bluuebunny/crossref_metadata_2025 huggingface dataset:
Trisomy 21 induces pericentrosomal crowding disrupting early stages of primary ciliogenesis and mouse cerebellar development
Authors: Jewett Cayla E , McCurdy Bailey L , O’Toole Eileen T , Given Katherine S , Lin Carrie H , Olsen Valerie , Martin Whitney , Reinholdt Laura G , Espinosa Joaquin M , Sullivan Kelly D , Macklin Wendy B , Prekeris Rytis , Pearson Chad G
Published: November 2021

Primary cilia are signaling organelles essential for development and homeostasis. Loss of primary cilia is lethal, and decreased or defective cilia cause multisystemic conditions called ciliopathies. Down syndrome shares clinical overlap with ciliopathies. We previously showed that trisomy 21 diminishes primary cilia formation and function due to elevated Pericentrin, a centrosome protein encoded on chromosome 21. Pericentrin is mislocalized, creating aggregates that disrupt pericentrosomal trafficking and microtubule organization. Here, we examine the cilia-related molecules and pathways disrupted in trisomy 21 and their in vivo phenotypic relevance. Utilizing ciliogenesis time course experiments, we reveal how Pericentrin, microtubule networks, and components of ciliary vesicles are reorganized for ciliogenesis in euploid cells. Early in ciliogenesis, chromosome 21 polyploidy results in elevated Pericentrin and microtubule networks away from the centrosome that ensnare MyosinVA and EHD1, blocking mother centriole uncapping that is essential for ciliogenesis. Ciliated trisomy 21 cells have persistent trafficking defects that reduce transition zone protein localization, which is critical for Sonic hedgehog signaling. Sonic hedgehog signaling is decreased and anticorrelates with Pericentrin levels in trisomy 21 primary mouse embryonic fibroblasts. Finally, we observe decreased ciliation in vivo. A mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers. Our work reveals that elevated Pericentrin in trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding results in signaling defects consistent with the neurological deficits found in individuals with Down syndrome.
CORSID enables de novo identification of transcription regulatory sequences and genes in coronaviruses
Authors: Zhang Chuanyi , Sashittal Palash , El-Kebir Mohammed
Published: November 2021

Genes in coronaviruses are preceded by transcription regulatory sequences (TRSs), which play a critical role in gene expression mediated by the viral RNA-dependent RNA-polymerase via the process of discontinuous transcription. In addition to being crucial for our understanding of the regulation and expression of coronavirus genes, we demonstrate for the first time how TRSs can be leveraged to identify gene locations in the coronavirus genome. To that end, we formulate the TRS AND Gene Identification (TRS-Gene-ID) problem of simultaneously identifying TRS sites and gene locations in unannotated coronavirus genomes. We introduce CORSID (CORe Sequence IDentifier), a computational tool to solve this problem. We also present CORSID-A, which solves a constrained version of the TRS-Gene-ID problem, the TRS Identification (TRS-ID) problem, identifying TRS sites in a coronavirus genome with specified gene annotations. We show that CORSID-A outperforms existing motif-based methods in identifying TRS sites in coronaviruses and that CORSID outperforms state-of-the-art gene finding methods in finding genes in coronavirus genomes. We demonstrate that CORSID enables de novo identification of TRS sites and genes in previously unannotated coronaviruses. CORSID is the first method to perform accurate and simultaneous identification of TRS sites and genes in coronavirus genomes without the use of any prior information.
Centrosomal Enrichment and Proteasomal Degradation of SYS-1/β-catenin Requires the Microtubule Motor Dynein
Authors: Thompson Joshua W. , Valdes Michel Maria F. , Phillips Bryan T.
Published: November 2021

The C. elegans Wnt/β-catenin Asymmetry (WβA) pathway utilizes asymmetric regulation of SYS- 1/β-catenin and POP-1/TCF coactivators. WβA differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased towards their appropriate cell fate. Despite the induction of asymmetry, β-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Due to the mitosis-specific localization of SYS-1 to centrosomes and enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNAi-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe depletion of microtubules by nocodazole treatment or RNAi of dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose a model whereby retrograde microtubule-mediated trafficking enables SYS-1 enrichment at centrosomes, enhancing its eventual proteasomal degradation. These studies support the link between centrosomal localization and enhancement of proteasomal degradation, particularly for proteins not generally considered ‘centrosomal’.
A large-scale genome and transcriptome sequencing analysis reveals the mutation landscapes induced by high-activity adenine base editors in plants
Authors: Li Shaofang , Liu Lang , Sun Wenxian , Zhou Xueping , Zhou Huanbin
Published: November 2021

Background

The high-activity adenine base editors (ABEs), engineered with the recently-developed tRNA adenosine deaminases (TadA8e and TadA9), show robust base editing activity but raise concerns about off-target effects.

