between the diffusive half time and the averaged mean curvature of the membrane shape. Our findings thus help to estimate the characteristic diffusive time scale based on the simple measure for membrane morphology.

Significance statement

Diffusion is an integral process for distributing proteins throughout biological membranes. These membranes can have complex shapes and structures, often featuring elongated shapes such as tubes and like a necklace of pearls. The diffusion process on these shapes is significantly different from the well studied planar substrate. We use numerical simulations to understand how the characteristic diffusion time is a function of membrane shape, where we find the diffusion of proteins on strongly curved shapes is significantly slower than on planar membranes. Our results provide a simple relationship to estimate the characteristic diffusion time of proteins on membranes based on its mean and Gaussian curvature.
Cyanobacterial NOS expression improves nitrogen use efficiency, nitrogen-deficiency tolerance and yield in Arabidopsis
Authors: Fiorella Del Castello , Noelia Foresi , Andrés Nejamkin , Christian Lindermayr , Franz Buegger , Lorenzo Lamattina , Natalia Correa-Aragunde
Published: October 2020

Developing strategies to improve nitrogen (N) use efficiency (NUE) in plants is a challenge to reduce environmental problems linked to over-fertilization. The nitric oxide synthase (NOS) enzyme from the cyanobacteriaSynechococcusPCC 7335 (SyNOS) has been recently identified and characterized. SyNOS catalyzes the conversion of arginine to citrulline and nitric oxide (NO), and then 70% of the produced NO is rapidly oxidized to nitrate by an unusual globin domain in its 5'-terminus. In this study, we assessed whether SyNOS expression in plants affects N metabolism improving NUE and yield. Our results showed that transgenic Arabidopsis plants had higher primary shoot length and shoot branching when grown in N-deficient conditions and higher seed production in N-sufficient and -deficient conditions. Moreover, transgenic plants showed significantly increased NUE in both N conditions. No differences were observed in N uptake for SyNOS lines. However, SyNOS lines presented an increase in N assimilation/remobilization under low N conditions. In addition, SyNOS lines had greater N-deficiency tolerance compared to wt plants. Our results support that SyNOS expression generates a positive effect on N metabolism and seed production in Arabidopsis, and it might be envisaged as a strategy to improve productivity in crops under adverse N environments.
The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein
Authors: do Nascimento Larissa Melo , Egler Franziska , Arnold Katharina , Papavisiliou Nina , Clayton Christine , Erben Esteban
Published: October 2020

Salivarian trypanosomes cause human sleeping sickness and economically important livestock diseases. The “bloodstream forms”, which replicate extracellularly in the blood and tissue fluids of mammals, are coated by a monolayer of Variant Surface Glycoprotein (VSG). Switching of the expressedVSGgene is central to parasite pathogenicity because it enables the parasites to evade adaptive immunity via antigenic variation. Adequate levels of VSG expression - 10% of total protein and 7% of mRNA - are attained through very active RNA polymerase I transcription, efficient mRNA processing (transsplicing of a capped leader and polyadenylation), and high mRNA stability. We here show howVSGmRNA stability is maintained. Purification of theVSGmRNA with associated proteins specifically selected CFB2, an F-box mRNA-binding protein that lacks known RNA-binding domains. CFB2 binds to a stabilizing complex (MKT1-PBP1-XAC1-LSM12) that recruits poly(A) binding protein and a specialized cap-binding translation initiation complex, EIF4E6-EIF4G5. The interaction of CFB2 with MKT1 is essential for CFB2’s expression-promoting activity, while the F-box auto-regulates CFB2 abundance via interaction with SKP1, a component of the ubiquitination machinery. The results of reporter experiments indicate that CFB2 acts via conserved sequences in theVSGmRNA 3’-untranslated region. Depletion of CFB2 leads to highly specific loss ofVSGmRNA. VSG expression is essential not only for antigenic variation but also for trypanosome cell division. Correspondingly, depletion of CFB2 causes cell cycle arrest, dramatic morphological abnormalities and trypanosome death.
Silencing of oncogenic KRAS by mutant-selective small interfering RNA
Authors: Papke Bjoern , Azam Salma H. , Feng Anne Y. , Van Swearingen Amanda E. D. , Gutierrez-Ford Christina , Pallan Pradeep S. , Egli Martin , Cox Adrienne D. , Der Channing J. , Pecot Chad V.
Published: October 2020

