bluuebunny/crossref_metadata_2025 huggingface dataset:
A Random Forest Classifier Uses Antibody Responses to Plasmodium Antigens to Reveal Candidate Biomarkers of the Intensity and Timing of Past Exposure to Plasmodium falciparum
Authors: Bérubé Sophie , Kobayashi Tamaki , Norris Douglas E. , Ruczinski Ingo , Moss William J. , Wesolowski Amy , Louis Thomas A.
Published: February 2022

1

Important goals of malaria surveillance efforts include accurately quantifying the burden of malaria over time, which can be useful to target and evaluate interventions. The majority of malaria surveillance methods capture active or recent infections which poses several challenges to achieving malaria surveillance goals. In high transmission settings, asymptomatic infections are common and therefore accurate measurement of malaria burden often demands active surveillance; in low transmission regions where infections are rare accurate surveillance requires sampling a large subset of the population; and in any context monitoring malaria burden over time necessitates serial sampling. Antibody responses to Plasmodium falciparum parasites persist after infection and therefore measuring antibodies has the potential to overcome several of the current difficulties associated with malaria surveillance. However, identifying which antibody responses are markers of the timing and intensity of past exposure to P. falciparum is challenging, particularly among adults who tend to be re-exposed multiple times over the course of their lifetime and therefore have similarly high antibody responses to many P. falciparum antigens. A previous analysis of 479 serum samples from individuals in three regions in southern Africa with different historical levels of P. falciparum malaria transmission (high, intermediate, and low) revealed regional differences in antibody responses to P. falciparum antigens among children under 5 years of age. Using a novel bioinformatic pipeline optimized for protein microarrays that minimizes between-sample technical variation, we used antibody responses to P. falciparum and P. vivax antigens as predictors in random forest models to classify adult samples into these three regions of differing historical malaria transmission with high accuracy. Many of the antigens that were most important for classification in these models do not overlap with previously published results and are therefore novel candidate markers for the timing and intensity of past exposure to P. falciparum. Measuring antibody responses to these antigens could potentially lead to improved malaria serosurveillance that captures the timing and intensity of past exposure.
Lens aquaporin-5 inserts into bovine fiber cell plasma membranes through mitochondria-associated lysosome secretion
Authors: Gletten Romell B. , Cantrell Lee S. , Bhattacharya Sujoy , Schey Kevin L.
Published: February 2022

PURPOSE

To spatially map aquaporin-5 (AQP5) expression in bovine lens, molecularly characterize cytoplasmic AQP5-containing vesicles in the outer cortex, and elucidate AQP5 membrane trafficking mechanisms.

METHODS

Immunofluorescence was performed on bovine lens cryosections using AQP5, TOMM20, COX IV, calnexin, LC3B, LIMP-2, and connexin-50 antibodies and the fluorescent lipid membrane dye CM-DiI. AQP5 plasma membrane insertion was defined via line expression profile analysis. Transmission electron microscopy (TEM) was performed on bovine lens tissue sections to define cytoplasmic organelle identity, morphology, and subcellular localization in cortical fiber cells. Bovine lenses were treated with 10 nM bafilomycin A1 or 0.1% dimethyl sulfoxide vehicle control in ex vivo culture to determine changes in AQP5 plasma membrane expression.

RESULTS

Immunofluorescence analysis revealed cytoplasmic AQP5 expression in bovine lens epithelial cells and differentiating fiber cells. In the bovine lens cortex, complete AQP5 plasma membrane insertion occurs at r/a 0.951 + 0.005. AQP5-containing cytoplasmic vesicles are spheroidal, tubular in morphology, express TOMM20, and contain LC3B and LIMP-2 as fiber cells mature. TEM analysis revealed spheroidal, tubular autophagosomes, autolysosomes, and lysosomes with degrading mitochondria. AQP5-containing cytoplasmic vesicles and autolysosomes dock and fuse with the plasma membrane. Bafiloymcin A1 treatment reduced AQP5 plasma membrane expression by 27%.

CONCLUSIONS

AQP5 localizes to spheroidal, tubular cytoplasmic vesicles in the differentiating bovine lens fiber cells. During fiber cell differentiation, these vesicles incorporate LC3B and fuse with LIMP-2-positive lysosomes. AQP5 trafficking to the plasma membrane occurs through lysosome secretion as a novel mechanism of AQP5 trafficking.
Autoantibodies linked to autoimmune diseases associate with COVID-19 outcomes
Authors: Baiocchi Gabriela Crispim , Vojdani Aristo , Rosenberg Avi Z , Vojdani Elroy , Halpert Gilad , Ostrinski Yuri , Zyskind Israel , Filgueiras Igor Salerno , Schimke Lena F. , Marques Alexandre H. C. , Giil Lasse M. , Lavi Yael Bublil , Silverberg Jonathan I. , Zimmerman Jason , Hill Dana Ashley , Thornton Amanda , Kim Myungjin , De Vito Roberta , Fonseca Dennyson Leandro M. , Plaça Desireé Rodrigues , Freire Paula Paccielli , Camara Niels Olsen Saraiva , Calich Vera Lúcia Garcia , Heidecke Harald , Lattin Miriam T. , Ochs Hans D. , Riemekasten Gabriela , Amital Howard , Cabral-Marques Otavio , Shoenfeld Yehuda
Published: February 2022