Results

In this study, we performed a comprehensive evaluation of ABE8e- and ABE9-induced DNA and RNA mutations in Oryza sativa. Whole-genome sequencing analysis of plants transformed with four ABEs, including SpCas9n-TadA8e, SpCas9n-TadA9, SpCas9n-NG-TadA8e, and SpCas9n-NG-TadA9, revealed that ABEs harboring TadA9 lead to a higher number of off-target A-to-G (A>G) single-nucleotide variants (SNVs), and that those harboring the CRISPR/SpCas9n-NG lead to a higher total number of off-target SNVs in the rice genome. An analysis of the T-DNAs carrying the ABEs indicated that the on-target mutations could be introduced before and/or after T-DNA integration into plant genomes, with more off-target A>G SNVs forming after the ABEs had integrated into the plant genome. Furthermore, we detected off-target A>G RNA mutations in plants with high expression of ABEs but not in plants with low expression of ABEs. The off-target A>G RNA mutations tended to cluster, while off-target A>G DNA mutations rarely clustered.

Conclusion

Our findings that Cas proteins, TadA variants, temporal expression of ABEs, and expression levels of ABEs contribute to ABE specificity in rice provide insight into the specificity of ABEs and suggest alternative ways to increase ABE specificity besides engineering TadA variants.
Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological and pathological disease in a Syrian hamster model of COVID-19
Authors: Esparza Thomas J. , Chen Yaozong , Martin Negin P. , Bielefeldt-Ohmann Helle , Bowen Richard A. , Tolbert William D. , Pazgier Marzena , Brody David L.
Published: November 2021

There remains an unmet need for globally deployable, low-cost therapeutics for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Previously, we reported on the isolation and in vitro characterization of a potent single-domain nanobody, NIH-CoVnb-112, specific for the receptor binding domain (RBD) of SARS-CoV-2. Here, we report on the molecular basis for the observed broad in vitro neutralization capability of NIH-CoVnb-112 against variant SARS-CoV-2 pseudoviruses, including the currently dominant Delta variant. The structure of NIH-CoVnb-112 bound to SARS-CoV-2 RBD reveals a large contact surface area overlapping the angiotensin converting enzyme 2 (ACE2) binding site, which is largely unencumbered by the common RBD mutations. In an in vivo pilot study, we demonstrate effective reductions in weight loss, viral burden, and lung pathology in a Syrian hamster model of COVID-19 following nebulized delivery of NIH-CoVnb-112. These findings support the further development of NIH-CoVnb-112 as a potential adjunct preventative therapeutic for the treatment of SARS-CoV-2 infection.
The Drosophila RNA binding protein Nab2 patterns dendritic arbors and axons via the planar cell polarity pathway
Authors: Corgiat Edwin B. , List Sara M. , Rounds J. Christopher , Yu Dehong , Chen Ping , Corbett Anita H. , Moberg Kenneth H.
Published: November 2021

RNA binding proteins support neurodevelopment by modulating numerous steps in post-transcriptional regulation, including splicing, export, translation, and turnover of mRNAs that can traffic into axons and dendrites. One such RBP is ZC3H14, which is lost in an inherited intellectual disability. The Drosophila melanogaster ZC3H14 ortholog, Nab2, localizes to neuronal nuclei and cytoplasmic ribonucleoprotein granules, and is required for olfactory memory and proper axon projection into brain mushroom bodies. Nab2 can act as a translational repressor in conjunction with the Fragile-X mental retardation protein homolog Fmr1 and shares target RNAs with the Fmr1-interacting RBP Ataxin-2. However, neuronal signaling pathways regulated by Nab2 and their potential roles outside of mushroom body axons remain undefined. Here, we demonstrate that Nab2 restricts branching and projection of larval sensory dendrites via the planar cell polarity pathway, and that this link may provide a conserved mechanism through which Nab2/ZC3H14 modulates projection of both axons and dendrites. Planar cell polarity proteins are enriched in a Nab2-regulated brain proteomic dataset. Complementary genetic data indicate that Nab2 guides dendrite and axon growth through the planar-cell-polarity pathway. Analysis of the core planar cell polarity protein Vang, which is depleted in the Nab2 mutant whole-brain proteome, uncovers selective and dramatic loss of Vang within axon/dendrite-enriched brain neuropil relative to brain regions containing cell bodies. Collectively, these data demonstrate that Nab2 regulates dendritic arbors and axon projection by a planar-cell-polarity-linked mechanism and identify Nab2 as required for accumulation of the core planar cell polarity factor Vang in distal neuronal projections.
Crystal structure of Arabidopsis thaliana HPPK/DHPS, a bifunctional enzyme and target of the herbicide asulam
Authors: Vadlamani Grishma , Sukhoverkov Kirill V. , Haywood Joel , Breese Karen J. , Fisher Mark F. , Stubbs Keith A. , Bond Charles S. , Mylne Joshua S.
Published: November 2021