Oncogenic mutations in theKRASgene are well-established drivers of cancer. While the recently developed KRASG12Cinhibitors offer a targeted KRAS therapy and have shown success in the clinic, KRASG12Crepresents only 11% of all KRAS mutations. Current therapeutic approaches for all other KRAS mutations are both indirect and non-mutant-selective, largely focusing on inhibition of downstream KRAS effectors such as MAP kinases. Inhibition of KRAS downstream signaling results in a system-wide down-modulation of the respective targets, raising concerns about systemic cell toxicity. Here, we describe a custom short interfering RNA (siRNA) oligonucleotide (EFTX-D1) designed to preferentially bind mRNA of the most commonly occurring KRAS missense mutations in codons 12 and 13. We determined that EFTX-D1 preferentially reduced the mutant KRAS sequence versus wild-type at the levels of both transcription and translation, and reversed oncogenic KRAS-induced morphologic and growth transformation. Furthermore, EFTX-D1 significantly impaired the proliferation of several KRAS mutant cancer cell lines in 2-D as well as 3-D assays. Taken together, our data indicate a novel use of RNA interference (RNAi) to target oncogenic KRAS-driven cancers specifically.
Discovery of novel putative tumor suppressors from CRISPR screens reveals rewired lipid metabolism in AML cells
Authors: Lenoir W. Frank , Morgado Micaela , DeWeirdt Peter C , McLaughlin Megan , Griffith Audrey L , Sangree Annabel K , Feeley Marissa N , Anvar Nazanin Esmaeili , Kim Eiru , Bertolet Lori L , Colic Medina , Dede Merve , Doench John G , Hart Traver
Published: October 2020

CRISPR knockout screens in hundreds of cancer cell lines have revealed a substantial number of context-specific essential genes that, when associated with a biomarker such as lineage or oncogenic mutation, offer candidate tumor-specific vulnerabilities for targeted therapies or novel drug development. Data-driven analysis of knockout fitness screens also yields many other functionally coherent modules that show emergent essentiality or, in rarer cases, the opposite phenotype of faster proliferation. We develop a systematic approach to classify these suppressors of proliferation, which are highly enriched for tumor suppressor genes, and define a network of 145 genes in 22 discrete modules. One surprising module contains several elements of the glycerolipid biosynthesis pathway and operates exclusively in a subset of AML lines, which we call Fatty Acid Synthesis/Tumor Suppressor (FASTS) cells. The proliferation suppressor activity of genes involved in the synthesis of saturated fatty acids, coupled with a more severe fitness phenotype for the desaturation pathway, suggests that these cells operate at the limit of their carrying capacity for saturated fatty acids, which we confirmed biochemically. Overexpression of genes in this module is associated with a survival advantage in an age-matched cohort of AML patients, suggesting the gene cluster driving anin vitrophenotype may be associated with a novel, clinically relevant subtype.
Social isolation impairs the prefrontal-nucleus accumbens circuit subserving social recognition in mice
Authors: Park Gaeun , Ryu Changhyeon , Kim Soobin , Lee Yong-Seok , Kim Sang Jeong
Published: October 2020