The SARS-CoV-2 infection is associated with increased levels of autoantibodies targeting immunological proteins such as cytokines and chemokines. Reports further indicate that COVID-19 patients may develop a wide spectrum of autoimmune diseases due to reasons not fully understood. Even so, the landscape of autoantibodies induced by SARS-CoV-2 infection remains uncharted territory. To gain more insight, we carried out a comprehensive assessment of autoantibodies known to be linked to diverse autoimmune diseases observed in COVID-19 patients, in a cohort of 248 individuals, of which171 were COVID-19 patients (74 with mild, 65 moderate, and 32 with severe disease) and 77were healthy controls. Dysregulated autoantibody serum levels, characterized mainly by elevated concentrations, occurred mostly in patients with moderate or severe COVID-19 infection, and was accompanied by a progressive disruption of physiologic IgG and IgA autoantibody signatures. A similar perturbation was found in patients with anosmia. Notably, autoantibody levels often accompanied anti-SARS-CoV-2 antibody concentrations, being both indicated by random forest classification as strong predictors of COVID-19 outcome, together with age. Moreover, higher levels of autoantibodies (mainly IgGs) were seen in the elderly with severe disease compared with young COVID-19 patients with severe disease. These findings suggest that the SARS-CoV-2 infection induces a broader loss of self-tolerance than previously thought, providing new ideas for therapeutic interventions.
Immune system and blood-brain barrier-wide biomarker analyses provide causal evidence for autoimmunity in dementia
Authors: Lindbohm Joni V. , Mars Nina , Sipilä Pyry N. , Singh-Manoux Archana , Runz Heiko ,   FinnGen, Livingston Gill , Seshadri Sudha , Xavier Ramnik , Hingorani Aroon D. , Ripatti Samuli , Kivimäki Mika
Published: February 2022

Immune system and blood brain barrier (BBB) dysfunction are implicated in the development of Alzheimer’s disease and other dementias, but their causal role remains unknown. We performed Mendelian randomization (MR) for over 43,643 immune system and BBB-related biomarkers and identified 126 potential causal risk factors for dementias. A phenome-wide analysis using MR-based polygenic risk score in FinnGen study (N=339,233) for these risk factors revealed a common genetic background for dementias and autoimmune diseases which was supported by further HLA analyses. Pathway analyses linked the 126 proteins to amyloid-β, tau and α-synuclein pathways, increased inflammatory responses, and altered self-tolerance mechanisms. In inverse-probability-weighted analyses simulating randomized controlled drug trials in observational data, anti-inflammatory and immunosuppressive medications were associated with reduced dementia risk (p<0.01 for methotrexate and TNF-α inhibitors). These converging results from different research lines suggest that autoimmunity is a modifiable component in diseases causing dementia.
Cross-kingdom metagenomic profiling of Lake Hillier reveals pigment-rich polyextremophiles and wide-ranging metabolic adaptations
Authors: Sierra Maria A. , Ryon Krista A. , Tierney Braden T. , Foox Jonathan , Bhattacharya Chandrima , Afshin Evan , Butler Daniel , Green Stefan J. , Thomas W. Kelley , Ramsdell Jordan , Bivens Nathan J. , McGrath Ken , Mason Christopher E. , Tighe Scott W.
Published: February 2022

Background Lake Hillier is a hypersaline lake known for its distinctive bright pink color. The cause of this phenomenon in other hypersaline sites has been attributed to halophiles, Dunaliella, and Salinibacter, however, a systematic analysis of the microbial communities, their functional features, and the prevalence of pigment-producing-metabolisms has not been previously studied.

Our results are evidence that Lake Hillier is composed of a diverse set of microorganisms including archaea, bacteria, algae, and viruses. Our data indicate a core microbiome in Lake Hillier composed of multiple pigment-producer microbes, many of which are cataloged as polyextremophiles. Additionally, we estimated the diversity of metabolic pathways in the lake and determined that many of these are related to pigment production. We reconstructed complete or partial genomes for 21 discrete bacteria (N = 14) and archaea (N = 7), only 2 of which could be taxonomically annotated to previously observed species.

Our findings provide the first metagenomic study to decipher the source of the pink color of Australia’s Lake Hillier. The study of this pink hypersaline environment is evidence of a microbial consortium of pigment producers, a repertoire of polyextremophiles, a core microbiome and potentially novel species.
The Mycobacterium tuberculosis protein O-phosphorylation landscape
Authors: Frando Andrew , Boradia Vishant , Gritsenko Marina , Beltejar Michael-Claude , Day Le , Sherman David R. , Ma Shuyi , Jacobs Jon M. , Grundner Christoph
Published: February 2022