Herbicides are vital for modern agriculture, but their utility is threatened by genetic or metabolic resistance in weeds as well as heightened regulatory scrutiny. Of the known herbicide modes of action, 6-hydroxymethyl-7,8-dihydropterin synthase (DHPS) which is involved in folate biosynthesis, is targeted by just one commercial herbicide, asulam. A mimic of the substrate para-aminobenzoic acid, asulam is chemically similar to sulfonamide antibiotics – and while still in widespread use, asulam has faced regulatory scrutiny. With an entire mode of action represented by just one commercial agrochemical, we sought to improve the understanding of its plant target. Here we solve a 2.6 Å resolution crystal structure for Arabidopsis thaliana DHPS that is conjoined to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and reveal a strong structural conservation with bacterial counterparts at the sulfonamide-binding pocket of DHPS. We demonstrate asulam and the antibiotics sulfacetamide and sulfamethoxazole have herbicidal as well as antibacterial activity and explore the structural basis of their potency by modelling these compounds in mitochondrial HPPK/DHPS. Our findings suggest limited opportunity for the rational design of plant selectivity from asulam and that pharmacokinetic or delivery differences between plants and microbes might be the best approaches to safeguard this mode of action.
Identification of Escherichia coli Genes Essential for High-Level, Clinically Relevant, Resistance to Antibiotics
Authors: Reyes-Fernández Esmeralda Z. , Cudkowicz Noemie Alon , Steiner-Mordoch Sonia , Schuldiner Shimon
Published: November 2021

Antibiotic resistance is one of the biggest public health challenges of our time. Here we present a novel approach to recognizing molecular mechanisms essential for maintaining high-level clinically relevant antibiotic resistance. To identify essential genes in this context, we subjected Escherichia coli EV18, a strain highly resistant to quinolones, to random transposon mutagenesis. This strain’s unique advantage is that the screen is performed at very high concentrations of the antibiotic, non-permissive for most strains. The transposon’s insertion affected the transcription of five genes required for the maintenance of resistance in EV18. Three of these genes (YihO, YhdP, and WaaY) have not been previously identified as essential for high-level antimicrobial resistance (AMR). Our work provides a new perspective to identify and characterize novel players crucial for maintaining AMR in E. coli.
Physical activity and health: Findings from Finnish monozygotic twin pairs discordant for physical activity
Authors: Kujala Urho M. , Leskinen Tuija , Rottensteiner Mirva , Aaltonen Sari , Ala-Korpela Mika , Waller Katja , Kaprio Jaakko
Published: November 2021

Genetic and early environmental differences including early health habits associate with future health. To provide insight on the causal nature of these associations, monozygotic (MZ) twin pairs discordant for health habits provide an interesting natural experiment. Twin pairs discordant for leisure-time physical activity (LTPA) in early adult life are thus a powerful study design to investigate the associations between long-term LTPA and indicators of health and wellbeing. We have used this study design by identifying 17 LTPA discordant twin pairs from two Finnish twin cohorts and summarize key findings of these studies in this paper. The carefully characterized rare long-term LTPA discordant MZ twin pairs have participated in multi-dimensional clinical examinations. The occurrence of type 2 diabetes and death has been evaluated on the basis of prospective questionnaire data and register follow-up among a larger number of twin pairs. Key findings highlight that, compared to less active twins in such MZ twin pairs, the twins with higher long-term LTPA have higher physical fitness, reduced body fat, reduced visceral fat, reduced liver fat, increased lumen diameters of conduit arteries to the lower limbs, increased bone mineral density in loaded bone areas, increased high-density lipoprotein cholesterol levels and reduced occurrence of type 2 diabetes. However, we have not been able to document differences in the life expectancy between the less and more active twin siblings of the LTPA discordant MZ twin pairs. The findings are in agreement with intervention studies but not with all observational studies in which genetic factors are not controlled for.
Machine Learning Based Prediction of COVID-19 Mortality Suggests Repositioning of Anticancer Drug for Treating Severe Cases
Authors: Linden Thomas , Hanses Frank , Domingo-Fernández Daniel , DeLong Lauren Nicole , Kodamullil Alpha Tom , Schneider Jochen , Vehreschild Maria J.G.T. , Lanznaster Julia , Ruethrich Maria Madeleine , Borgmann Stefan , Hower Martin , Wille Kai , Feldt Thorsten , Rieg Siegbert , Hertenstein Bernd , Wyen Christoph , Roemmele Christoph , Vehreschild Jörg Janne , Jakob Carolin E. M. , Stecher Melanie , Kuzikov Maria , Zaliani Andrea , Fröhlich Holger ,   LEOSS study group
Published: November 2021