The medial prefrontal cortex (mPFC) plays important roles in social behaviors, but it is not clear how early social experiences affect the mPFC and its subcortical circuit. We report that mice singly housed for 8 weeks immediately after weaning (SH mice) show a deficit in social recognition, even after 4 weeks of re-socialization. In SH mice, prefrontal infralimbic (IL) neurons projecting to the shell region of nucleus accumbens (NAcSh) showed decreased excitability compared to normally group housed (GH) mice. Furthermore, NAcSh-projecting IL neurons were activated when the mice encountered a familiar conspecific, which was not shown in SH mice. Chemogenetic inhibition of NAcSh-projecting IL neurons in normal mice selectively impaired social recognition without affecting social interaction, whereas activation of these neurons reversed social recognition deficit in SH mice. Therefore, mPFC IL-NAcSh projection is a novel brain circuit affected by early social experience; its activation is required for the social recognition.
Cytonemes with complex geometries and composition extend into invaginations of target cells
Authors: Wood Brent M. , Baena Valentina , Huang Hai , Terasaki Mark , Kornberg Thomas B.
Published: October 2020

Cytonemes are specialized filopodia that mediate paracrine signaling in Drosophila and other animals. Studies using fluorescence confocal microscopy (CM) established their general paths, cell targets, and essential roles in signaling. To investigate details unresolvable by CM, we used high pressure freezing and electron microscopy to visualize cytoneme structures, paths, contents, and contacts. We observed cytonemes previously seen by CM in the Drosophila wing imaginal disc system, including in disc, tracheal air sac primordium (ASP), and myoblast cytonemes, and identified cytonemes extending into invaginations of target cells and cytonemes connecting ASP cells and connecting myoblasts. Diameters of cytoneme shafts vary between wide (206 ± 51.8 nm) and thin (55.9 ±16.2 nm) segments at regular intervals. Actin, ribosomes, and membranous compartments are present throughout; rough endoplasmic reticulum and mitochondria are in wider proximal sections. These results reveal novel structural features of filopodia and provide a basis for understanding cytoneme cell biology and function.

Cytoneme signaling filopodia of Drosophila cells have regions defined by oscillating diameters, and contents that include ER, mitochondria and ribosomes, and form contacts along invaginations of target cell membranes.
Ultrasound-mediated mechanical forces selectively kill tumor cells
Authors: Tijore Ajay , Margadant Felix , Yao Mingxi , Hariharan Anushya , Chew Claire Alexandra Zhen , Powell Simon , Bonney Glenn Kunnath , Sheetz Michael
Published: October 2020

Ultrasound has been used to target tumors either through local heating or local nanobubbles but these methods damage surrounding normal cells in the target area. Recent studies show that tumor cells are susceptible to mechanical stresses and undergo calcium-dependent apoptosis under conditions that promote normal cell growth. Here we report that low-frequency ultrasound causes apoptosis of tumor cells by activating a calpain-dependent mitochondrial pathway that depends upon calcium entry through the mechanosensitive Piezo1 channels. This is a general property of all tumor cell lines tested so far irrespective of tissue origin. In animals, ultrasound irradiation causes tumor killing in the chick chorioallantoic membrane (CAM) model with relatively little damage to the chick embryos. Further, patient-derived pancreatic tumor organoids are killed by ultrasound treatment. Because low-level ultrasound causes apoptosis of tumor cells from many different tissues in different microenvironments, it may offer a safe non-invasive approach to augment tumor treatments.
Cancer methylomes characterization enabled by Rocker-meth
Authors: Benelli Matteo , Franceschini Gian Marco , Magi Alberto , Romagnoli Dario , Biagioni Chiara , Migliaccio Ilenia , Malorni Luca , Di Leo Angelo , Demichelis Francesca
Published: October 2020