Bacterial phosphosignaling has long been synonymous with the histidine kinases of the two component systems, but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignaling enzymes in eukaryotes, but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria remains unclear. Here, we explored the global signaling capacity of the STPKs in Mtb. We generated STPK loss-and gain-of-function strains and measured the resulting O-phosphorylation and transcriptional changes. This deep phosphoproteome shows that O-phosphorylation in Mtb is an underexplored protein modification that affects >70% of the proteome. The substrate-STPK interactions show an extensive interface with the transcriptional machinery, resulting in regulation of gene expression of ∼30% of Mtb genes. Mtb O-phosphorylation gives rise to an expansive, distributed, and cooperative network of a complexity that has previously only been associated with eukaryotic phosphosignaling networks.
Molecular architecture of the human caveolin-1 complex
Authors: Porta Jason C. , Han Bing , Gulsevin Alican , Chung Jeongmin , Peskova Yelena , Connolly Sarah , Mchaourab Hassane S. , Meiler Jens , Karakas Erkan , Kenworthy Anne K. , Ohi Melanie D.
Published: February 2022

Membrane sculpting proteins shape the morphology of cell membranes and facilitate remodeling in response to physiological and environmental cues. Complexes of the monotopic membrane protein caveolin function as essential curvature-generating components of caveolae, flask-shaped invaginations that sense and respond to plasma membrane tension. However, the structural basis for caveolin’s membrane remodeling activity is currently unknown. Here, we show, using cryo-electron microscopy, that the human caveolin-1 complex is composed of 11 protomers organized into a tightly packed disc with a flat membrane-embedded surface. The structural insights suggest a new mechanism for how membrane sculpting proteins interact with membranes and reveal how key regions of caveolin-1, including its scaffolding, oligomerization, and intramembrane domains, contribute to its function.

One-Sentence Summary

Cryo-electron microscopy reveals that Caveolin-1 oligomerizes into a tightly packed disc with a flat membrane-binding surface.
Minus one frameshifted puromycin N-acetyltransferase is targeted to the nucleolus: sporadic but reproducible chimera formation by a transfected Lc3b construct
Authors: Thekkinghat Arvind A. , Rangarajan Pundi N.
Published: February 2022

Map1Lc3b is a protein that has pivotal functions in cellular autophagy. At least three groups in the past decade have reported its presence in the nucleoli of cells, but its functions in that organelle remain unknown. We isolated a few clonal populations of cells stably expressing V5-tagged mouse Lc3b highly enriched in the nucleoli, but the frequency of occurrence of such clones was strikingly low. The phenomenon was readily reproducible, though the protein in the nucleolus puzzlingly had varying molecular masses in different clones but consistently displayed a very strong interaction with the mitochondrial protein C1qbp, which has well-documented functions in the nucleolus. We investigated further and discovered that, in at least one of the clones, Lc3b had formed a chimera with the puromycin resistance gene in the plasmid, plausibly by illegitimate recombination during or after integration of the construct into the cellular genomic DNA. The -1 shifted reading frame of puromycin N-acetyltransferase (pac) can encode a protein that is equally long as the one encoded by the complete pac ORF, but is targeted to the nucleoli due to a drastic shift in the isoelectric point (pI). Notably, this set of events again brings into focus the low threshold often reported for recombination events to occur in eukaryotic cells, the multiple factors influencing them, and calls for increased vigilance in experiments involving DNA transfection and gene targeting.
mRNA 5′ terminal sequences drive 200-fold differences in expression through effects on synthesis, translation and decay
Authors: van den Elzen Antonia M.G. , Watson Maegan J. , Thoreen Carson C.
Published: February 2022

mRNA regulatory sequences control gene expression at multiple levels including translation initiation and mRNA decay. The 5′ terminal sequences of mRNAs have unique regulatory potential because of their proximity to key post-transcriptional regulators. Here we have systematically probed the function of 5′ terminal sequences in gene expression in human cells. Using a library of reporter mRNAs initiating with all possible 7-mer sequences at their 5′ ends, we find an unexpected impact on transcription that underlies 200-fold differences in mRNA expression. Library sequences that promote high levels of transcription mirrored those found in native mRNAs and define two basic classes with similarities to classic Initiator (Inr) and TCT core promoter motifs. By comparing transcription, translation and decay rates, we identify sequences that are optimized for both efficient transcription and growth-regulated translation and stability, including variants of terminal oligopyrimidine (TOP) motifs. We further show that 5′ sequences of endogenous mRNAs are enriched for multi-functional TCT/TOP hybrid sequences. Together, our results reveal how 5′ sequences define two general classes of mRNAs with distinct growth-responsive profiles of expression across synthesis, translation and decay.
Azithromycin possesses biofilm–inhibitory activity and potentiates non-bactericidal colistin methanesulfonate against Klebsiella pneumonia
Authors: Moshynets Olena V , Baranovskyi Taras P , Cameron Scott , Iugin Olga S , Pokholenko Ianina , Jordan Robyn , Kamyshnyi Aleksandr , Krikunov Alexey A , Potochilova Viktoria V , Rudnieva Kateryna L , Spiers Andrew
Published: February 2022