Despite available vaccinations COVID-19 case numbers around the world are still growing, and effective medications against severe cases are lacking. In this work, we developed a machine learning model which predicts mortality for COVID-19 patients using data from the multi-center ‘Lean European Open Survey on SARS-CoV-2-infected patients’ (LEOSS) observational study (>100 active sites in Europe, primarily in Germany), resulting into an AUC of almost 80%. We showed that molecular mechanisms related to dementia, one of the relevant predictors in our model, intersect with those associated to COVID-19. Most notably, among these molecules was tyrosine kinase 2 (TYK2), a protein that has been patented as drug target in Alzheimer’s Disease but also genetically associated with severe COVID-19 outcomes. We experimentally verified that anti-cancer drugs Sorafenib and Regorafenib showed a clear anti-cytopathic effect in Caco2 and VERO-E6 cells and can thus be regarded as potential treatments against COVID-19. Altogether, our work demonstrates that interpretation of machine learning based risk models can point towards drug targets and new treatment options, which are strongly needed for COVID-19.
Quantification and progress over time of specific antibodies against SARS-CoV-2 in breast milk of lactating women vaccinated with BNT162b2 Pfizer-BioNTech COVID-19 vaccine (LacCOVID)
Authors: Esteve-Palau Erika , Gonzalez-Cuevas Araceli , Guerrero M. Eugenia , Garcia-Terol Clara , Alvarez M. Carmen , Garcia Geneva , Moreno Encarna , Medina Francisco , Casadevall David , Diaz-Brito Vicens
Published: November 2021

Importance

To our knowledge, this is the first study to analyze long–term passage (6 months after immunization) of specific antibodies induced by BNT162b2 COVID-19 vaccine through breast milk.

Objectives

Main objective: to determine SARS-CoV-2 vaccine induced antibody levels in the breast milk of lactating women 4 weeks after mRNA BNT162b2 Pfizer-BioNTech COVID-19 complete vaccination.

Secondary objectives: to analyze SARS-CoV-2 antibody levels (breast milk and serum) at different time-points after vaccination, examine the correlation of SARS-CoV-2 antibody levels between serum and breast milk, describe adverse events related to vaccination (AErV) in both mothers and infants and determine the rate of COVID-19 infections.

Design

Prospective cohort study between February and September 2021.

Setting

Parc Sanitari Sant Joan de Déu, an urban hospital in Spain.

Participants

During our health worker vaccination campaign at our hospital between January and March 2, we recruited 33 lactating women vaccinated with BNT162b2 Pfizer-BioNTech COVID-19.

Results

A total of 33 volunteers were included in the study. The median (IQR) age of mothers was 38 (36-39) years and 15 (10-22) months for the infants.

Primary end-point: at 4 w after second dose median (IQR) IgG-S1 levels for serum–milk pairs were 12,478 (6,870-20,801) to 50.4 (24.3-104) arbitrary units (AU) per mL.

Secondary end-points

SARS-CoV-2 antibody levels at different time-points were (serum-milk): 519 (234-937) to 1 (0-2.9) AU/mL at 2w after first dose, 18,644 (9,923-29,264) to 78 (33.7-128) AU/mL at 2w, 4,094 (2,413-8,480) to 19.9 (10.8-51.9) AU/mL at 12w, and 1,350 (831-2,298) to 8.9 (7.8-31.5) at 24w after second dose.

We found a positive correlation of SARS-CoV-2 antibody levels between serum and breast milk (Pearson correlation coefficient 0.68).

No serious AErV were observed.

We found two (6%) COVID-19 vaccine breakthrough infections.

Conclusions

Pfizer-BioNTech COVID-19 vaccination is safe during breastfeeding and it transmits antibodies into breast milk with a positive correlation with serum levels, and both decrease over time in a 6-month follow-up. Infants of breastfeeding vaccinated women could be protected for at least six months after vaccination and serum determination of SARS-CoV-2 IgG-S1 could indicate the breastmilk levels of antibodies during this period.
ANALYSIS OF IMMUNE ESCAPE VARIANTS FROM ANTIBODY-BASED THERAPEUTICS AGAINST COVID-19
Authors: Focosi Daniele , Maggi Fabrizio , Franchini Massimo , McConnell Scott , Casadevall Arturo
Published: November 2021

Accelerated SARS-CoV-2 evolution under selective pressure by massive deployment of neutralizing antibody-based therapeutics is a concern with potentially severe implications for public health. We review here reports of documented immune escape after treatment with monoclonal antibodies and COVID19 convalescent plasma (CCP). While the former is mainly associated with specific single amino acid mutations at residues within the receptor-binding domain (e.g., E484K/Q, Q493R, and S494P), the few cases of immune evasion after CCP were associated with recurrent deletions within the N-terminal domain of Spike protein (e.g, ΔHV69-70, ΔLGVY141-144 and ΔAL243-244). Continuous genomic monitoring of non-responders is needed to better understand immune escape frequencies and fitness of emerging variants.
50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA
Authors: Laughlin Zane T. , Nandi Suparno , Dey Debayan , Zelinskaya Natalia , Witek Marta A. , Srinivas Pooja , Nguyen Ha An , Kuiper Emily G. , Comstock Lindsay R. , Dunham Christine M. , Conn Graeme L.
Published: November 2021

Changes in bacterial ribosomal RNA methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2’-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a post-catalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144 which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.