Differentially DNA methylated regions (DMRs) inform on the role of epigenetic changes in cancer. We present Rocker-meth, a computational method exploiting a heterogeneous hidden Markov model to detect DMRs across multiple experimental platforms. Its application to more than 6,000 methylation profiles across 14 tumor types provides a comprehensive catalog of tumor type-specific and shared DMRs, also amenable to single-cell DNA-methylation data. In depth integrative analysis including orthogonal omics shows the enhanced ability of Rocker-meth in recapitulating known associations, further uncovering the pan-cancer relationship between DNA hypermethylation and transcription factor deregulation depending on the baseline chromatin state.
E-cadherin is a structuring component of invadopodia in pancreatic cancer
Authors: Dobric Aurélie , Germain Sébastien , Silvy Françoise , Bonier Rénaté , Audebert Stéphane , Camoin Luc , Dusetti Nelson , Soubeyran Philippe , Iovanna Juan , Rigot Véronique , André Frédéric
Published: October 2020

Graphical abstract

Background

The appearance of hybrid epithelial-mesenchymal (E/M) cells expressing E-cadherin is favourable for the establishment of pro-invasive function. However, the molecular mechanism and potential roles of E-cadherin in cancer cell invasion stay unexplored.

Methods

We used models of E/M hybrid cell lines, tissues sections and patient-derived xenografts from a multi-center clinical trial. E-cadherin involvement in invadopodia formation was assessed using a gelatin-FITC degradation assay. Mechanistic studies were performed by using proteomic analysis, siRNA strategy and proximity ligation assay.

Results

We showed that E-cadherin is a critical component of invadopodia. This unexpected localization results from a synergistic trafficking of E-cadherin and MT1-MMP through Rab vesicle-dependent pathway. Modulation of E-cadherin expression or activation impacted invadopodia formation.Moreover, colocalization of E-cadherin and Actin in ―ring structures‖as precursor of invadopodia reveals that E-cadherin is required for invadopodia structuration.

Conclusion

E-cadherin, initially localized in the adherens junctions could be recycled to nascent invadopodia where it will interact with several components such as Arp2/3, Cortactin or MT1-MMP.The trans-adhesive properties of E-cadherin are therefore essential for structuringinvadopodia.
Origin and microenvironment contribute to the sexually dimorphic phenotype and function of peritoneal macrophages
Authors: Bain Calum C. , Gibson Douglas A. , Steers Nicholas , Boufea Katarina , Louwe Pieter A. , Docherty Catherine , Huici Victor , Gentek Rebecca , Magalhaes-Pinto Marlene , Bajenoff Marc , Benezech Cecile , Dockrell David , Saunders Philippa TK , Batada Nizar , Jenkins Stephen J
Published: October 2020

Macrophages reside in the body cavities where they maintain serosal homeostasis and provide immune surveillance. Peritoneal macrophages are implicated in the aetiology of pathologies including peritonitis, endometriosis and metastatic cancer thus understanding the factors that govern their behaviour is vital. Using a combination of fate mapping techniques, we have investigated the impact of sex and age on murine peritoneal macrophage differentiation, turnover and function. We demonstrate that the sexually dimorphic replenishment of peritoneal macrophages from the bone marrow, which is high in males and very low in females, is driven by changes in the local microenvironment that arise upon sexual maturation. Population and single cell RNAseq revealed striking dimorphisms in gene expression between male and female peritoneal macrophages that was in part explained by differences in composition of these populations. By estimating the time of residency of different subsets within the cavity and assessing development of dimorphisms with age and in monocytopenic Ccr2−/− mice, we demonstrate that key sex-dependent features of peritoneal macrophages are a function of the differential rate of replenishment from the bone marrow while others are reliant on local microenvironment signals. Importantly, we demonstrate that the dimorphic turnover of peritoneal macrophages contributes to differences in the ability to protect against pneumococcal peritonitis between the sexes. These data highlight the importance of considering both sex and age in susceptibility to inflammatory and infectious disease.
Improving the soft X-ray reflectivity of Cr/Ti multilayers by co-deposition of B4C
Authors: Zhu Jingtao , Zhang Jiayi , Li Haochuan , Tu Yuchun , Chen Jinwen , Wang Hongchang , Dhesi Sarnjeet S. , Cui Mingqi , Zhu Jie , Jonnard Philippe
Published: October 2020