Novel antibiotic combinations may act synergistically to inhibit the growth of multidrug-resistant bacterial pathogens but predicting which will be successful is difficult, and standard antimicrobial susceptibility testing may not identify important physiological differences between planktonic free-swimming and biofilm-protected surface-attached sessile cells. Using a nominally macrolide-resistant model Klebsiella pneumoniae strain (ATCC 10031) we demonstrate the effectiveness of several macrolides in inhibiting biofilm growth in multi-well plates, and the ability of azithromycin (AZM) to improve the effectiveness of the antibacterial last-agent-of-choice for K. pneumoniae infections, colistin methanesulfonate (CMS), against biofilms. This synergistic action was also seen in biofilm tests of several K. pneumoniae hospital isolates and could also be identified in polymyxin B disc-diffusion assays on azithromycin plates. Our work highlights the complexity of antimicrobial-resistance in bacterial pathogens and the need to test antibiotics with biofilm models where potential synergies might provide new therapeutic opportunities not seen in liquid culture or colony-based assays.
Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity
Authors: Czarna Anna , Plewka Jacek , Kresik Leanid , Matsuda Alex , Karim Abdulkarim , Robinson Colin , O’Byrne Sean , Cunningham Fraser , Georgiou Irene , Pachota Magdalena , Popowicz Grzegorz , Wyatt Paul Graham , Dubin Grzegorz , Pyrć Krzysztof
Published: February 2022

During the RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10 mediated modulation remains unclear in the absence of nsp14 structure. Here we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10 bound structures explain the modulatory role of the co-factor protein. Further, the structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.
CRISPR-mediated correction of skeletal muscle Ca2+handling in a novel DMD patient-derived pluripotent stem cell model
Authors: Morera Cristina , Kim Jihee , Paredes-Redondo Amaia , Nobles Muriel , Rybin Denis , Moccia Robert , Kowala Anna , Meng Jinhong , Garren Seth , Liu Pentao , Morgan Jennifer E , Muntoni Francesco , Christoforou Nicolas , Owens Jane , Tinker Andrew , Lin Yung-Yao
Published: February 2022

Mutations in thedystrophingene cause the most common and currently incurable Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting. Although abnormal Ca2+handling is a pathological feature of DMD, mechanisms underlying defective Ca2+homeostasis remain unclear. Here we generate a novel DMD patient-derived pluripotent stem cell (PSC) model of skeletal muscle with an isogenic control using clustered regularly interspaced short palindromic repeat (CRISPR)- mediated precise gene correction. Transcriptome analysis identifies dysregulated gene sets in the absence of dystrophin, including genes involved in Ca2+handling, excitation-contraction coupling and muscle contraction. Specifically, analysis of intracellular Ca2+transients and mathematical modeling of Ca2+dynamics reveal significantly reduced cytosolic Ca2+clearance rates in DMD-PSC derived myotubes. Pharmacological assays demonstrate Ca2+flux in myotubes is determined by both intracellular and extracellular sources. DMD-PSC derived myotubes display significantly reduced velocity of contractility. Compared with a non-isogenic wild type PSC line, these pathophysiological defects could be rescued by CRISPR-mediated precise gene correction. Our study provides new insights into abnormal Ca2+homeostasis in DMD and suggests that Ca2+signaling pathways amenable to pharmacological modulation are potential therapeutic targets. Importantly, we have established a human physiology-relevantin vitromodel enabling rapid pre-clinical testing of potential therapies for DMD.
Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces robust neutralizing salivary IgA
Authors: Stolovich-Rain Miri , Kumari Sujata , Friedman Ahuva , Kirillov Saveliy , Socol Yakov , Billan Maria , Pal Ritesh Ranjan , Golding Peretz , Oiknine-Djian Esther , Sirhan Salim , Sagie Michal Bejerano , Cohen-Kfir Einav , Elgrably-Weiss Maya , Zhou Bing , Ravins Miriam , Gatt Yair E , Das Kathakali , Zelig Orly , Wiener Reuven , Wolf Dana G , Elinav Hila , Strahilevitz Jacob , Padawer Dan , Baraz Leah , Rouvinski Alexander
Published: February 2022

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable ‘sterilizing immunity’ at the mucosal level. Our study uncovers, strong neutralizing mucosal component (NT50 ≤ 50pM), emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the Receptor-Binding-Domain (RBD) of SARS-CoV-2 spike protein and demonstrate that these IgAs are key mediators of potent neutralization. RBD-targeting IgAs were found to associate with the Secretory Component, indicating their bona-fide transcytotic origin and their dimeric tetravalent nature. The mechanistic understanding of the exceptionally high neutralizing activity provided by mucosal IgA, acting at the first line of defence, will advance vaccination design and surveillance principles, pointing to novel treatment approaches, and to new routes of vaccine administration and boosting.