Significance Statement

The bacterial ribosome is an important target for antibiotics used to treat infection. However, resistance to these essential drugs can arise through changes in ribosomal RNA (rRNA) modification patterns through the action of intrinsic or acquired rRNA methyltransferase enzymes. How these antibiotic resistance-associated enzymes recognize their ribosomal targets for site-specific modification is currently not well defined. Here, we uncover the molecular basis for large ribosomal (50S) subunit substrate recognition and modification by the Mycobacterium tuberculosis methyltransferase TlyA, necessary for optimal activity of the antitubercular drug capreomycin. From this work, recognition of complex rRNA structures distant from the site of modification and “flipping” of the target nucleotide base both emerge as general themes in ribosome recognition for bacterial rRNA modifying enzymes.
Intestinal Transgene Delivery with Native E. coli Chassis Allows Persistent Physiological Changes
Authors: Russell Baylee J. , Brown Steven D. , Saran Anand R. , Mai Irene , Lingaraju Amulya , Siguenza Nicole , Maissy Erica , Dantas Machado Ana C. , Pinto Antonio F. M. , Miyamoto Yukiko , Richter R. Alexander , Ho Samuel B. , Eckmann Lars , Hasty Jeff , Saghatelian Alan , Knight Rob , Zarrinpar Amir
Published: November 2021

Live bacterial therapeutics (LBT) could reverse disease by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally-raised (CR) hosts have been unsuccessful, because engineered microbial organisms (i.e., chassis) cannot colonize the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli isolated from stool cultures of CR mice were modified to express functional bacterial (bile salt hydrolase) and eukaryotic (Interleukin-10) genes. Reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect host physiology and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to “knock-in” specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts, and enables LBT with curative intent.
Structural basis for assembly of TRAPPII complex and specific activation of GTPase Ypt31/32
Authors: Mi Chenchen , Zhang Li , Sun Shan , Huang Guoqiang , Shao Guangcan , Yang Fan , You Xin , Dong Meng-Qiu , Sui Sen-Fang
Published: November 2021

Transport protein particle (TRAPP) complexes belong to the multiprotein tethering complex and have three forms- TRAPPI, TRAPPII and TRAPPIII, which share a core of six TRAPPI proteins. TRAPPII facilitates intra-Golgi and endosome-to-Golgi transports by activating GTPase Ypt31/Ypt32 as the guanine nucleotide exchange factor (GEF) in yeast. Here we present cryo-EM structures of yeast TRAPPII in apo and Ypt32-bound states. All the structures show a dimeric architecture assembled by two triangle shaped monomers, while the monomer in the apo structure exhibits both open and closed conformations, and the monomer in the Ypt32-bound form only captures the closed conformation. Located in the interior of the monomer, Ypt32 binds with both TRAPPI and Trs120 via its nucleotide binding domain and binds with Trs31 of TRAPPI via its hypervariable domain. Combined with functional analysis, the structures provide insights into the assembly of TRAPPII and the mechanism of the specific activation of Ypt31/Ypt32 by TRAPPII.

One Sentence Summary

Structures of TRAPPII in different states reveal the mechanism of the specific activation of Ypt32 by TRAPPII.
POTATO: An automated pipeline for batch analysis of optical tweezers data
Authors: Buck Stefan , Pekarek Lukas , Caliskan Neva
Published: November 2021

Optical tweezers is a single-molecule technique that allows probing of intra- and intermolecular interactions that govern complex biological processes involving molecular motors, protein–nucleic acid interactions and protein/RNA folding. Recent developments in instrumentation eased and accelerated optical tweezers data acquisition, but analysis of the data remains challenging. Here, to enable high-throughput data analysis, we developed an automated python-based analysis pipeline called POTATO (Practical Optical Tweezers Analysis TOol). POTATO automatically processes the high-frequency raw data generated by force-ramp experiments and identifies (un)folding events using predefined parameters. After segmentation of the force-distance trajectories at the identified (un)folding events, sections of the curve can be fitted independently to worm-like chain and freely-jointed chain models, and the work applied on the molecule can be calculated by numerical integration. Furthermore, the tool allows plotting of constant force data and fitting of the Gaussian distance distribution over time. All these features are wrapped in a user-friendly graphical interface (https://github.com/REMI-HIRI/POTATO), which allows researchers without programming knowledge to perform sophisticated data analysis.