The `water window', covering 2.4–4.4 nm, is an important wavelength range particularly essential to biology research. Cr/Ti multilayers are one of the promising reflecting elements in this region because the near-normal-incidence reflectivity is theoretically as high as 64% at 2.73 nm. However, due to multilayer imperfections, the reported reflectivity is lower than 3% for near-normal incidence. Here, B and C were intentionally incorporated into ultra-thin Cr/Ti soft X-ray multilayers by co-deposition of B4C at the interfaces. The effect on the multilayer structure and composition has been investigated using X-ray reflectometry, X-ray photoelectron spectroscopy, and cross-section electron microscopy. It is shown that B and C are mainly bonded to Ti sites, forming a nonstoichiometric TiB
x
C
y
composition, which hinders the interface diffusion, supresses the crystallization of the Cr/Ti multilayer and dramatically improves the interface quality of Cr/TiB
x
C
y
multilayers. As a result, the near-normal-incidence reflectivity of soft X-rays increases from 4.48% to 15.75% at a wavelength of 2.73 nm.
Practical design and performance of a new merged APPLE-Knot undulator
Authors: Zhang Fayuan , Sun Zhipeng , Qiao Yuxi , Qiao Shan
Published: October 2020

Constructing vacuum-ultraviolet beamlines at synchrotron radiation facilities with giga-electron volt storage ring results in serious heat load on the beamlines which can reduce their performance. To solve this problem, an APPLE-Knot undulator with eight magnet rows has been built at the Shanghai Synchrotron Radiation Facility and has achieved very good performance. However, its performance in vertical polarization mode is imperfect. Here, a new configuration of a magnet-merged APPLE-Knot undulator that has achieved a better performance is reported.
Crystal structure of diammonium bis[tris(oxamide dioxime-κ2N,N′)nickel(II)] bis[tris(oxalato-κ2O,O′)chromate(III)] 6.76-hydrate
Authors: Mbiangué Yves Alain , Bijvédé Alfred , Kenfack Tsobnang Patrice , Wenger Emmanuel , Lecomte Claude
Published: October 2020

The asymmetric unit of the title compound, (NH4)2[Ni(C2H6N4O2)3]2[Cr(C2O4)3]2·6.76H2O, comprises two NH4+cations, two [Ni(C2H6N4O2)3]2+cations and two [Cr(C2O4)3]3–anions, as well as eight water molecules of crystallization of which only one is fully occupied. In the cationic and anionic complexes, the central atoms (NiIIand CrIII) are each surrounded by three bidentate ligands (N-chelating oxamide dioxime and O-chelating oxalate, respectively), resulting in distorted octahedral coordination spheres. In the crystal, O—H...O hydrogen bonds between the oxamide dioxime ligands as donor groups and the oxalate ligands as acceptor groups alternately connect the cationic and anionic complexes into infinite pillars extending parallel to [100]. Moreover, N—H...O hydrogen bonds between the same ligands connect neighboring pillars, thus delineating channels that accommodate the charge-balancing NH4+cations as well as the water molecules of crystallization. Although the H atoms could not be localized for these two species, the corresponding N...O and O...O distances indicate hydrogen bonds of medium strength.
4-(Dimethylamino)benzohydrazide
Authors: Kelley Steven P. , Mossine Valeri V. , Mawhinney Thomas P.
Published: October 2020

The title compound, C9H13N3O, crystallizes in the monoclinic space group C2/c and all non-hydrogen atoms are within 0.1 Å of the molecular mean plane. In the crystal, the hydrogen-bonding pattern results in [001] chains built up from fused R
2
2(6) and R
2
2(10) rings; the former consists of N—H...N bonds and the latter N—H...O bonds. Electrostatic and dispersion forces are major contributors to the lattice energy, which was estimated by DFT calculations to be −215.7 kJ mol−1.