Significance statement

We unveiled powerful mucosal neutralization upon BNT162b2 vaccination, mediated by temporary polymeric IgA and explored its longitudinal properties. We present a model, whereby the molecular architecture of polymeric mucosal IgA and its spatial properties are responsible for the outstanding SARS-CoV-2 neutralization potential. We established a methodology for quantitative comparison of immunoreactivity and neutralization for IgG and IgAs in serum and saliva in molar equivalents for standardization in diagnostics, surveillance of protection and for vaccine evaluations.
LncRNA RUS shapes the gene expression program towards neurogenesis
Authors: Schneider Marius F. , Müller Veronika , Müller Stephan A. , Lichtenthaler Stefan F. , Becker Peter B. , Scheuermann Johanna C.
Published: February 2022

The evolution of brain complexity correlates with an increased expression of long, non- coding (lnc) RNAs in neural tissues. Although prominent examples illustrate the potential of lncRNAs to scaffold and target epigenetic regulators to chromatin loci, only few cases have been described to function during brain development. We present a first functional characterization of the lncRNA LINC01322, which we term RUS for ‘RNA upstream of Slitrk3’. The RUS gene is well conserved in mammals by sequence and synteny next to the neurodevelopmental gene Slitrk3. RUS is exclusively expressed in neural cells and its expression increases along with neuronal markers during neuronal differentiation of mouse embryonic cortical neural stem cells. Depletion of RUS locks neuronal precursors in an intermediate state towards neuronal differentiation resulting in arrested cell cycle and increased apoptosis. RUS associates with chromatin in the vicinity of genes involved in neurogenesis, most of which change their expression upon RUS depletion. The identification of a range of epigenetic regulators as specific RUS interactors suggests that the lncRNA may mediate gene activation and repression in a highly context-dependent manner.
Multisensory integration of social signals by a pathway from the basal amygdala to the auditory cortex in maternal mice
Authors: Nowlan Alexandra C. , Choe Jane , Ansari Hoda J. , Kelahan Clancy , Shea Stephen D.
Published: February 2022

Social encounters are inherently multimodal events, yet how and where social cues of distinct sensory modalities merge and interact in the brain is poorly understood. When their pups wander away from the nest, mother mice use a combination of vocal and olfactory signals emitted by the pups to locate and retrieve them. Previous work revealed the emergence of multisensory interactions in the auditory cortex (AC) of both dams and virgins who co-habitate with pups (‘surrogates’). Here we identify a neural pathway that integrates information about odors with responses to sound. We found that a scattered population of glutamatergic neurons in the basal amygdala (BA) projects to the AC and responds to odors, including the smell of pups. These neurons exhibit increased activity when the female is searching for pups that terminates upon contact. Finally, we show that selective optogenetic activation of BA-AC neurons modulates responses to pup calls, and that this modulation switches from predominantly suppressive to predominantly excitatory after maternal experience. This supports an underappreciated role for the amygdala in directly shaping sensory representations in an experience-dependent manner. We propose that the BA-AC pathway integrates olfaction and audition to facilitate maternal care, and speculate that it may carry valence information to the AC.
The giant Mimivirus 1.2 Mb genome is elegantly organized into a 30 nm helical protein shield
Authors: Villalta Alejandro , Schmitt Alain , Estrozi Leandro F. , Quemin Emmanuelle R. J. , Alempic Jean-Marie , Lartigue Audrey , Pražák Vojtěch , Belmudes Lucid , Vasishtan Daven , Colmant Agathe M. G. , Honoré Flora A. , Couté Yohann , Grünewald Kay , Abergel Chantal
Published: February 2022

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ∼0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography and proteomics revealed that it is encased into a ∼30 nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.

One-Sentence Summary

Mimivirus genome organization in the icosahedral virion.
Mechanistic model of MAPK signaling reveals how allostery and rewiring contribute to drug resistance
Authors: Fröhlich Fabian , Gerosa Luca , Muhlich Jeremy , Sorger Peter K.
Published: February 2022

BRAFV600E is prototypical of oncogenic mutations that can be targeted therapeutically and treatment of BRAF-mutant melanomas with RAF and MEK inhibitors results in rapid tumor regression. However, drug-induced rewiring causes BRAFV600E melanoma cells to rapidly acquire a drug-adapted state. In patients this is thought to promote acquisition or selection for resistance mutations and disease recurrence. In this paper we use an energy-based implementation of ordinary differential equations in combination with proteomic, transcriptomic and imaging data from melanoma cells, to model the precise mechanisms responsible for adaptive rewiring. We demonstrate the presence of two parallel MAPK (RAF-MEK-ERK kinase) reaction channels in BRAFV600E melanoma cells that are differentially sensitive to RAF and MEK inhibitors. This arises from differences in protein oligomerization and allosteric regulation induced by oncogenic mutations and drug binding. As a result, the RAS-regulated MAPK channel can be active under conditions in which the BRAFV600E-driven channel is fully inhibited. Causal tracing demonstrates that this provides a sufficient quantitative explanation for initial and acquired responses to multiple different RAF and MEK inhibitors individually and in combination.