SIGNIFICANCE

Studying (un)folding of biopolymer structures with optical tweezers under different conditions generates very large datasets for statistical data analysis. Recent technical improvements accelerated data acquisition by coupling modern instruments with microfluidic systems, at the same time creating the need for a high-throughput, and unbiased data analysis. We developed Practical Optical Tweezers Analysis TOol (POTATO); an open-source python-based tool that can process data gathered by any OT force-ramp experiment in an automated fashion. POTATO is principally designed for data preprocessing, identification of (un)folding events and the fitting of the force-distance curves. In addition, all parameters for preprocessing, statistical analysis and fitting of the curves can be adapted to suit the dataset under analysis in an easy-to-use graphical user interface.
Evolution of a new testis-specific functional promotor within the highly conservedMap2k7gene of the mouse
Authors: Heinen Tobias , Xie Chen , Keshavarz Maryam , Stappert Dominik , Künzel Sven , Tautz Diethard
Published: November 2021

Map2k7(synonymMkk7) is a conserved regulatory kinase gene and a central component of the JNK signaling cascade with key functions during cellular differentiation. It shows complex transcription patterns and different transcript isoforms are known in the mouse (Mus musculus). We have previously identified a newly evolved testis specific transcript for theMap2k7gene in the subspeciesM. m. domesticus. Here, we identify the new promotor that drives this transcript and find that its transcript codes for an open reading frame (ORF) of 50 amino acids. The new promotor was gained in the stem lineage of closely related mouse species, but was secondarily lost in the subspeciesM. m. musculusandM. m. castaneus. A single mutation can be correlated with its transcriptional activity inM. m. domesticusand cell culture assays demonstrate the capability of this mutation to drive expression. A mouse knock-out line in which the promotor region of the new transcript is deleted reveals a functional contribution of the newly evolved promotor to sperm motility and to the spermatid transcriptome. Our data show that a new functional transcript (and possibly protein) can evolve within an otherwise highly conserved gene, supporting the notion of regulatory changes contributing to the emergence of evolutionary novelties.
Are island plants really poorly defended? No support for the loss of defense hypothesis in domatia-bearing plants
Authors: Biddick M.
Published: November 2021

1.

The loss of defense hypothesis posits that island colonizers experience a release from predation on the mainland and subsequently lose their defensive adaptations. While support for the hypothesis is abundant, it has never been tested in domatia-bearing plants. Leaf domatia are cave-like structures produced on the underside of leaves that facilitate a defensive mutualism with predatory and fungivorous mites. I tested the loss of defense hypothesis in six domatia-bearing taxa inhabiting New Zealand and its offshore islands. No support for the loss of defense hypothesis was found. Changes in domatia investment were instead associated with changes in leaf size – a trait that has been repeatedly observed to undergo rapid evolution on islands. Overall results demonstrate that not all types of defense are lost on islands, suggesting a higher-resolution approach is needed when studying the evolution of defense on islands.
MICS1 is the Ca2+/H+ antiporter of mammalian mitochondria
Authors: Austin Shane , Mekis Ronald , Mohammed Sami E. M. , Scalise Mariafrancesca , Pfeiffer Christina , Galluccio Michele , Borovec Tamara , Parapatics Katja , Vitko Dijana , Dinhopl Nora , Bennett Keiryn L. , Indiveri Cesare , Nowikovsky Karin
Published: November 2021

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics, cell death pathways and cytosolic Ca2+ homeostasis. Mitochondrial Ca2+ content strictly depends on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+/H+ exchanger (CHE). Originally identified as the mitochondrial K+/H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identified MICS1, the only mitochondrial member of the TMBIM family. Applying cell-based and cell-free biochemical assays, here we demonstrate that MICS1 is responsible for the Na+- and permeability transition pore-independent mitochondrial Ca2+ release and identify MICS1 as the long-sought mitochondrial CHE. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.
Cells use molecular working memory to navigate in changing chemoattractant fields
Authors: Nandan Akhilesh , Das Abhishek , Lott Robert , Koseska Aneta
Published: November 2021

In order to migrate over large distances, cells within tissues and organisms rely on sensing local gradient cues. These cues however are multifarious, irregular or conflicting, changing both in time and space. Here we find that single cells utilize a molecular mechanism akin to a working memory, to generate persistent directional migration when signals are disrupted by temporally memorizing their position, while still remaining adaptive to spatial and temporal changes of the signal source. Using dynamical systems theory, we derive that these information processing capabilities are inherent for protein networks whose dynamics is maintained away from steady state through organization at criticality. We demonstrate experimentally using the Epidermal growth factor receptor (EGFR) signaling network, that the memory is maintained in the prolonged receptor’s activity via a slow-escaping remnant, a dynamical ”ghost” of the attractor of the polarized signaling state, that further results in memory in migration. As this state is metastable, it also enables continuous adaptation of the migration direction when the signals vary in space and time. We therefore show that cells implement real-time computations without stable-states to navigate in changing chemoattractant fields by memorizing position of disrupted signals while maintaining sensitivity to novel chemical cues.
Physiological and molecular mechanism of tolerance of two maize genotypes under multiple abiotic stresses
Authors: Rafique Suphia
Published: November 2021

Abiotic stresses are the major threat to crops regardless of their nature, duration, and frequency, their occurrence either singly, and or combination is deleterious for the plant growth and development. Maize is most important crop largely grown in tropical region in summer rainy season, often face a stress combination of drought and waterlogging. We previously showed under multiple stresses up-regulated leaf proteins of maize plants were involved to enhance the tolerance mechanism of tolerant genotype. Whereas, in susceptible genotypes up-regulated proteins ameliorate to survive the stressful condition. Further to understand the response of roots proteome under multiple stresses was determined using the 2DE technique. The results of the root proteome show the up-regulated proteins of CML49 genotype (tolerant) are involved in enhancing the N content, cell wall remodeling, and acclimatization during the stresses. Up-regulated proteins of CML100 genotype (sensitive) are stressed marker of roots primary and secondary metabolism. However, the root proteome of both genotypes correlates with the leaf proteome (previous). Therefore, the present study and our previous results provide comprehensive insight into the molecular mechanisms of tolerance in multiple abiotic stresses of maize plants.
Nucleolar-based Dux repression is essential for 2-cell stage exit
Authors: Xie Sheila Q. , Leeke Bryony J. , Whidling Chad , Wagner Ryan T. , Garcia-Llagostera Ferran , Chammas Paul , Cheung Nathan T-F. , Dormann Dirk , McManus Michael T. , Percharde Michelle
Published: November 2021

Upon fertilisation, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice involves the transient upregulation of MERVL transposons and MERVL-driven genes at the 2-cell stage. The mechanisms and requirement for MERVL and 2-cell (2C) gene upregulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow 2C exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and 2C embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking Pol I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper Dux silencing and leads to 2-4 cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation and gene repression during early development.
Self-organization of kinetochore-fibers in human mitotic spindles
Authors: Conway William , Kiewisz Robert , Fabig Gunar , Kelleher Colm P. , Wu Hai-Yin , Anjur-Dietrich Maya , Müller-Reichert Thomas , Needleman Daniel
Published: November 2021

During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent electron tomography reconstructions (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that many KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of the electron tomography reconstructions, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, continually decreasing in speed as they approach the poles. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.
METTL3 promotes homologous recombination repair and modulates chemotherapeutic response by regulating the EGF/Rad51 axis
Authors: Li Enjie , Xia Mingyue , Du Yu , Long Kaili , Ji Feng , Pan Feiyan , He Lingfeng , Hu Zhigang , Guo Zhigang
Published: November 2021

METTL3 and N6-methyladenosine (m6A) are involved in many types of biological and pathological processes, including DNA repair. However, the function and mechanism of METTL3 in DNA repair and chemotherapeutic response remain largely unknown. In present study, we identified that METTL3 participates in the regulation of homologous recombination repair (HR), which further influences chemotherapeutic response in breast cancer (BC) cells. Knockdown of METTL3 sensitized BC cells to Adriamycin (ADR) treatment and increased accumulation of DNA damage. Mechanically, we demonstrated that inhibition of METTL3 impaired HR efficiency and increased ADR-induced DNA damage by regulating m6A modification of EGF/RAD51 axis. METTL3 promoted EGF expression through m6A modification, which further upregulated RAD51 expression, resulting in enhanced HR activity. We further demonstrated that the m6A “reader,” YTHDC1, bound to the m6A modified EGF transcript and promoted EGF synthesis, which enhanced HR and cell survival during ADR treatment. Our findings reveal a pivotal mechanism of METTL3-mediated HR and chemotherapeutic drug response, which may contribute to cancer therapy.
Computational design of nanoscale rotational mechanics in de novo protein assemblies
Authors: Courbet A. , Hansen J. , Hsia Y. , Bethel N. , Park YJ. , Xu C. , Moyer A. , Boyken S.E. , Ueda G. , Nattermann U. , Nagarajan D. , Silva D. , Sheffler W. , Quispe J. , King N. , Bradley P. , Veesler D. , Kollman J. , Baker D.
Published: November 2021

Natural nanomachines like the F1/F0-ATPase contain protein components that undergo rotation relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein rotary machinery from designed axle and ring components. Using cryoelectron microscopy, we find that axle-ring systems assemble as designed and populate diverse rotational states depending on symmetry match or mismatch and the designed interface energy landscape. These mechanical systems with internal rotational degrees of freedom are a step towards the systematic design of genetically encodable nanomachines.