Highlights

A thermodynamic framework enables structure-based description of allosteric interactions in the EGFR and MAPK pathways

Causal decomposition of efficacy of targeted drugs elucidates rewiring of MAPK channels

Model-based extrapolation from type I½ RAF inhibitors to type II RAF inhibitors

A unified mechanistic explanation for adaptive and genetic resistance across BRAF-cancers
LabGym: quantification of user-defined animal behaviors using learning-based holistic assessment
Authors: Hu Yujia , Ferrario Carrie R. , Maitland Alexander D. , Ionides Rita B. , Ghimire Anjesh , Watson Brendon , Iwasaki Kenichi , White Hope , Xi Yitao , Zhou Jie , Ye Bing
Published: February 2022

Quantifying animal behavior is important for many branches of biological research. Current computational tools for behavioral quantification typically rely on a few pre-defined, simplified features to identify a behavior. However, such an approach restricts the information used and the tool’s applicability to a limited range of behavior types or species. Here we report a new tool, LabGym, for quantifying animal behaviors without such limitations. Combining a novel approach for effective evaluation of animal motion with customizable convolutional recurrent networks for capturing spatiotemporal details, LabGym provides holistic behavioral assessment and accurately identify user-defined animal behaviors without restrictions on behavior types or animal species. It then provides quantitative measurements of each behavior, which quantify the behavior intensity and the body kinematics during the behavior. LabGym requires neither any intermediate step for processing features that causes information loss nor programming knowledge from users for post-hoc analysis. It tracks multiple animals simultaneously in various experimental settings for high-throughput and versatile analysis. It also provides users a way to generate visualizable behavioral datasets that are valuable resources for the research community. We demonstrate its efficacy in capturing subtle behavioral changes in animals ranging from soft-bodied invertebrates to mammals.
The probability of chromatin to be at the nuclear lamina has no systematic effect on its transcription level in fruit fly
Authors: Afanasyev Alexander Y. , Kim Yoonjin , Tolokh Igor S. , Sharakhov Igor V. , Onufriev Alexey V.
Published: February 2022

Multiple studies have demonstrated a negative correlation between gene expression and the positioning of genes at the nuclear envelope (NE) lined by nuclear lamina. In this paper, we ask whether there is a causal, systematic relationship between the expression level of the groups of genes in topologically associating domains (TADs) of Drosophila nuclei and the probabilities of TADs to be found at the NE. To investigate the nature of this possible relationship, we combine a coarse-grained dynamic model of the entire Drosophila nucleus with genome-wide gene expression data; we analyze the TAD averaged transcription levels of genes against the probabilities of individual TADs to be near the NE in the control and lamins-depleted nuclei. Our findings demonstrate that, within the statistical error margin, the stochastic positioning ofDrosophila melanogasterTADs at the NE does not, by itself, systematically affects the mean level of gene expression in these TADs, while the expected negative correlation is confirmed. The correlation is weak and disappears completely for TADs not containing lamina-associated domains (LADs) or TADs containing LADs, considered separately. Verifiable hypotheses of the underlying mechanism for the presence of correlation without causality are discussed.
On Correlation between Structural Properties and Viral Escape Measurements from Deep Mutational Scanning
Authors: Zhang Leili , Domeniconi Giacomo , Yang Chih-Chieh
Published: February 2022

Encouraged by recent efforts to map responses of SARS-CoV-2 mutations to various antibody treatments with deep mutational scanning, we explored the possibility of tying measurable structural contact information from the binding complexes of antibodies and their targets to experimentally determined viral escape responses. With just a single crystal structure for each binding complex, we find that the average correlation coefficient R is surprisingly high at 0.76. Our two methods for calculating contact information use binary contacts measured between all residues of two proteins. By varying the parameters to obtain binary contacts, we find that 3.6 Å and 7 Å are pivotal distances to toggle the binary step function when tallying the contacts for each method. The correlations are improved by short simulations (∼25 ns), which increase average R to 0.78. With blind tests using the random forest model, we can further improve average R to 0.84. These easy-to-implement measurements can be utilized in computational screening of viral mutations that escape antibody treatments and potentially other protein-protein interaction problems.
Single-Cell Multi-Omic Roadmap of Human Fetal Pancreatic Development
Authors: Sean de la O , Liu Zhe , Sun Han , Yu Shengyang K. , Wong Daniel M. , Chu Emily , Rao Sneha A. , Eng Nicolas , Peixoto Gabriel , Bouza Jacquelyn , Shen Yin , Knox Sarah M. , Tward Aaron D. , Gloyn Anna L. , Sneddon Julie B.
Published: February 2022

The critical cellular transitions that govern human pancreas development are largely unknown. We performed large-scale single-cell RNA-sequencing (scRNA-Seq) to interrogate human fetal pancreas development from 8-20 weeks post conception. We identified 103 distinct cell types, including four novel endocrine progenitor subtypes displaying unique transcriptional features and differentiation potency. Integration with single-nucleus Assay for Transposase Accessible Chromatin Sequencing (snATAC-Seq) identified candidate regulators of human endocrine cell fate and revealed development-specific regulatory annotation at diabetes risk loci. Comparison of in vitro stem cell-derived and endogenous endocrine cells predicted aberrant genetic programs leading to the generation of off-target cells. Finally, knock-out studies revealed that the gene FEV regulates human endocrine differentiation. This work establishes a roadmap of human pancreatic development, highlights previously unappreciated cellular diversity and lineage dynamics, and provides a blueprint for understanding pancreatic disease and physiology, as well as generating human stem cell-derived islet cells in vitro for regenerative medicine purposes.
Atypical MAPK regulates translocation of GATA transcription factor in response to chemoattractant stimulation
Authors: Hadwiger Jeffrey A. , Cai Huaqing , Aranda Ramee G. , Fatima Saher
Published: February 2022