One-Sentence Summary

Computationally designed self-assembling protein rotary machines sample internal degrees of freedom sculpted within the energy landscape.
Multi-omic rejuvenation and lifespan extension upon exposure to youthful circulation
Authors: Zhang Bohan , Lee David E. , Trapp Alexandre , Tyshkovskiy Alexander , Lu Ake T. , Bareja Akshay , Kerepesi Csaba , Katz Lauren H. , Shindyapina Anastasia V. , Dmitriev Sergey E. , Baht Gurpreet S. , Horvath Steve , Gladyshev Vadim N. , White James P.
Published: November 2021

Heterochronic parabiosis (HPB) is known for its functional rejuvenation effects across several mouse tissues. However, its impact on the biological age of organisms and their long-term health remains unknown. Here, we performed extended (3-month) HPB, followed by a 2-month detachment period of anastomosed pairs. Old detached mice exhibited improved physiological parameters and lived longer than control isochronic mice. HPB drastically reduced the biological age of blood and liver based on epigenetic analyses across several clock models on two independent platforms; remarkably, this rejuvenation effect persisted even after 2 months of detachment. Transcriptomic and epigenomic profiles of anastomosed mice showed an intermediate phenotype between old and young, suggesting a comprehensive multi-omic rejuvenation effect. In addition, old HPB mice showed transcriptome changes opposite to aging, but akin to several lifespan-extending interventions. Altogether, we reveal that long-term HPB can decrease the biological age of mice, in part through long-lasting epigenetic and transcriptome remodeling, culminating in the extension of lifespan and healthspan.
The abiotic and biotic environment together predict plant, mammal, and bird diversity and turnover across the United States
Authors: Gallinat Amanda S. , Pearse William D.
Published: November 2021

The distribution of taxonomic, phylogenetic, and functional biodiversity results from a combination of abiotic and biotic drivers which are scale dependent. Parsing the relative influence of these drivers is critical to understanding the processes underlying species assembly and generating predictions of biodiversity across taxonomic groups and for novel sites. However, doing so requires data that capture a spatial extent large enough to reflect broad-scale dynamics such as speciation and biogeography, and a spatial grain fine enough to detect local-scale dynamics like environmental filtering and biotic interactions. We used species inventories of vascular plants, birds, and mammals collected by the U.S. National Ecological Observatory Network (NEON) at 38 terrestrial field sites, to explore the processes underlying taxonomic, phylogenetic, and functional diversity and turnover. We found that, for both species richness (alpha-diversity) and turnover (beta-diversity), taxonomic, phylogenetic, and functional diversity are weak proxies for one-another, and thus may capture different species assembly processes. All diversity metrics were best predicted by a combination of abiotic and biotic variables. Taxonomic and phylogenetic richness tended to be higher at warmer, wetter sites, reflecting the role energy inputs play in driving broad-scale diversity. However, plant diversity was negatively correlated with bird phylogenetic and mammal functional diversity, implying trait conservation in plant communities may limit niche availability for consumer species. Equally, turnover in bird and mammal species across sites were associated with plant turnover. That the biodiversity of one taxon is predictive of another across these North American sites, even when controlling for environment, supports a role for the cross-clade biotic environment in driving species assembly.
Multiscale profiling of enzyme activity in cancer
Authors: Soleimany Ava P. , Kirkpatrick Jesse D. , Wang Cathy S. , Jaeger Alex M. , Su Susan , Naranjo Santiago , Zhong Qian , Cabana Christina M. , Jacks Tyler , Bhatia Sangeeta N.
Published: November 2021

Diverse processes in cancer are mediated by enzymes, which most proximally exert their function through their activity. Methods to quantify enzyme activity, rather than just expression, are therefore critical to our ability to understand the pathological roles of enzymes in cancer and to harness this class of biomolecules as diagnostic and therapeutic targets. Here we present an integrated set of methods for measuring specific enzyme activities across the organism, tissue, and cellular levels, which we unify into a methodological hierarchy to facilitate biological discovery. We focus on proteases for method development and validate our approach through the study of tumor progression and treatment response in an autochthonous model of Alk-mutant lung cancer. To quantitatively measure activity dynamics over time, we engineered multiplexed, peptide-based nanosensors to query protease activity in vivo. Machine learning analysis of sensor measurements revealed dramatic protease dysregulation in lung cancer, including significantly enhanced proteolytic cleavage of one peptide, S1 (Padj < 0.0001), which returned to healthy levels within three days after initiation of targeted therapy. Next, to link these organism-level observations to the in situ context, we established a multiplexed assay for on-tissue localization of enzyme activity and pinpointed S1 cleavage to endothelial cells and pericytes of the tumor vasculature. Lastly, to directly link enzyme activity measurements to cellular phenotype, we designed a high-throughput method to isolate and characterize proteolytically active cells, uncovering profound upregulation of pro-angiogenic transcriptional programs in S1-positive cells. Together, these methods allowed us to discover that protease production by angiogenic vasculature responds rapidly to targeted therapy against oncogene-addicted tumor cells, identifying a highly dynamic interplay between tumor cells and their microenvironment. This work provides a generalizable framework to functionally characterize enzyme activity in cancer.