The Dictyostelium atypical MAPK Erk2 (a.k.a. ErkB) is required for chemotactic responses to external cAMP as individual amoeba aggregate and undergo a multicellular developmental program. External cAMP also stimulates the translocation of the GATA transcription factor, GtaC, a key regulator of developmental gene expression and a developmental timer of repeated cAMP stimulation of developing cells. In this study, atypical MAPK Erk2 was found to be essential for the translocation of GtaC in response to external cAMP as indicated by the cellular distribution of GFP-GtaC reporters. Erk2 was also demonstrated to mediate the translocation of GtaC in response to external folate, a signal that directs the foraging of amoeba for bacterial food sources. Erk1, the only other Dictyostelium MAPK, was not required for the GtaC translocation to either chemoattractant, indicating that GFP-GtaC is a kinase translocation reporter specific for atypical MAPKs. The translocation of GFP-GtaC in response to folate was absent in mutants lacking the folate receptor, Far1 (a.k.a. GrlL) or the coupled G protein, Gα4 subunit. Loss of GtaC function resulted in enhanced chemotactic movement to folate. Inspection of the GtaC primary sequence revealed four sites recognized as Erk2 preferred phosphorylation sites. The creation and analysis of GFP-GtaC mutants with alterations at these sites determined that these sites are required for translocation in response to folate. Consistent with the role of translocation for GtaC function certain combinations of these phosphorylation site alterations interfered with the ability of GFP-GtaC reporter to rescue aggregation of gtaC- cells. These findings provide the first evidence that atypical MAPKs can regulate transcription factors through specific phosphorylation sites in response to chemoattractants. The ability of different stimuli to regulate GtaC translocation through Erk2 indicates atypical MAPKs can regulate different cell fates in part through the translocation of transcription factors.
MafB, WDR77, and ß-catenin interact with each other and have similar genome association profiles
Authors: He Lizhi , Gao Mingshi , Pratt Henry , Weng Zhiping , Struhl Kevin
Published: February 2022

MafB (a bZIP transcription factor), ß-catenin (the ultimate target of the Wnt signal transduction pathway that acts as a transcriptional co-activator of LEF/TCF proteins), and WDR77 (a transcriptional co-activator of multiple hormone receptors) are important for breast cellular transformation. Unexpectedly, these proteins interact directly with each other, and they have similar genomic binding profiles. Furthermore, while some of these common target sites coincide with those bound by LEF/TCF, the majority are located just downstream of transcription initiation sites at a position near paused RNA polymerase (Pol II) and the +1 nucleosome. Occupancy levels of these factors at these promoter-proximal sites are strongly correlated with the level of paused Pol II and transcriptional activity.
Mechanism of Voltage Gating in the Voltage-Sensing Phosphatase Ci-VSP
Authors: Shen Rong , Meng Yilin , Roux Benoît , Perozo Eduardo
Published: February 2022

The conformational changes in voltage-sensing domain (VSD) are driven by the transmembrane electric field acting on charges and countercharges. Yet, the overall energetics and detailed mechanism of this process are not fully understood. Here, we determined free energy and displacement charge landscapes, as well as major conformations corresponding to a complete functional gating cycle in the isolated voltage-sensing domain of the phosphatase Ci-VSP (Ci-VSD) comprising four transmembrane helices (segments S1-S4). Molecular dynamics simulations highlight the extent of S4 movements. In addition to the crystallographically determined activated ‘Up’ and resting ‘Down’ states, the simulations predict two novel Ci-VSD conformations: a deeper resting state (‘Down-minus’) and an extended activated (‘Up-plus’) state. These additional conformations were experimentally probed via systematic cysteine mutagenesis with metal-ion bridges and the engineering of proton conducting mutants at hyperpolarizing voltages. These results show that voltage activation involves sequentially populating these four states in a stepwise way, translating one arginine across the membrane electric field per step, transferring ~3 e0 charges.
Tangled piecewise-linear embeddings of trivalent graphs
Authors: O'Keeffe Michael , Treacy Michael M. J.
Published: February 2022

A method is described for generating and exploring tangled piecewise-linear embeddings of trivalent graphs under the constraints of point-group symmetry. It is shown that the possible vertex-transitive tangles are either graphs of vertex-transitive polyhedra or bipartite vertex-transitive nonplanar graphs. One tangle is found for 6 vertices, three for 8 vertices (tangled cubes), seven for 10 vertices, and 21 for 12 vertices. Also described are four isogonal embeddings of pairs of cubes and 12 triplets of tangled cubes (16 and 24 vertices, respectively). Vertex 2-transitive embeddings are obtained for tangled trivalent graphs with 6 vertices (two found) and 8 vertices (45 found). Symmetrical tangles of the 10-vertex Petersen graph and the 20-vertex Desargues graph are also described. Extensions to periodic tangles are indicated. These are all interesting and viable targets for molecular synthesis.
Highlighting the factors governing transglycosylation in the GH5_5 endo-1,4-β-glucanase RBcel1
Authors: Collet Laetitia , Vander Wauven Corinne , Oudjama Yamina , Galleni Moreno , Dutoit Raphaël
Published: February 2022

Transglycosylating glycoside hydrolases (GHs) offer great potential for the enzymatic synthesis of oligosaccharides. Although knowledge is progressing, there is no unique strategy to improve the transglycosylation yield. Obtaining efficient enzymatic tools for glycan synthesis with GHs remains dependent on an improved understanding of the molecular factors governing the balance between hydrolysis and transglycosylation. This enzymatic and structural study of RBcel1, a transglycosylase from the GH5_5 subfamily isolated from an uncultured bacterium, aims to unravel such factors. The size of the acceptor and donor sugars was found to be critical since transglycosylation is efficient with oligosaccharides at least the size of cellotetraose as the donor and cellotriose as the acceptor. The reaction pH is important in driving the balance between hydrolysis and transglycosylation: hydrolysis is favored at pH values below 8, while transglycosylation becomes the major reaction at basic pH. Solving the structures of two RBcel1 variants, RBcel1_E135Q and RBcel1_Y201F, in complex with ligands has brought to light some of the molecular factors behind transglycosylation. The structure of RBcel1_E135Q in complex with cellotriose allowed a +3 subsite to be defined, in accordance with the requirement for cellotriose as a transglycosylation acceptor. The structure of RBcel1_Y201F has been obtained with several transglycosylation intermediates, providing crystallographic evidence of transglycosylation. The catalytic cleft is filled with (i) donors ranging from cellotriose to cellohexaose in the negative subsites and (ii) cellobiose and cellotriose in the positive subsites. Such a structure is particularly relevant since it is the first structure of a GH5 enzyme in complex with transglycosylation products that has been obtained with neither of the catalytic glutamate residues modified.
High-resolution structures of the bound effectors avadomide (CC-122) and iberdomide (CC-220) highlight advantages and limitations of the MsCI4 soaking system
Authors: Heim Christopher , Hartmann Marcus D.
Published: February 2022

Cereblon (CRBN) is the substrate receptor of the CRL4CRBN E3 ubiquitin ligase and is a central player in targeted protein degradation. It is the target of the thalidomide-derived immunomodulatory drugs (IMiDs) and is one of the most widely employed receptors for proteolysis-targeting chimeras (PROTACs), both of which induce the ubiquitination and subsequent proteasomal degradation of target proteins. Structural studies of ligand binding to CRBN are crucial to elucidate the mechanisms of action and for mediation of side effects, ultimately aiding the development of next-generation IMiDs and PROTACs. With this aim, a crystal-soaking system based on the single-domain bacterial homologue MsCI4 has previously been established and used to delineate the binding modes of several classes of small molecules, including FDA-approved drugs, at the molecular level. Here, this system was used to characterize the binding of the next-generation IMiDs avadomide (CC-122) and iberdomide (CC-220) at high resolution, highlighting the advantages and limitations of the MsCI4 system and its implications for the development of future cereblon effectors.
The parallel-stranded d(CGA) duplex is a highly predictable structural motif with two conformationally distinct strands
Authors: Luteran Emily M. , Paukstelis Paul J.
Published: February 2022

DNA can adopt noncanonical structures that have important biological functions while also providing structural diversity for applications in nanotechnology. Here, the crystal structures of two oligonucleotides composed of d(CGA) triplet repeats in the parallel-stranded duplex form are described. The structure determination of four unique d(CGA)-based parallel-stranded duplexes across two crystal structures has allowed the structural parameters of d(CGA) triplets in the parallel-stranded duplex form to be characterized and established. These results show that d(CGA) units are highly uniform, but that each strand in the duplex is structurally unique and has a distinct role in accommodating structural asymmetries induced by the C–CH+ base pair.
A recurring packing contact in crystals of InlB pinpoints functional binding sites in the internalin domain and the B repeat
Authors: Geerds Christina , Bleymüller Willem M. , Meyer Timo , Widmann Christiane , Niemann Hartmut H.
Published: February 2022

InlB, a bacterial agonist of the human receptor tyrosine kinase MET, consists of an N-terminal internalin domain, a central B repeat and three C-terminal GW domains. In all previous structures of full-length InlB or an InlB construct lacking the GW domains (InlB392), there was no interpretable electron density for the B repeat. Here, three InlB392 crystal structures in which the B repeat is resolved are described. These are the first structures to reveal the relative orientation of the internalin domain and the B repeat. A wild-type structure and two structures of the T332E variant together contain five crystallographically independent molecules. Surprisingly, the threonine-to-glutamate substitution in the B repeat substantially improved the crystallization propensity and crystal quality of the T332E variant. The internalin domain and B repeat are quite rigid internally, but are flexibly linked to each other. The new structures show that inter-domain flexibility is the most likely cause of the missing electron density for the B repeat in previous InlB structures. A potential binding groove between B-repeat strand β2 and an adjacent loop forms an important crystal contact in all five crystallographically independent chains. This region may represent a hydrophobic `sticky patch' that supports protein–protein interactions. This assumption agrees with the previous finding that all known inactivating point mutations in the B repeat lie within strand β2. The groove formed by strand β2 and the adjacent loop may thus represent a functionally important protein–protein interaction site in the B repeat.